The number and timing of time -dependent covariate measurements for the Cox regression model

The problem of analyzing data with updated measurements in the time-dependent proportional hazards model arises frequently in practice. One available option is to reduce the number of intervals (or updated measurements) to be included in the Cox regression model. We empirically investigated the bias of the estimator of the time-dependent covariate while varying the effect of failure rate, sample size, true values of the parameters and the number of intervals. We also evaluated how often a time-dependent covariate needs to be collected and assessed the effect of sample size and failure rate on the power of testing a time-dependent effect.^ A time-dependent proportional hazards model with two binary covariates was considered. The time axis was partitioned into k intervals. The baseline hazard was assumed to be 1 so that the failure times were exponentially distributed in the ith interval. A type II censoring model was adopted to characterize the failure rate. The factors of interest were sample size (500, 1000), type II censoring with failure rates of 0.05, 0.10, and 0.20, and three values for each of the non-time-dependent and time-dependent covariates (1/4,1/2,3/4).^ The mean of the bias of the estimator of the coefficient of the time-dependent covariate decreased as sample size and number of intervals increased whereas the mean of the bias increased as failure rate and true values of the covariates increased. The mean of the bias of the estimator of the coefficient was smallest when all of the updated measurements were used in the model compared with two models that used selected measurements of the time-dependent covariate. For the model that included all the measurements, the coverage rates of the estimator of the coefficient of the time-dependent covariate was in most cases 90% or more except when the failure rate was high (0.20). The power associated with testing a time-dependent effect was highest when all of the measurements of the time-dependent covariate were used. An example from the Systolic Hypertension in the Elderly Program Cooperative Research Group is presented. ^

Biologic and molecular analysis of tumor suppressor loci in human glioblastoma multiforme

Molecular and cytogenetic analyses of human glioblastomas have revealed frequent genetic alterations, including major deletions in chromosomes 9, 10, and 17, suggesting the presence of glioma-associated tumor suppressor genes on these chromosomes. To examine this hypothesis, copies of chromosomes 2, 4, and 10 derived from a human fibroblast cell line were independently introduced into a human glioma cell line, U251, by microcell-mediated chromosomal transfer. Successful transfer of chromosomes in each case was confirmed by resistance to the drug G418, indicating the presence of the neomycin-resistance gene previously integrated into each transferred chromosome. The presence of novel chromosomes and or chromosomal fragments was also demonstrated by molecular and karyotypic analyses. The hybrid clones containing either a novel chromosome 4 or chromosome 10 displayed suppression of the tumorigenic phenotype in vivo and suppression of the transformed phenotype in vitro, while cells containing a transferred chromosome 2 failed to alter their tumorigenic phenotype. The hybrid cells containing chromosome 4 or 10 exhibited a significant decrease in their saturation density, altered cellular morphology at high cell density, but only a slight decrease in their exponential growth rate. A dramatic decrease was observed in growth of cells with chromosome 4 or 10 in soft agarose, with the number and size of the colonies being greatly reduced, compared to the parental or chromosome 2 containing cells. The introduction of chromosome 4 or 10 also completely suppressed tumor formation in nude mice. These studies indicate that chromosome 10, as hypothesized, and chromosome 4, a novel finding for gliomas, harbor tumor suppressor loci that may be directly involved in the initiation or progression of normal glial precursors to human glioblastoma multiforme. ^

Metastatic lung cancer in a new mouse model inheriting p53 and latent K-ras mutations

Lung cancer is a devastating disease with very poor prognosis. The design of better treatments for patients would be greatly aided by mouse models that closely resemble the human disease. The most common type of human lung cancer is adenocarcinoma with frequent metastasis. Unfortunately, current models for this tumor are inadequate due to the absence of metastasis. Based on the molecular findings in human lung cancer and metastatic potential of osteosarcomas in mutant p53 mouse models, I hypothesized that mice with both K-ras and p53 missense mutations might develop metastatic lung adenocarcinomas. Therefore, I incorporated both K-rasLA1 and p53RI72HΔg alleles into mouse lung cells to establish a more faithful model for human lung adenocarcinoma and for translational and mechanistic studies. Mice with both mutations ( K-rasLA1/+ p53R172HΔg/+) developed advanced lung adenocarcinomas with similar histopathology to human tumors. These lung adenocarcinomas were highly aggressive and metastasized to multiple intrathoracic and extrathoracic sites in a pattern similar to that seen in lung cancer patients. This mouse model also showed gender differences in cancer related death and developed pleural mesotheliomas in 23.2% of them. In a preclinical study, the new drug Erlotinib (Tarceva) decreased the number and size of lung lesions in this model. These data demonstrate that this mouse model most closely mimics human metastatic lung adenocarcinoma and provides an invaluable system for translational studies. ^ To screen for important genes for metastasis, gene expression profiles of primary lung adenocarcinomas and metastases were analyzed. Microarray data showed that these two groups were segregated in gene expression and had 79 highly differentially expressed genes (more than 2.5 fold changes and p<0.001). Microarray data of Bub1b, Vimentin and CCAM1 were validated in tumors by quantitative real-time PCR (QPCR). Bub1b , a mitotic checkpoint gene, was overexpressed in metastases and this correlated with more chromosomal abnormalities in metastatic cells. Vimentin, a marker of epithelial-mesenchymal transition (EMT), was also highly expressed in metastases. Interestingly, Twist, a key EMT inducer, was also highly upregulated in metastases by QPCR, and this significantly correlated with the overexpression of Vimentin in the same tumors. These data suggest EMT occurs in lung adenocarcinomas and is a key mechanism for the development of metastasis in K-ras LA1/+ p53R172HΔg/+ mice. Thus, this mouse model provides a unique system to further probe the molecular basis of metastatic lung cancer.^

Effect of patient accrual patterns on power and size of log-rank test for time-to-event outcome in clinical trials

The determination of size as well as power of a test is a vital part of a Clinical Trial Design. This research focuses on the simulation of clinical trial data with time-to-event as the primary outcome. It investigates the impact of different recruitment patterns, and time dependent hazard structures on size and power of the log-rank test. A non-homogeneous Poisson process is used to simulate entry times according to the different accrual patterns. A Weibull distribution is employed to simulate survival times according to the different hazard structures. The current study utilizes simulation methods to evaluate the effect of different recruitment patterns on size and power estimates of the log-rank test. The size of the log-rank test is estimated by simulating survival times with identical hazard rates between the treatment and the control arm of the study resulting in a hazard ratio of one. Powers of the log-rank test at specific values of hazard ratio (≠1) are estimated by simulating survival times with different, but proportional hazard rates for the two arms of the study. Different shapes (constant, decreasing, or increasing) of the hazard function of the Weibull distribution are also considered to assess the effect of hazard structure on the size and power of the log-rank test. ^

Heterotrimeric G protein -mediated signal transduction in Neurospora crassa

Heterotrimeric G protein-mediated signal transduction is one of numerous means that cells utilize to respond to external stimuli. G proteins consist of α, β andγ subunits. Extracellular ligands bind to seven-transmembrane helix receptors, triggering conformational changes. This is followed by activation of coupled G proteins through the exchange of GDP for GTP on the Gα subunit. Once activated, Gα-GTP dissociates from the βγ dimer. Both of these two moieties can interact with downstream effectors, such as adenylyl cyclase, phospholipase C, phosphodiesterases, or ion channels, leading to a series of changes in cellular metabolism and physiology. ^ Neurospora crassa is a eukaryotic multicellular filamentous fungus, with asexual/vegetative and sexual phases to its life cycle. Three Gα (GNA-1, GNA-2, GNA-3) and one Gβ (GNB-1) proteins have been identified in this organism. This dissertation investigates GNA-1 and GNB-1 mediated signaling pathways in N. crassa. ^ GNA-1 was the first identified microbial Gα that belongs to a mammalian superfamily (Gαi). Deletion of GNA-1 leads to multiple defects in N. crassa. During the asexual cycle, Δgna-1 strains display a slower growth rate and delayed conidiation on solid medium. In the sexual cycle, the Δgna-1 mutant is male-fertile but female-sterile. Biochemical studies have shown that Δ gna-1 strains have lower adenosine 3′–5 ′ cyclic monophosphate (cAMP) levels than wild type under conditions where phenotypic defects are observed. In this thesis work, strains containing one of two GTPase-deficient gna-1 alleles (gna-1 R178C, gna-1Q204L) leading to constitutive activation of GNA-1 have been constructed and characterized. Activation of GNA-1 causes uncontrolled aerial hyphae proliferation, elevated sensitivity to heat and oxidative stresses, and lower carotenoid synthesis. To further study the function of GNA-1, constructs to enable expression of mammalian Gαi superfamily members were transformed into a Δ gna-1 strain, and complementation of Δgna-1 defects investigated. Gαs, which is not a member of Gα i superfamily was used as a control. These mammalian Gα genes were able to rescue the vegetative growth rate defect of the Δ gna-1 strain in the following order: Gαz > Gα o > Gαs > Gαt > Gαi. In contrast, only Gαo was able to complement the sexual defect of a Δgna-1 strain. With regard to the thermotolerance phenotype, none of the mammalian Gα genes restored the sensitivity to a wild type level. These results suggest that GNA-1 regulates two independent pathways during the vegetative and sexual cycles in N. crassa. ^ GNB-1, a G protein β subunit from N. crassa, was identified and its functions investigated in this thesis work. The sequence of the gnb-1 gene predicts a polypeptide of 358 residues with a molecular mass of 39.7 kDa. GNB-1 exhibits 91% identity to Cryphonectria parasitica CPGB-1, and also displays significant homology with human and Dictyostelium Gβ genes (∼66%). A Δ gnb-1 strain was constructed and shown to exhibit defects in asexual spore germination, vacuole number and size, mass accumulation and female fertility. A novel role for GNB-1 in regulation of GNA-1 and GNA-2 protein levels was also demonstrated. ^