Robert Lagier

Thrombophilias and adverse pregnancy outcomes: results from the Danish National Birth Cohort

Summary.  Background:  Inherited thrombophilias have inconsistently been linked to adverse pregnancy outcomes. Differences in study design, size and population could explain this heterogeneity.

Role of a cirrhosis risk score for the early prediction of fibrosis progression in hepatitis C patients with minimal liver disease.

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.

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

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

Neither Replication nor Simulation Supports a Role for the Axon Guidance Pathway in the Genetics of Parkinson's Disease

Susceptibility to sporadic Parkinsons disease (PD) is thought to be influenced by both genetic and environmental factors and their interaction with each other. Statistical models including multiple variants in axon guidance pathway genes have recently been purported to be capable of predicting PD risk, survival free of the disease and age at disease onset; however the specific models have not undergone independent validation. Here we tested the best proposed risk panel of 23 single nucleotide polymorphisms (SNPs) in two PD sample sets, with a total of 525 cases and 518 controls. By single marker analysis, only one marker was significantly associated with PD risk in one of our sample sets (rs6692804: P = 0.03). Multi-marker analysis using the reported model found a mild association in one sample set (two sided P = 0.049, odds ratio for each score change = 1.07) but no significance in the other (two sided P = 0.98, odds ratio = 1), a stark contrast to the reported strong association with PD risk (P = 4.64×10−38, odds ratio as high as 90.8). Following a procedure similar to that used to build the reported model, simulated multi-marker models containing SNPs from randomly chosen genes in a genome wide PD dataset produced P-values that were highly significant and indistinguishable from similar models where disease status was permuted (3.13×10−23 to 4.90×10−64), demonstrating the potential for overfitting in the model building process. Together, these results challenge the robustness of the reported panel of genetic markers to predict PD risk in particular and a role of the axon guidance pathway in PD genetics in general.

The cirrhosis risk score predicts liver fibrosis progression in patients with initially mild chronic hepatitis C.

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Vascular associated gene variants in patients with preeclampsia: results from the Danish National Birth Cohort

Objective. Preeclampsia has been linked to subsequent vascular disease with many shared predisposing factors. We investigated the association between severe preeclampsia, and its subtypes, and specific vascular‐related polymorphisms. Design. The study was a retrospective nested case‐cohort design. Setting. Pregnant Danish women participating in the Danish National Birth Cohort. Population. 263 cases of severe preeclampsia and 1851 random controls were selected from the Danish National Birth Cohort. Methods. We validated all cases of severe preeclampsia and genotyped for 108 single nucleotide polymorphisms (SNPs) that were selected based on previous publications on the association with vascular disease. Logistic models were used for statistical analyses. Main outcome measures. Maternal polymorphisms in genomic models. Results. We found 17 of 108 SNPs associated with severe preeclampsia (p < 0.05). Women homozygous for the rs1799983 in NOS3 were 1.6‐fold [95% confidence interval (CI) 1.0–2.4] more likely to develop severe preeclampsia. Women homozygous for the rs1010 SNP in VAMP8 were twofold (95%CI 1.1–3.5) more likely to deliver preterm when preeclampsia was present. Women homozygous for the rs10811661 SNP were 2.1‐fold (95%CI 1.1–3.9) more likely to develop severe preeclampsia and 3.7‐fold (95%CI 1.1–12.4) more likely to deliver a small‐for‐gestational age child when preeclampsia was present. All associations are available as Supporting Information. Conclusion. We found several vascular‐associated SNPs linked to severe preeclampsia; however, most of these associations are probably by pure chance, which warrants replication and further translational research. To date, no specific SNP has yet proven valuable in a clinical setting in predicting preeclampsia.

Abstract 5356: Validation of a clinically actionable cancer core gene test for solid tumors facilitating targeted molecular therapy and immunotherapy

 This study included formalin-fixed, paraffin-embedded (FFPE) tumor samples and whole blood samples from lung, colorectal, melanoma, and breast cancer tumors.  Four major types of mutations were assessed: single nucleotide variations (SNVs), insertion/deletions (INDELs), whole gene copy number variations (CNVs), and structural rearrangements (translocations).  The test analyzed exons from 49 core cancer genes, introns from a subset of genes known to have prevalent gene rearrangements, and the TERT (telomerase reverse transcriptase) promoter region.  Targeted DNA regions were captured by hybridization with complementary biotinylated RNA baits, and next-generation sequencing was performed.  Microsatellite instability (MSI) status was evaluated when paired whole blood samples were available.

DEVELOPMENT OF A NOVEL MULTI-BIOMARKER ASSESSMENT SCORE FOR ALZHEIMER’S DISEASE

TDP had lower levels of sAPPb and higher levels of YKL-40 in CSF compared to controls (Fig. 1). The sAPPb/YKL-40 ratio was lower in both FTLD groups compared to controls. In the ROC analysis (Fig. 2), the sAPPb/YKL-40 ratio had an area under the curve of 0.91 (95%CI 0.86-0.96) to distinguish FTLD subjects from controls, but lower values to distinguish FTLD from AD (AUC 0.70; 95%CI 0.61-0.79) and to discriminate FTLDTau from FTLD-TDP (AUC 0.67; 95%CI 0.51-0.82). Conclusions:These findings suggest that the sAPPb/YKL-40 ratio could be of interest to distinguish patients with FTLD from those with similar clinical phenotypes. Although the ratio cannot be used to discriminate between the main pathologies underlying FTLD, it might be useful to interrogate the neuropathological substrate in some clinical scenarios and, eventually, to select potential candidates for clinical trials.

Abstract P2-09-19: Expression Profiling of a Metastasis Score and a “Glycolytic Index” in Genetically-Defined Human Breast Isogenic Cell Lines Treated with a PI3K Inhibitor

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.

Abstract B116: Molecular profiling of breast cancer cell lines using signatures reported in prognostic and predictive patient studies

Background: Breast cancer cell lines recapitulate complex tumors in humans and have been widely used to shed light on oncogenesis and characterize the response to established and new therapeutics. Gene signatures may be used to assess which oncogenic pathways are deregulated in breast cancer cells and to guide targeted therapy. Browser‐based access to CGH and expression array profiles has begun to provide systems biology information on these cell lines. Since RT‐PCR is increasingly being used to profile gene signatures in patient breast cancer tumors and due to often observed discordance between arrays and RT‐PCR, we embarked on a translational study to profile the expression levels of constituent genes and derived scores of reported signatures in commonly used breast cancer cell lines. Methods: RNA from 47 cell lines with substantial overlaps with cell line collections reported by Neve et al. (36/51), Kao et al. (36/52) and the Integrated Cancer Biology Program (ICBP‐45) (43/45) was profiled using RT‐PCR for individual genes of the signatures reported by Paik et al. (21‐gene Recurrence Score and subset gene groups), Tutt et al (14‐gene metastasis score), and Teschendorff and Caldas (7‐gene immune response score). The relative changes in gene expression were calculated as previously described. Individual gene and group weightings were as reported. Results: The 14‐gene metastasis signature was highly correlated with the Proliferation Group of the Recurrence Score (r2=0.78). In ER positive cell line subset analysis, this same comparison resulted in r2=0.72. The ER Group of the Recurrence Score was correlated to the overall Recurrence Score (r2=0.65) and highly correlated to the level of the progesterone receptor (r2=0.82). The HER2 mRNA level alone was highly correlated to the HER2 Group of the Recurrence Score (r2=0.73). The metastasis score differentially‐weighted with the hormone receptor levels was correlated with the Recurrence Score (r2=0.70). The 7‐gene immune response gene set was not correlated with proliferation indices or composite scores in the combined or ER‐negative cell line collections. Correlations with the luminal and basal tumor subtypes required composite scores of proliferation, HER2 and hormone receptor status. Conclusion: Breast cancer cell lines of commonly used collections have an elevated and continuous range of molecular proliferation indices as determined by groups of genes in signatures reported in community and randomized control trial settings. Even though sharing only a limited number of genes, composite scores encompassing proliferation, hormone receptors and growth factor RNA levels are similar. Gene sets focused on immune response may provide discrete additional information. Knowledge of pharmacologic responses of these cell lines to established and new therapeutics with associated subgroup and derived overall scores may contribute to our understanding of the discrete oncologic pathways involved and inform future individualized patient management. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B116.