A Bayesian approach to determine the composition of heterogeneous cancer tissue.

BACKGROUND: Cancer Tissue Heterogeneity is an important consideration in cancer research as it can give insights into the causes and progression of cancer. It is known to play a significant role in cancer cell survival, growth and metastasis. Determining the compositional breakup of a heterogeneous cancer tissue can also help address the therapeutic challenges posed by heterogeneity. This necessitates a low cost, scalable algorithm to address the challenge of accurate estimation of the composition of a heterogeneous cancer tissue. METHODS: In this paper, we propose an algorithm to tackle this problem by utilizing the data of accurate, but high cost, single cell line cell-by-cell observation methods in low cost aggregate observation method for heterogeneous cancer cell mixtures to obtain their composition in a Bayesian framework. RESULTS: The algorithm is analyzed and validated using synthetic data and experimental data. The experimental data is obtained from mixtures of three separate human cancer cell lines, HCT116 (Colorectal carcinoma), A2058 (Melanoma) and SW480 (Colorectal carcinoma). CONCLUSION: The algorithm provides a low cost framework to determine the composition of heterogeneous cancer tissue which is a crucial aspect in cancer research.

An in-silico study examining the induction of apoptosis by Cryptotanshinone in metastatic melanoma cell lines.

BACKGROUND: Metastatic melanoma is an aggressive form of skin cancer that evades various anti-cancer treatments including surgery, radio-,immuno- and chemo-therapy. TRAIL-induced apoptosis is a desirable method to treat melanoma since, unlike other treatments, it does not harm non-cancerous cells. The pro-inflammatory response to melanoma by nF κB and STAT3 pathways makes the cancer cells resist TRAIL-induced apoptosis. We show that due to to its dual action on DR5, a death receptor for TRAIL and on STAT3, Cryptotanshinone can be used to increase sensitivity to TRAIL. METHODS: The development of chemoresistance and invasive properties in melanoma cells involves several biological pathways. The key components of these pathways are represented as a Boolean network with multiple inputs and multiple outputs. RESULTS: The possible mutations in genes that can lead to cancer are captured by faults in the combinatorial circuit and the model is used to theoretically predict the effectiveness of Cryptotanshinone for inducing apoptosis in melanoma cell lines. This prediction is experimentally validated by showing that Cryptotanshinone can cause enhanced cell death in A375 melanoma cells. CONCLUSION: The results presented in this paper facilitate a better understanding of melanoma drug resistance. Furthermore, this framework can be used to detect additional drug intervention points in the pathway that could amplify the action of Cryptotanshinone.

Dynamical modeling of uncertain interaction-based genomic networks.

BACKGROUND: Most dynamical models for genomic networks are built upon two current methodologies, one process-based and the other based on Boolean-type networks. Both are problematic when it comes to experimental design purposes in the laboratory. The first approach requires a comprehensive knowledge of the parameters involved in all biological processes a priori, whereas the results from the second method may not have a biological correspondence and thus cannot be tested in the laboratory. Moreover, the current methods cannot readily utilize existing curated knowledge databases and do not consider uncertainty in the knowledge. Therefore, a new methodology is needed that can generate a dynamical model based on available biological data, assuming uncertainty, while the results from experimental design can be examined in the laboratory. RESULTS: We propose a new methodology for dynamical modeling of genomic networks that can utilize the interaction knowledge provided in public databases. The model assigns discrete states for physical entities, sets priorities among interactions based on information provided in the database, and updates each interaction based on associated node states. Whenever uncertainty in dynamics arises, it explores all possible outcomes. By using the proposed model, biologists can study regulation networks that are too complex for manual analysis. CONCLUSIONS: The proposed approach can be effectively used for constructing dynamical models of interaction-based genomic networks without requiring a complete knowledge of all parameters affecting the network dynamics, and thus based on a small set of available data.

Identifying mechanistic similarities in drug responses.

MOTIVATION: In early drug development, it would be beneficial to be able to identify those dynamic patterns of gene response that indicate that drugs targeting a particular gene will be likely or not to elicit the desired response. One approach would be to quantitate the degree of similarity between the responses that cells show when exposed to drugs, so that consistencies in the regulation of cellular response processes that produce success or failure can be more readily identified. RESULTS: We track drug response using fluorescent proteins as transcription activity reporters. Our basic assumption is that drugs inducing very similar alteration in transcriptional regulation will produce similar temporal trajectories on many of the reporter proteins and hence be identified as having similarities in their mechanisms of action (MOA). The main body of this work is devoted to characterizing similarity in temporal trajectories/signals. To do so, we must first identify the key points that determine mechanistic similarity between two drug responses. Directly comparing points on the two signals is unrealistic, as it cannot handle delays and speed variations on the time axis. Hence, to capture the similarities between reporter responses, we develop an alignment algorithm that is robust to noise, time delays and is able to find all the contiguous parts of signals centered about a core alignment (reflecting a core mechanism in drug response). Applying the proposed algorithm to a range of real drug experiments shows that the result agrees well with the prior drug MOA knowledge. AVAILABILITY: The R code for the RLCSS algorithm is available at http://gsp.tamu.edu/Publications/supplementary/zhao12a.

Analytical Study of Performance of Linear Discriminant Analysis in Stochastic Settings.

This paper provides exact analytical expressions for the first and second moments of the true error for linear discriminant analysis (LDA) when the data are univariate and taken from two stochastic Gaussian processes. The key point is that we assume a general setting in which the sample data from each class do not need to be identically distributed or independent within or between classes. We compare the true errors of designed classifiers under the typical i.i.d. model and when the data are correlated, providing exact expressions and demonstrating that, depending on the covariance structure, correlated data can result in classifiers with either greater error or less error than when training with uncorrelated data. The general theory is applied to autoregressive and moving-average models of the first order, and it is demonstrated using real genomic data.

Assessing the efficacy of molecularly targeted agents on cell line-based platforms by using system identification.

BACKGROUND: Molecularly targeted agents (MTAs) are increasingly used for cancer treatment, the goal being to improve the efficacy and selectivity of cancer treatment by developing agents that block the growth of cancer cells by interfering with specific targeted molecules needed for carcinogenesis and tumor growth. This approach differs from traditional cytotoxic anticancer drugs. The lack of specificity of cytotoxic drugs allows a relatively straightforward approach in preclinical and clinical studies, where the optimal dose has usually been defined as the "maximum tolerated dose" (MTD). This toxicity-based dosing approach is founded on the assumption that the therapeutic anticancer effect and toxic effects of the drug increase in parallel as the dose is escalated. On the contrary, most MTAs are expected to be more selective and less toxic than cytotoxic drugs. Consequently, the maximum therapeutic effect may be achieved at a "biologically effective dose" (BED) well below the MTD. Hence, dosing study for MTAs should be different from cytotoxic drugs. Enhanced efforts to molecularly characterize the drug efficacy for MTAs in preclinical models will be valuable for successfully designing dosing regimens for clinical trials. RESULTS: A novel preclinical model combining experimental methods and theoretical analysis is proposed to investigate the mechanism of action and identify pharmacodynamic characteristics of the drug. Instead of fixed time point analysis of the drug exposure to drug effect, the time course of drug effect for different doses is quantitatively studied on cell line-based platforms using system identification, where tumor cells' responses to drugs through the use of fluorescent reporters are sampled over a time course. Results show that drug effect is time-varying and higher dosages induce faster and stronger responses as expected. However, the drug efficacy change along different dosages is not linear; on the contrary, there exist certain thresholds. This kind of preclinical study can provide valuable suggestions about dosing regimens for the in vivo experimental stage to increase productivity.

Multiple-rule bias in the comparison of classification rules.

MOTIVATION: There is growing discussion in the bioinformatics community concerning overoptimism of reported results. Two approaches contributing to overoptimism in classification are (i) the reporting of results on datasets for which a proposed classification rule performs well and (ii) the comparison of multiple classification rules on a single dataset that purports to show the advantage of a certain rule. RESULTS: This article provides a careful probabilistic analysis of the second issue and the 'multiple-rule bias', resulting from choosing a classification rule having minimum estimated error on the dataset. It quantifies this bias corresponding to estimating the expected true error of the classification rule possessing minimum estimated error and it characterizes the bias from estimating the true comparative advantage of the chosen classification rule relative to the others by the estimated comparative advantage on the dataset. The analysis is applied to both synthetic and real data using a number of classification rules and error estimators. AVAILABILITY: We have implemented in C code the synthetic data distribution model, classification rules, feature selection routines and error estimation methods. The code for multiple-rule analysis is implemented in MATLAB. The source code is available at http://gsp.tamu.edu/Publications/supplementary/yousefi11a/. Supplementary simulation results are also included.

Anti-tumor effects of cryptotanshinone (C19H20O3) in human osteosarcoma cell lines.

Osteosarcoma is the most prevalent malignant bone tumor and occurs most commonly in the adolescent and young adult population. Despite the recent advances in surgeries and chemotherapy, the overall survival in patients with resectable metastases is around 20%. This challenge in osteosarcoma is often attributed to the drastic differences in the tumorigenic profiles and mutations among patients. With diverse mutations and multiple oncogenes, it is necessary to identify the therapies that can attack various mutations and simultaneously have minor side-effects. In this paper, we constructed the osteosarcoma pathway from literature and modeled it using ordinary differential equations. We then simulated this network for every possible gene mutation and their combinations and ranked different drug combinations based on their efficacy to drive a mutated osteosarcoma network towards cell death. Our theoretical results predict that drug combinations with Cryptotanshinone (C19H20O3), a traditional Chinese herb derivative, have the best overall performance. Specifically, Cryptotanshinone in combination with Temsirolimus inhibit the JAK/STAT, MAPK/ERK, and PI3K/Akt/mTOR pathways and induce cell death in tumor cells. We corroborated our theoretical predictions using wet-lab experiments on SaOS2, 143B, G292, and HU03N1 human osteosarcoma cell lines, thereby demonstrating the potency of Cryptotanshinone in fighting osteosarcoma.

In Silico Modeling of the Induction of Apoptosis by Cryptotanshinone in Osteosarcoma Cell Lines.

Osteosarcoma (OS) is the most common primary malignant bone tumor of both children and pet canines. Its characteristic genomic instability and complexity coupled with the dearth of knowledge about its etiology has made improvement in the current treatment difficult. We use the existing literature about the biological pathways active in OS and combine it with the current research involving natural compounds to identify new targets and design more effective drug therapies. The key components of these pathways are modeled as a Boolean network with multiple inputs and multiple outputs. The combinatorial circuit is employed to theoretically predict the efficacies of various drugs in combination with Cryptotanshinone. We show that the action of the herbal drug, Cryptotanshinone on OS cell lines induces apoptosis by increasing sensitivity to TNF-related apoptosis-inducing ligand (TRAIL) through its multi-pronged action on STAT3, DRP1 and DR5. The Boolean framework is used to detect additional drug intervention points in the pathway that could amplify the action of Cryptotanshinone.

Reporting bias when using real data sets to analyze classification performance.

MOTIVATION: It is commonplace for authors to propose a new classification rule, either the operator construction part or feature selection, and demonstrate its performance on real data sets, which often come from high-dimensional studies, such as from gene-expression microarrays, with small samples. Owing to the variability in feature selection and error estimation, individual reported performances are highly imprecise. Hence, if only the best test results are reported, then these will be biased relative to the overall performance of the proposed procedure. RESULTS: This article characterizes reporting bias with several statistics and computes these statistics in a large simulation study using both modeled and real data. The results appear as curves giving the different reporting biases as functions of the number of samples tested when reporting only the best or second best performance. It does this for two classification rules, linear discriminant analysis (LDA) and 3-nearest-neighbor (3NN), and for filter and wrapper feature selection, t-test and sequential forward search. These were chosen on account of their well-studied properties and because they were amenable to the extremely large amount of processing required for the simulations. The results across all the experiments are consistent: there is generally large bias overriding what would be considered a significant performance differential, when reporting the best or second best performing data set. We conclude that there needs to be a database of data sets and that, for those studies depending on real data, results should be reported for all data sets in the database. AVAILABILITY: Companion web site at http://gsp.tamu.edu/Publications/supplementary/yousefi09a/

Small-sample precision of ROC-related estimates.

MOTIVATION: The receiver operator characteristic (ROC) curves are commonly used in biomedical applications to judge the performance of a discriminant across varying decision thresholds. The estimated ROC curve depends on the true positive rate (TPR) and false positive rate (FPR), with the key metric being the area under the curve (AUC). With small samples these rates need to be estimated from the training data, so a natural question arises: How well do the estimates of the AUC, TPR and FPR compare with the true metrics? RESULTS: Through a simulation study using data models and analysis of real microarray data, we show that (i) for small samples the root mean square differences of the estimated and true metrics are considerable; (ii) even for large samples, there is only weak correlation between the true and estimated metrics; and (iii) generally, there is weak regression of the true metric on the estimated metric. For classification rules, we consider linear discriminant analysis, linear support vector machine (SVM) and radial basis function SVM. For error estimation, we consider resubstitution, three kinds of cross-validation and bootstrap. Using resampling, we show the unreliability of some published ROC results. AVAILABILITY: Companion web site at http://compbio.tgen.org/paper_supp/ROC/roc.html CONTACT: edward@mail.ece.tamu.edu.

Targeting oncogenic mutations in colorectal cancer using cryptotanshinone.

Colorectal cancer (CRC) is one of the most prevalent types of cancer in the world and ranks second in cancer deaths in the US. Despite the recent improvements in screening and treatment, the number of deaths associated with CRC is still very significant. The complexities involved in CRC therapy stem from multiple oncogenic mutations and crosstalk between abnormal pathways. This calls for using advanced molecular genetics to understand the underlying pathway interactions responsible for this cancer. In this paper, we construct the CRC pathway from the literature and using an existing public dataset on healthy vs tumor colon cells, we identify the genes and pathways that are mutated and are possibly responsible for the disease progression. We then introduce drugs in the CRC pathway, and using a boolean modeling technique, we deduce the drug combinations that produce maximum cell death. Our theoretical simulations demonstrate the effectiveness of Cryptotanshinone, a traditional Chinese herb derivative, achieved by targeting critical oncogenic mutations and enhancing cell death. Finally, we validate our theoretical results using wet lab experiments on HT29 and HCT116 human colorectal carcinoma cell lines.

Comprehensive live-cell imaging analysis of cryptotanshinone and synergistic drug-screening effects in various human and canine cancer cell lines.

BACKGROUND: Several studies have highlighted both the extreme anticancer effects of Cryptotanshinone (CT), a Stat3 crippling component from Salvia miltiorrhiza, as well as other STAT3 inhibitors to fight cancer. METHODS: Data presented in this experiment incorporates 2 years of in vitro studies applying a comprehensive live-cell drug-screening analysis of human and canine cancer cells exposed to CT at 20 µM concentration, as well as to other drug combinations. As previously observed in other studies, dogs are natural cancer models, given to their similarity in cancer genetics, epidemiology and disease progression compared to humans. RESULTS: Results obtained from several types of human and canine cancer cells exposed to CT and varied drug combinations, verified CT efficacy at combating cancer by achieving an extremely high percentage of apoptosis within 24 hours of drug exposure. CONCLUSIONS: CT anticancer efficacy in various human and canine cancer cell lines denotes its ability to interact across different biological processes and cancer regulatory cell networks, driving inhibition of cancer cell survival.

A Gaussian Mixture-Model Exploiting Pathway Knowledge for Dissecting Cancer Heterogeneity.

In this work, we develop a systematic approach for applying pathway knowledge to a multivariate Gaussian mixture model for dissecting a heterogeneous cancer tissue. The downstream transcription factors are selected as observables from available partial pathway knowledge in such a way that the subpopulations produce some differential behavior in response to the drugs selected in the upstream. For each subpopulation, each unique (drug, observable) pair is considered as a unique dimension of a multivariate Gaussian distribution. Expectation-maximization (EM) algorithm with hill-climbing is then used to rank the most probable estimates of the mixture composition based on the log-likelihood value. A major contribution of this work is to examine the efficacy of the EM based approach in estimating the composition of experimental mixture sets from cell-by-cell measurements collected on a dynamic cell imaging platform. Towards this end, we apply the algorithm on hourly data collected for two different mixture compositions of A2058, HCT116, and SW480 cell lines for three scenarios: untreated, Lapatinib-treated, and Temsirolimus-treated. Additionally, we show how this methodology can provide a basis for comparing the killing rate of different drugs for a heterogeneous cancer tissue. This obviously has important implications for designing efficient drugs for treating heterogeneous malignant tumors.

In Silico Design and Experimental Validation of Combination Therapy for Pancreatic Cancer.

The number of deaths associated with Pancreatic Cancer has been on the rise in the United States making it an especially dreaded disease. The overall prognosis for pancreatic cancer patients continues to be grim because of the complexity of the disease at the molecular level involving the potential activation/inactivation of several diverse signaling pathways. In this paper, we first model the aberrant signaling in pancreatic cancer using a multi-fault Boolean Network. Thereafter, we theoretically evaluate the efficacy of different drug combinations by simulating this boolean network with drugs at the relevant intervention points and arrive at the most effective drug(s) to achieve cell death. The simulation results indicate that drug combinations containing Cryptotanshinone, a traditional Chinese herb derivative, result in considerably enhanced cell death. These in silico results are validated using wet lab experiments we carried out on Human Pancreatic Cancer (HPAC) cell lines.

Bayesian Inference Identifies Combination Therapeutic Targets in Breast Cancer.

OBJECTIVE: Breast cancer is the second leading cause of cancer death among US women; hence, identifying potential drug targets is an ever increasing need. In this paper, we integrate existing biological information with graphical models to deduce the significant nodes in the breast cancer signaling pathway. METHODS: We make use of biological information from the literature to develop a Bayesian network. Using the relevant gene expression data we estimate the parameters of this network. Then, using a message passing algorithm, we infer the network. The inferred network is used to quantitatively rank different interventions for achieving a desired phenotypic outcome. The particular phenotype considered here is the induction of apoptosis. RESULTS: Theoretical analysis pinpoints to the role of Cryptotanshinone, a compound found in traditional Chinese herbs, as a potent modulator for bringing about cell death in the treatment of cancer. CONCLUSION: Using a mathematical framework, we showed that the combination therapy of mTOR and STAT3 genes yields the best apoptosis in breast cancer. SIGNIFICANCE: The computational results we arrived at are consistent with the experimental results that we obtained using Cryptotanshinone on MCF-7 breast cancer cell lines and also by the past results of others from the literature, thereby demonstrating the effectiveness of our model.

DNA methylation changes associated with Parkinson's disease progression: outcomes from the first longitudinal genome-wide methylation analysis in blood.

Parkinson's Disease (PD) is a common neurodegenerative disorder currently diagnosed based on the presentation of characteristic movement symptoms. Unfortunately, patients exhibiting these symptoms have already undergone significant dopaminergic neuronal loss. Earlier diagnosis, aided by molecular biomarkers specific to PD, would improve overall patient care. Epigenetic mechanisms, which are modified by both environment and disease pathophysiology, are emerging as important components of neurodegeneration. Alterations to the PD methylome have been reported in epigenome-wide association studies. However, the extent to which methylation changes correlate with disease progression has not yet been reported; nor the degree to which methylation is affected by PD medication. We performed a longitudinal genome-wide methylation study surveying ~850,000 CpG sites in whole blood from 189 well-characterized PD patients and 191 control individuals obtained at baseline and at a follow-up visit ~2 y later. We identified distinct patterns of methylation in PD cases versus controls. Importantly, we identified genomic sites where methylation changes longitudinally as the disease progresses. Moreover, we identified methylation changes associated with PD pathology through the analysis of PD cases that were not exposed to anti-parkinsonian therapy. In addition, we identified methylation sites modulated by exposure to dopamine replacement drugs. These results indicate that DNA methylation is dynamic in PD and changes over time during disease progression. To the best of our knowledge, this is the first longitudinal epigenome-wide methylation analysis for Parkinson's disease and reveals changes associated with disease progression and in response to dopaminergic medications in the blood methylome.

SNiPer-HD: improved genotype calling accuracy by an expectation-maximization algorithm for high-density SNP arrays.

MOTIVATION: The technology to genotype single nucleotide polymorphisms (SNPs) at extremely high densities provides for hypothesis-free genome-wide scans for common polymorphisms associated with complex disease. However, we find that some errors introduced by commonly employed genotyping algorithms may lead to inflation of false associations between markers and phenotype. RESULTS: We have developed a novel SNP genotype calling program, SNiPer-High Density (SNiPer-HD), for highly accurate genotype calling across hundreds of thousands of SNPs. The program employs an expectation-maximization (EM) algorithm with parameters based on a training sample set. The algorithm choice allows for highly accurate genotyping for most SNPs. Also, we introduce a quality control metric for each assayed SNP, such that poor-behaving SNPs can be filtered using a metric correlating to genotype class separation in the calling algorithm. SNiPer-HD is superior to the standard dynamic modeling algorithm and is complementary and non-redundant to other algorithms, such as BRLMM. Implementing multiple algorithms together may provide highly accurate genotyping calls, without inflation of false positives due to systematically miss-called SNPs. A reliable and accurate set of SNP genotypes for increasingly dense panels will eliminate some false association signals and false negative signals, allowing for rapid identification of disease susceptibility loci for complex traits. AVAILABILITY: SNiPer-HD is available at TGen's website: http://www.tgen.org/neurogenomics/data.

Classification of State Trajectories in Gene Regulatory Networks.

Gene-expression-based phenotype classification is used for disease diagnosis and prognosis relating to treatment strategies. The present paper considers classification based on sequential measurements of multiple genes using gene regulatory network (GRN) modeling. There are two networks, original and mutated, and observations consist of trajectories of network states. The problem is to classify an observation trajectory as coming from either the original or mutated network. GRNs are modeled via probabilistic Boolean networks, which incorporate stochasticity at both the gene and network levels. Mutation affects the regulatory logic. Classification is based upon observing a trajectory of states of some given length. We characterize the Bayes classifier and find the Bayes error for a general PBN and the special case of a single Boolean network affected by random perturbations (BNp). The Bayes error is related to network sensitivity, meaning the extent of alteration in the steady-state distribution of the original network owing to mutation. Using standard methods to calculate steady-state distributions is cumbersome and sometimes impossible, so we provide an efficient algorithm and approximations. Extensive simulations are performed to study the effects of various factors, including approximation accuracy. We apply the classification procedure to a p53 BNp and a mammalian cell cycle PBN.

Phenotype Classification Using Moment Features of Single-Cell Data.

Features for standard expression microarray and RNA-Seq classification are expression averages over collections of cells. Single cell provides expression measurements for individual cells in a collection of cells from a particular tissue sample. Hence, it can yield feature vectors consisting of higher order and mixed moments. This article demonstrates the advantage of using these expression moments in cancer-related classification. We use synthetic data generated from 2 real networks, the mammalian cell cycle network and a melanoma-related pathway network, and real single-cell data generated via fluorescent protein reporters from 2 cell lines, HT-29 and HCT-116. The networks consist of hidden binary regulatory networks with Gaussian observations. The steady-state distributions of both the original and mutated networks are found, and data are drawn from these for moment-based classification using the mean, variance, skewness, and mixed moments. For the real data, we only observe 1 gene at a time, so that only the mean, variance, and skewness are considered, the analysis being done for 2 genes, EGFR and ERRB2. For the synthetic data, classification improves as we move from just the mean to mean, variance, and skewness and then to these plus the mixed moments. Comparisons are done with 3, 4, or 5 features, using feature selection. Sample size effects are considered. For the real data, we only consider mean, variance, and skewness, with results improving when the higher order moments are used as features.