Methods for the Analysis of High Precision Differential Hydrogen Deuterium Exchange Data.

Hydrogen/deuterium exchange (HDX) mass spectrometry has been widely applied to the characterization of protein dynamics. More recently, differential HDX has been shown to be effective for the characterization of ligand binding. Previously we have described a fully automated HDX system for use as a ligand screening platform. Here we describe and validate the required data analysis workflow to facilitate the use of HDX as a robust approach for ligand screening. Following acquisition of HDX data at a single on-exchange time point (n ≥ 3), one way analysis of variance in conjunction with the Tukey multiple comparison procedure is used to establish the significance of any measured difference. Analysis results are graphed with respect to a single peptide, ligand or group of ligands, or displayed as an overview within a heat map. For the heat map display, only Δ%D values with a Tukey-adjusted P value less than 0.05 are colored. Hierarchical clustering is used to bin compounds with highly similar HDX signatures. The workflow is evaluated with a small data set showing the ligand binding domain (LDB) of the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) screened against 10 functionally selective ligands. More significantly, data for the vitamin D receptor (VDR) in complex with 87 ligands are presented. To highlight the robustness and precision of our automated HDX platform we analyzed the data from 4191 replicate HDX measurements acquired over an eight month timeframe. Ninety six percent of these measurements were within 10 percent of the mean value. Work has begun to integrate these analysis and graphing components within our HDX software suite.

A method for obtaining randomized block designs in preclinical studies with multiple quantitative blocking variables.

A method is proposed for block randomization of treatments to experimental units that can accommodate both multiple quantitative blocking variables and unbalanced designs. Hierarchical clustering in conjunction with leaf-order optimization is used to block experimental units in multivariate space. The method is illustrated in the context of a diabetic mouse assay. A simulation study is presented to explore the utility of the proposed randomization method relative to that of a completely randomized approach, both in the presence and absence of covariate adjustment. An example R function is provided to illustrate the implementation of the method.

Identification and characterization of noncalcemic, tissue-selective, nonsecosteroidal vitamin D receptor modulators.

Vitamin D receptor (VDR) ligands are therapeutic agents for the treatment of psoriasis, osteoporosis, and secondary hyperparathyroidism. VDR ligands also show immense potential as therapeutic agents for autoimmune diseases and cancers of skin, prostate, colon, and breast as well as leukemia. However, the major side effect of VDR ligands that limits their expanded use and clinical development is hypercalcemia that develops as a result of the action of these compounds mainly on intestine. In order to discover VDR ligands with less hypercalcemia liability, we sought to identify tissue-selective VDR modulators (VDRMs) that act as agonists in some cell types and lack activity in others. Here, we describe LY2108491 and LY2109866 as nonsecosteroidal VDRMs that function as potent agonists in keratinocytes, osteoblasts, and peripheral blood mononuclear cells but show poor activity in intestinal cells. Finally, these nonsecosteroidal VDRMs were less calcemic in vivo, and LY2108491 exhibited more than 270-fold improved therapeutic index over the naturally occurring VDR ligand 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] in an in vivo preclinical surrogate model of psoriasis.

Statistical inference for relative potency in bivariate dose-response assays with correlated responses.

The analysis of dose-response assays measuring two correlated responses is considered. Attention is given to statistical inference for the potency ratio. Results from a simulation study suggest that a post hoc adjustment for the correlation in parameter estimates obtained from univariate fits provides nearly as much power to detect differences in potency as a bivariate response model fit.

Bilateral oophorectomy and breast cancer risk reduction among women with a family history.

PURPOSE: We investigated whether removal of the ovaries is an appropriate risk reduction option for women at elevated risk of breast cancer based on family history of breast cancer. PATIENTS AND METHODS: This question was investigated among a group of 851 women less than age 60 who underwent bilateral oophorectomy between 1970 and 1994 for various reasons. Questionnaire information was collected from 680 (80%) and women were grouped into family risk categories. Reported occurrences of breast cancer were compared to expected rates based on the Gail model. RESULTS: The number of observed breast cancers among women in the cohort was lower than expected for all levels of familial risk, with women in the highest risk groups experiencing about half to one-fourth the number of cancers expected. The apparent protective effect of oophorectomy was stronger among women who were both premenopausal and less than age 50 at time of surgery. CONCLUSION: These data support oophorectomy as a valid breast cancer prevention option for women of all risk levels.

Her-2/neu expression in ovarian cancer: pre- and postexposure to platinum chemotherapy.

OBJECTIVE: Our objective was to determine if the level of Her-2/neu expression in advanced ovarian cancer changed after platinum-based chemotherapy. METHODS: Tissue samples from 43 patients who had surgery for ovarian cancer between 1991 and 2001 at the Mayo Clinic were stained for Her-2/neu expression using the DAKO kit and reviewed independently by two pathologists. Patient charts were reviewed for demographic data, clinical course, chemotherapy, and survival times. RESULTS: Her-2/neu expression was 0 in 30 patients (69.76%), 1+ in 12 patients (27.9%), and 3+ in 1 patient (2.32%) before chemotherapy. After platinum chemotherapy, Her-2/neu expression changed from 0 to 1+ in 7 patients, from 1+ to 0 in 4 patients, 0 to 2+ in 1 patient, and 1+ to 2+ in 2 patients and no change was seen in 29 patients. Both pathologists agreed in all instances when the score was 0 or 1+ and disagreed in two instances between a negative and a weakly positive staining. CONCLUSIONS: Our findings indicate a low level of overexpression of Her-2/neu at the time of primary diagnosis of epithelial ovarian cancer. Relapsing tumors show no significant change in the intensity of Her-2/neu expression after platinum-based chemotherapy. Further prospective studies are needed to confirm these findings and to ascertain whether platinum chemotherapy indeed has no effect on Her-2/neu expression.

Identification of a global gene expression signature of B-chronic lymphocytic leukemia.

B-chronic lymphocytic leukemia (B-CLL) is an adult-onset leukemia characterized by significant accumulation of apoptosis-resistant monoclonal B lymphocytes. In this study, we performed gene expression profiling on B cells obtained from 10 healthy age-matched individuals and CLL B cells from 38 B-CLL patients to identify key genetic differences between CLL and normal B cells. In addition, we leveraged recent independent studies to assess the reproducibility of our molecular B-CLL signature. We used a novel combination of several methods of data analysis including our own software and identified 70 previously unreported genes that differentiate leukemic cells from normal B cells, as well as confirmed recently reported B-CLL specific expression levels of an additional 10 genes. Importantly, many of these genes have previously been linked with other cancers, thus lending further support to their importance as candidate genes leading to B-CLL pathogenesis. We have also validated a subset of these genes using independent methodologies. Moreover, we show that our genes can be used to create a diagnostics signature that performs with perfect sensitivity and specificity in an independent cohort of 21 B-CLL and 20 normal subjects, thus strongly validating the informative nature of our set of genes. Finally, we identified a group of 31 genes that distinguish between low (Rai stage 0) and high (Rai stage 4) risk patients, suggesting that there may also be a gene expression signature that associates with disease progression.

Genome-wide linkage analysis of systolic blood pressure: a comparison of two approaches to phenotype definition.

Problem 1 of the Genetic Analysis Workshop 13(GAW13) contains longitudinal data of cardiovascular measurements from 330 pedigrees. The longitudinal data complicates the phenotype definition because multiple measurements are taken on each individual. To address this complication, we propose an approach that uses generalized estimating equations to obtain residuals for each time point for each person. The mean residual is then taken as the new phenotype with which to use in a variance components linkage analysis. We compare our phenotype definition approach to an approach that first reduces the multiple measurements to a single measurement and then models these summary statistics as regression terms in a variance components analysis. For each approach, multipoint linkage analysis was performed using the residuals and the SOLAR computer program. Our results show little difference between the methods based on the LOD scores.

Differential gene expression of TGF-beta family members and osteopontin in breast tumor tissue: analysis by real-time quantitative PCR.

Several cytokines including members of the transforming growth factor-beta (TGF-beta) and tumor necrosis factor (TNF) families have been implicated in the homing mechanism of breast cancer metastasis. We hypothesize that primary breast tumor tissues differentially express modulators of bone cell function and that this expression pattern contributes to their aggressive and metastatic potential and to their capacity to establish and grow in bone. We, therefore, examined the gene expression pattern of the TGF-beta family members (inhibin/activin betaA subunit (activin betaA), inhibin alpha subunit, and bone morphogenetic protein-2 (BMP-2)), the TNF family members (receptor activator of NF-KB ligand (RANKL) and osteoprotegerin (OPG)), and osteopontin (OPN) in normal, non-invasive, invasive, and metastatic human breast cancer specimens. The mRNA transcript levels of these genes were quantified by reverse transcription (RT) and fluorescent-based kinetic PCR in 18 normal breast tissues, five ductal carcinoma in situ (DCIS). 24 primary breast tumor tissue, and five distant metastases. The mRNA transcript level of each gene was normalized to the amount of beta-actin present in the samples. We observed differential gene expression of the selected TGF-beta family members as well as OPN in breast cancer progression. The average gene expression of the putative tumor suppressor, inhibin alpha, did not significantly change in any of the tumor tissues examined compared to normal breast tissue. The mRNA level of BMP-2, a protein with anti-proliferative effects in breast cancer cell lines and involved in bone formation, significantly decreased in non-invasive, invasive, and liver metastatic breast tumor tissue compared to normal breast tissue. The gene expression of activin betaA, a protein involved in cell proliferation and osteoclast induction, increased in invasive and bone metastatic tumor tissue compared to normal breast tissue. The mRNA level of OPN, a bone matrix protein associated with enhanced malignancy, increased in non-invasive, invasive, and liver and bone metastatic breast tumor tissue compared to normal breast tissue. In contrast, the average gene expressions of the TNF family members, RANKL and OPG, proteins involved in the regulation of osteoclastogenesis, were only slightly if at all changed in the different stage breast tumor tissues. These results suggest that differential gene expression of bone-related proteins, especially OPN, activin betaA, and BMP-2, by primary breast tumor tissues may play a significant role in the invasiveness and metastatic potential of breast cancer.

Host genetic polymorphism analysis in cervical cancer.

BACKGROUND: The natural history of cervical cancer comprises a latency period that probably involves long-term immunologic tolerance of human papillomavirus infection. Identifying host determinants of viral persistence may help to better understand the mechanisms of tolerance and may lead to the development of tests that can allow more focused follow-up of high-risk individuals. METHODS: Genotypic frequencies of 12 polymorphic loci in four candidate genes from 127 cervical cancer patients were compared with a control group of 108 female blood donors. Genotypes were determined by PCR amplification and direct sequencing of isolated genomic DNA. RESULTS: The tumor necrosis factor-alpha (TNFalpha) -238 polymorphism was significantly underrepresented in cervical cancer patients [heterozygotes (HETs), odds ratio (OR) = 0.33; 95% confidence interval (CI), 0.11-0.96], as was the TNFalpha -376 polymorphism (P = 0.02; 0% for any variant genotype in cases vs 4.7% in controls). The NRAMP1 3' untranslated region STP+86 polymorphism also appeared to be inversely associated with cervical cancer, but this result did not reach statistical significance (HET, OR = 0.57; 95% CI, 0.32-1.02). The p53 codon 72 arginine allele showed a suggestive negative association with cervical cancer (HET, OR = 0.49; 95% CI, 0.14-1.63; homozygotes, OR = 0.35; 95% CI, 0.11-1.17). The remaining alleles tested showed no association with cervical cancer. CONCLUSIONS: We identified host genetic polymorphisms that may be associated with cervical cancer risk, some of which have been linked to potential functional effects on cellular immune responses or antigen processing. We failed to confirm earlier reports of increased cervical cancer susceptibility in women who harbor the p53 P72R allele. Although our findings support the general hypothesis that host immunogenetic determinants other than class II MHC may be important in the development of cervical cancer, further analysis of the HLA gene cluster comprising the implicated TNFalpha single-nucleotide polymorphisms will be required to determine whether their association is linkage independent.