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Supplementary Tables 1-2 from Multiple Gene Expression Classifiers from Different Array Platforms Predict Poor Prognosis of Colorectal Cancer

Purpose: This study aimed to develop gene classifiers to predict colorectal cancer recurrence. We investigated whether gene classifiers derived from two tumor series using different array platforms could be independently validated by application to the alternate series of patients.Experimental Design: Colorectal tumors from New Zealand (n = 149) and Germany (n = 55) patients had a minimum follow-up of 5 years. RNA was profiled using oligonucleotide printed microarrays (New Zealand samples) and Affymetrix arrays (German samples). Classifiers based on clinical data, gene expression data, and a combination of the two were produced and used to predict recurrence. The use of gene expression information was found to improve the predictive ability in both data sets. The New Zealand and German gene classifiers were cross-validated on the German and New Zealand data sets, respectively, to validate their predictive power. Survival analyses were done to evaluate the ability of the classifiers to predict patient survival.Results: The prediction rates for the New Zealand and German gene-based classifiers were 77% and 84%, respectively. Despite significant differences in study design and technologies used, both classifiers retained prognostic power when applied to the alternate series of patients. Survival analyses showed that both classifiers gave a better stratification of patients than the traditional clinical staging. One classifier contained genes associated with cancer progression, whereas the other had a large immune response gene cluster concordant with the role of a host immune response in modulating colorectal cancer outcome.Conclusions: The successful reciprocal validation of gene-based classifiers on different patient cohorts and technology platforms supports the power of microarray technology for individualized outcome prediction of colorectal cancer patients. Furthermore, many of the genes identified have known biological functions congruent with the predicted outcomes.

Recent reports have suggested a reproducible association between the rs11121615 SNP, located within an intron of the castor zinc finger 1 (CASZ1) gene, and varicose veins. This study aimed to determine if this variant is also differentially associated with the various clinical classifications of chronic venous disease (CVD). The rs11121615 SNP was genotyped in two independent cohorts from New Zealand (n = 1876 controls /1606 CVD cases) and the Netherlands (n = 1626/2966). Participants were clinically assessed using well-established CVD criteria. The association between the rs11121615 C-allele and varicose veins was validated in both cohorts. This was strongest in those with higher clinical severity classes and was not significant in those with non-varicose vein CVD. Functional analysis of the rs11121615 variant demonstrated that the risk allele was associated with increased enhancer activity. This study demonstrates that the CASZ1 gene associated C-allele of rs11121615 has a significant, reproducible, association with CVD (CEAP C ≥ 2 meta-odds ratio 1.31, 95% CI 1.27–1.34, P = 1 × 10−98, PHet = 0.25), but not with non-varicose vein (CEAP C1, telangiectasia or reticular veins) forms of venous disease. The effect size of this association therefore appears to be susceptible to influence by phenotypic heterogeneity, particularly if a cohort includes a large number of cases with lower severity CVD.

Aortic valve stenosis (AS) is a major cause of morbidity and mortality, with no effective medical therapies. Investigation into the underlying biology of AS in humans is limited by difficulties in obtaining healthy valvular tissue for use as a control group. However, micro-ribonucleic acids (miRNAs) are stable in post-mortem tissue. We compared valve specimens from patients undergoing aortic valve replacement for AS to non-diseased cadaveric valves. We found 106 differentially expressed miRNAs (p

Objective Cholesterol crystals have been shown to cause inflammation, and ultimately atherosclerotic lesions through the activation of the NLRP3 inflammasome. As cholesterol crystals have also been found in the walls of patients with abdominal aortic aneurysms (AAA), it is possible that the NLRP3 inflammasome is involved in AAA and genetic variability within this protein complex could alter disease risk. The primary objective of this study was to assess whether there is genetic evidence for a role of the NLRP3 inflammasome in AAA by testing for association of AAA with functional single nucleotide polymorphisms (SNPs) in the CARD8 and NLRP3 genes. Methods AAA patients ( n =1151) and controls ( n =727) were genotyped for CARD8 SNP rs2043211 and NLRP3 SNP rs35829419 using TaqMan SNP assays. IL1-β, C-reactive protein (CRP), and lipoprotein (a) [Lp(a)] were measured in the plasma of a subset of study participants. The Kruskal–Wallis Rank test was conducted to test for differences in mean concentration of IL1-β, CRP and Lp(a). Logistic regression was used to test for interaction between CARD8 and NLRP3. Results Significantly higher mean concentration of plasma IL1-β was observed in study participants who were homozygous for the common C allele of NLRP3 rs35829419 ( p =0.010). Interaction between rs2043211 and rs35829419 was observed in this dataset ( χ 2 =6.22; p =0.044), which strengthened when adjusted for age, gender, smoking, diabetes, hypertension, and dyslipidemia ( χ 2 =14.75; p =0.012); and separately for NOD2 genotype ( χ 2 =14.06; p =0.015). Conclusion Our finding suggests genetic variability within the NLRP3 inflammasome may be important in the pathophysiology of AAA.

Background Aortic stenosis (AS) is a progressive condition leading to heart failure and death without treatment. No medical therapy currently exists for AS, and a major management challenge is deciding on the correct timing of aortic valve replacement. MicroRNAs (mi RNA s) are short noncoding RNA s that are stable in the circulation. We wished to use mi RNA s as biomarkers of disease in AS . Methods and Results We performed microarray‐based whole mi RN ome profiling of 24 participants with AS and 27 control participants. After adjustment for age and multiple testing, we identified 4 mi RNA s significantly different between groups. These findings were then examined using quantitative polymerase chain reaction in a larger validation cohort of 101 controls and 94 participants with AS, stratified in a prespecified analysis by presence of coexisting coronary artery disease ( CAD ). We obtained mixed results for miR‐22‐3p, miR‐24‐3p, miR‐382‐5p, and miR‐451a in the validation cohort, with differing associations according to CAD status. miR‐21‐5p was increased in AS patients without CAD, but there was no difference between groups with CAD . Conclusion Despite holding great promise, circulating mi RNA profiling requires further refinement before translation into clinical use as a biomarker in aortic stenosis.

Colorectal cancer is one of the five leading causes of cancer mortality worldwide. The mechanisms of pathogen clearance, inflammation and regulation by T cells in the healthy bowel are also important in controlling tumor growth. The majority of studies analyzing T cells and their relationship to colorectal tumor growth have focused on individual T cell markers or gene clusters and thus the complexity of the T cell response contributing to the growth of the tumor is not clear. We have studied the T cells in colorectal cancer patients and have defined a unique T cell signature for colorectal tumor tissue. Using a novel analytical flow cytometric approach in concert with confocal microscopy, we have shown that the tumor has a lower frequency of effector T cells (CD69+), but a higher frequency of both regulatory (CD25hi Foxp3+) and inflammatory T cells (IL-17+) compared with associated nontransformed bowel tissue. We have also identified minor populations of T cells expressing conventional markers of both inflammatory and regulatory T cells (CD4+IL-17+Foxp3+) in the tumor tissue. These cells may represent intermediate populations or they may dictate an inflammatory versus regulatory function in surrounding T cells. Together, these data describe an immune microenvironment in colorectal cancer unique to the tumor tissue and distinct from the surrounding healthy bowel tissue, and this distinct environment is reflected by a gradient of T cells expressing markers of multiple T cell populations. These findings may be used to improve diagnosis and prognosis of colorectal cancer patients.

Contains fulltext : 97601.pdf (Publisher’s version ) (Closed access) Abdominal aortic aneurysm (AAA) is a common cause of morbidity and mortality and has a significant heritability. We carried out a genome-wide association discovery study of 1866 patients with AAA and 5435 controls and replication of promising signals (lead SNP with a p value < 1 x 10(-5)) in 2871 additional cases and 32,687 controls and performed further follow-up in 1491 AAA and 11,060 controls. In the discovery study, nine loci demonstrated association with AAA (p < 1 x 10(-5)). In the replication sample, the lead SNP at one of these loci, rs1466535, located within intron 1 of low-density-lipoprotein receptor-related protein 1 (LRP1) demonstrated significant association (p = 0.0042). We confirmed the association of rs1466535 and AAA in our follow-up study (p = 0.035). In a combined analysis (6228 AAA and 49182 controls), rs1466535 had a consistent effect size and direction in all sample sets (combined p = 4.52 x 10(-10), odds ratio 1.15 [1.10-1.21]). No associations were seen for either rs1466535 or the 12q13.3 locus in independent association studies of coronary artery disease, blood pressure, diabetes, or hyperlipidaemia, suggesting that this locus is specific to AAA. Gene-expression studies demonstrated a trend toward increased LRP1 expression for the rs1466535 CC genotype in arterial tissues; there was a significant (p = 0.029) 1.19-fold (1.04-1.36) increase in LRP1 expression in CC homozygotes compared to TT homozygotes in aortic adventitia. Functional studies demonstrated that rs1466535 might alter a SREBP-1 binding site and influence enhancer activity at the locus. In conclusion, this study has identified a biologically plausible genetic variant associated specifically with AAA, and we suggest that this variant has a possible functional role in LRP1 expression. 9 p.

This study aimed to investigate whether the endogenous active levels of MMP-9 or tissue inhibitor of metalloproteinases-1 (TIMP-1) were related to indices of diastolic dysfunction (DD) in the setting of contemporary treatment of coronary artery disease (CAD).We prospectively studied 116 patients with CAD and preserved left ventricular LV systolic function (ejection fraction ≥ 45%). All patients were free of heart failure symptoms at recruitment and underwent percutaneous intervention (PCI) of culprit lesions. Demographic and angiographic characteristics were collected. Plasma samples were analysed for the active form of MMP-9 and TIMP-1 using enzyme-linked immunosorbent assay-based isoform sensitive assays. Conventional and tissue Doppler-echocardiographic assessment of diastolic filling was undertaken with measurements of maximal early (E) and late (A) transmitral velocities in diastole, E/A ratio, E-wave deceleration time, isovolumic relaxation time, peak systolic (S), diastolic (D) and atrial reversal velocities of pulmonary venous flow, S/D fraction, time difference between A and duration of atrial reversal flow, early diastolic peak velocities of the lateral mitral annulus (E') and E/E'. Active MMP-9 level was higher in patients with more severe phases of DD (normal [n=22]: median 0.57 ng/ml; mild [n=19] 0.83 ng/ml; mild-moderate [n=41] 0.64 ng/ml; moderate or severe [n=34] 1.63 ng/ml; p0.0001 for trend). Three month post-PCI elevated levels of active MMP-9 had an adjusted odds ratio of 11.2 (2.3-56.0, p0.004) for association with moderate or severe DD.Elevated active MMP-9 level is associated with more severe DD in patients with CAD and preserved systolic function, which may indicate abnormal extracellular matrix metabolism in myocardial ischaemia.

Compromised quality of formalin-fixed paraffin-embedded (FFPE)-derived DNA has compounded the use of archival specimens for array-based genomic studies. Recent technological advances have led to first successes in this field; however, there is currently no general agreement on the most suitable platform for the array-based analysis of FFPE DNA. In this study, FFPE and matched fresh-frozen (FF) specimens were separately analyzed with Affymetrix single nucleotide polymorphism (SNP) 6.0 and Agilent 4x44 K oligonucleotide arrays to compare the genomic profiles from the two tissue sources and to assess the relative performance of the two platforms on FFPE material. Genomic DNA was extracted from matched FFPE–FF pairs of normal intestinal epithelium from four patients and were applied to the SNP and oligonucleotide platforms according to the manufacturer-recommended protocols. On the Affymetrix platform, a substantial increase in apparent copy number alterations was observed in all FFPE tissues relative to their matched FF counterparts. In contrast, FFPE and matched FF genomic profiles obtained via the Agilent platform were very similar. Both the SNP and the oligonucleotide platform performed comparably on FF material. This study demonstrates that Agilent oligonucleotide array comparative genomic hybridization generates reliable results from FFPE extracted DNA, whereas the Affymetrix SNP-based array seems less suitable for the analysis of FFPE material.

Purpose: To investigate the maximal physiological responses between aquatic and land-based graded exercise tests in overweight women. Methods: Twenty healthy, overweight (body mass index (BMI) ≥ 25 kg·m- 2 ), Caucasian women (mean ± SD; age 48 ± 7 yr, BMI 30 ± 4 kg.m- 2 ) completed a deep water running (DWR) and treadmill walking (TMW) graded exercise test. Maximal responses during the DWR and TMW graded exercise tests were compared using paired t-tests. Comparisons were made in the incidence of achievement of maximal oxygen consumption (VO 2max ) criteria between DWR and TMW protocols. Criteria were a plateau in VO 2 (change < 2.1 mL·kg·min -1 ), heart rate (HR) equal to or above the age-adjusted maximum, and respiratory exchange ratio (RER) ≥ 1.15. Results: Maximal responses for VO 2max (22.5 ± 4.86 vs 27.7 ± 4.73 mL·kg·min -1 ), HR max (159 ± 16 vs 170 ± 12 bpm), and RER (1.03 ± 0.06 vs 1.10 ± 0.06) were significantly lower (P < 0.01) for the DWR test compared with the TMW test, respectively. Achievement of various VO 2max criteria was demonstrated more consistently during the TMW test than the DWR test. Conclusion: Maximal physiological responses of overweight women to DWR and TMW are significantly different but are comparable with other populations. As the maximal responses for DWR compared with TMW differ, the use of land-based criteria for VO 2max is not recommended for a graded DWR exercise test.

Purpose: This study aimed to develop gene classifiers to predict colorectal cancer recurrence. We investigated whether gene classifiers derived from two tumor series using different array platforms could be independently validated by application to the alternate series of patients. Experimental Design: Colorectal tumors from New Zealand (n = 149) and Germany (n = 55) patients had a minimum follow-up of 5 years. RNA was profiled using oligonucleotide printed microarrays (New Zealand samples) and Affymetrix arrays (German samples). Classifiers based on clinical data, gene expression data, and a combination of the two were produced and used to predict recurrence. The use of gene expression information was found to improve the predictive ability in both data sets. The New Zealand and German gene classifiers were cross-validated on the German and New Zealand data sets, respectively, to validate their predictive power. Survival analyses were done to evaluate the ability of the classifiers to predict patient survival. Results: The prediction rates for the New Zealand and German gene-based classifiers were 77% and 84%, respectively. Despite significant differences in study design and technologies used, both classifiers retained prognostic power when applied to the alternate series of patients. Survival analyses showed that both classifiers gave a better stratification of patients than the traditional clinical staging. One classifier contained genes associated with cancer progression, whereas the other had a large immune response gene cluster concordant with the role of a host immune response in modulating colorectal cancer outcome. Conclusions: The successful reciprocal validation of gene-based classifiers on different patient cohorts and technology platforms supports the power of microarray technology for individualized outcome prediction of colorectal cancer patients. Furthermore, many of the genes identified have known biological functions congruent with the predicted outcomes.

Background The immune response has been proposed to be an important factor in determining patient outcome in colorectal cancer (CRC). Previous studies have concentrated on characterizing T cell populations in the primary tumour where T cells with regulatory effect (Foxp3+ Tregs) have been identified as both enhancing and diminishing anti-tumour immune responses. No previous studies have characterized the T cell response in the regional lymph nodes in CRC. Methods Immunohistochemistry was used to analyse CD4, CD8 or Foxp3+ T cell populations in the regional lymph nodes of patients with stage II CRC (n = 31), with (n = 13) or without (n = 18) cancer recurrence after 5 years of follow up, to determine if the priming environment for anti-tumour immunity was associated with clinical outcome. Results The proportions of CD4, CD8 or Foxp3+ cells in the lymph nodes varied widely between and within patients, and there was no association between T cell populations and cancer recurrence or other clinicopathological characteristics. Conclusions These data indicate that frequency of these T cell subsets in lymph nodes may not be a useful tool for predicting patient outcome.