Computational models applied to metabolomics data hints at the relevance of glutamine metabolism in breast cancer.

BACKGROUND: Metabolomics has a great potential in the development of new biomarkers in cancer and it has experiment recent technical advances. METHODS: In this study, metabolomics and gene expression data from 67 localized (stage I to IIIB) breast cancer tumor samples were analyzed, using (1) probabilistic graphical models to define associations using quantitative data without other a priori information; and (2) Flux Balance Analysis and flux activities to characterize differences in metabolic pathways. RESULTS: On the one hand, both analyses highlighted the importance of glutamine in breast cancer. Moreover, cell experiments showed that treating breast cancer cells with drugs targeting glutamine metabolism significantly affects cell viability. On the other hand, these computational methods suggested some hypotheses and have demonstrated their utility in the analysis of metabolomics data and in associating metabolomics with patient's clinical outcome. CONCLUSIONS: Computational analyses applied to metabolomics data suggested that glutamine metabolism is a relevant process in breast cancer. Cell experiments confirmed this hypothesis. In addition, these computational analyses allow associating metabolomics data with patient prognosis.

Biological molecular layer classification of muscle-invasive bladder cancer opens new treatment opportunities.

BACKGROUND: Muscle-invasive bladder tumors are associated with a high risk of relapse and metastasis even after neoadjuvant chemotherapy and radical cystectomy. Therefore, further therapeutic options are needed and molecular characterization of the disease may help to identify new targets. The aim of this study was to characterize muscle-invasive bladder tumors at the molecular level using computational analyses. METHODS: The TCGA cohort of muscle-invasive bladder cancer patients was used to describe these tumors. Probabilistic graphical models, layer analyses based on sparse k-means coupled with Consensus Cluster, and Flux Balance Analysis were applied to characterize muscle-invasive bladder tumors at a functional level. RESULTS: Luminal and Basal groups were identified, and an immune molecular layer with independent value was also described. Luminal tumors showed decreased activity in the nodes of epidermis development and extracellular matrix, and increased activity in the node of steroid metabolism leading to a higher expression of the androgen receptor. This fact points to the androgen receptor as a therapeutic target in this group. Basal tumors were highly proliferative according to Flux Balance Analysis, which makes these tumors good candidates for neoadjuvant chemotherapy. The Immune-high group showed a higher degree of expression of immune biomarkers, suggesting that this group may benefit from immune therapy. CONCLUSIONS: Our approach, based on layer analyses, established a Luminal group candidate for therapy with androgen receptor inhibitors, a proliferative Basal group which seems to be a good candidate for chemotherapy, and an immune-high group candidate for immunotherapy.

A new method for identifying bivariate differential expression in high dimensional microarray data using quadratic discriminant analysis.

BACKGROUND: One of the drawbacks we face up when analyzing gene to phenotype associations in genomic data is the ugly performance of the designed classifier due to the small sample-high dimensional data structures (n ≪ p) at hand. This is known as the peaking phenomenon, a common situation in the analysis of gene expression data. Highly predictive bivariate gene interactions whose marginals are useless for discrimination are also affected by such phenomenon, so they are commonly discarded by state of the art sequential search algorithms. Such patterns are known as weak/marginal strong bivariate interactions. This paper addresses the problem of uncovering them in high dimensional settings. RESULTS: We propose a new approach which uses the quadratic discriminant analysis (QDA) as a search engine in order to detect such signals. The choice of QDA is justified by a simulation study for a benchmark of classifiers which reveals its appealing properties. The procedure rests on an exhaustive search which explores the feature space in a blockwise manner by dividing it in blocks and by assessing the accuracy of the QDA for the predictors within each pair of blocks; the block size is determined by the resistance of the QDA to peaking. This search highlights chunks of features which are expected to contain the type of subtle interactions we are concerned with; a closer look at this smaller subset of features by means of an exhaustive search guided by the QDA error rate for all the pairwise input combinations within this subset will enable their final detection. The proposed method is applied both to synthetic data and to a public domain microarray data. When applied to gene expression data, it leads to pairs of genes which are not univariate differentially expressed but exhibit subtle patterns of bivariate differential expression. CONCLUSIONS: We have proposed a novel approach for identifying weak marginal/strong bivariate interactions. Unlike standard approaches as the top scoring pair (TSP) and the CorScor, our procedure does not assume a specified shape of phenotype separation and may enrich the type of bivariate differential expression patterns that can be uncovered in high dimensional data.

Bayesian networks established functional differences between breast cancer subtypes.

Breast cancer is a heterogeneous disease. In clinical practice, tumors are classified as hormonal receptor positive, Her2 positive and triple negative tumors. In previous works, our group defined a new hormonal receptor positive subgroup, the TN-like subtype, which had a prognosis and a molecular profile more similar to triple negative tumors. In this study, proteomics and Bayesian networks were used to characterize protein relationships in 96 breast tumor samples. Components obtained by these methods had a clear functional structure. The analysis of these components suggested differences in processes such as mitochondrial function or extracellular matrix between breast cancer subtypes, including our new defined subtype TN-like. In addition, one of the components, mainly related with extracellular matrix processes, had prognostic value in this cohort. Functional approaches allow to build hypotheses about regulatory mechanisms and to establish new relationships among proteins in the breast cancer context.

A novel approach to triple-negative breast cancer molecular classification reveals a luminal immune-positive subgroup with good prognoses.

Triple-negative breast cancer is a heterogeneous disease characterized by a lack of hormonal receptors and HER2 overexpression. It is the only breast cancer subgroup that does not benefit from targeted therapies, and its prognosis is poor. Several studies have developed specific molecular classifications for triple-negative breast cancer. However, these molecular subtypes have had little impact in the clinical setting. Gene expression data and clinical information from 494 triple-negative breast tumors were obtained from public databases. First, a probabilistic graphical model approach to associate gene expression profiles was performed. Then, sparse k-means was used to establish a new molecular classification. Results were then verified in a second database including 153 triple-negative breast tumors treated with neoadjuvant chemotherapy. Clinical and gene expression data from 494 triple-negative breast tumors were analyzed. Tumors in the dataset were divided into four subgroups (luminal-androgen receptor expressing, basal, claudin-low and claudin-high), using the cancer stem cell hypothesis as reference. These four subgroups were defined and characterized through hierarchical clustering and probabilistic graphical models and compared with previously defined classifications. In addition, two subgroups related to immune activity were defined. This immune activity showed prognostic value in the whole cohort and in the luminal subgroup. The claudin-high subgroup showed poor response to neoadjuvant chemotherapy. Through a novel analytical approach we proved that there are at least two independent sources of biological information: cellular and immune. Thus, we developed two different and overlapping triple-negative breast cancer classifications and showed that the luminal immune-positive subgroup had better prognoses than the luminal immune-negative. Finally, this work paves the way for using the defined classifications as predictive features in the neoadjuvant scenario.

Probabilistic graphical models relate immune status with response to neoadjuvant chemotherapy in breast cancer.

Breast cancer is the most frequent tumor in women and its incidence is increasing. Neoadjuvant chemotherapy has become standard of care as a complement to surgery in locally advanced or poor-prognosis early stage disease. The achievement of a complete response to neoadjuvant chemotherapy correlates with prognosis but it is not possible to predict who will obtain an excellent response. The molecular analysis of the tumor offers a unique opportunity to unveil predictive factors. In this work, gene expression profiling in 279 tumor samples from patients receiving neoadjuvant chemotherapy was performed and probabilistic graphical models were used. This approach enables addressing biological and clinical questions from a Systems Biology perspective, allowing to deal with large gene expression data and their interactions. Tumors presenting complete response to neoadjuvant chemotherapy had a higher activity of immune related functions compared to resistant tumors. Similarly, samples from complete responders presented higher expression ​​of lymphocyte cell lineage markers, immune-activating and immune-suppressive markers, which may correlate with tumor infiltration by lymphocytes (TILs). These results suggest that the patient's immune system plays a key role in tumor response to neoadjuvant treatment. However, future studies with larger cohorts are necessary to validate these hypotheses.

Molecular characterization of breast cancer cell response to metabolic drugs.

Metabolic reprogramming is a hallmark of cancer. It has been described that breast cancer subtypes present metabolism differences and this fact enables the possibility of using metabolic inhibitors as targeted drugs in specific scenarios. In this study, breast cancer cell lines were treated with metformin and rapamycin, showing a heterogeneous response to treatment and leading to cell cycle disruption. The genetic causes and molecular effects of this differential response were characterized by means of SNP genotyping and mass spectrometry-based proteomics. Protein expression was analyzed using probabilistic graphical models, showing that treatments elicit various responses in some biological processes such as transcription. Moreover, flux balance analysis using protein expression values showed that predicted growth rates were comparable with cell viability measurements and suggesting an increase in reactive oxygen species response enzymes due to metformin treatment. In addition, a method to assess flux differences in whole pathways was proposed. Our results show that these diverse approaches provide complementary information and allow us to suggest hypotheses about the response to drugs that target metabolism and their mechanisms of action.

Urothelial cancer proteomics provides both prognostic and functional information.

Traditionally, bladder cancer has been classified based on histology features. Recently, some works have proposed a molecular classification of invasive bladder tumors. To determine whether proteomics can define molecular subtypes of  muscle invasive urothelial cancer (MIUC) and allow evaluating the status of biological processes and its clinical value. 58 MIUC patients who underwent curative surgical resection at our institution between 2006 and 2012 were included. Proteome was evaluated by high-throughput proteomics in routinely archive FFPE tumor tissue. New molecular subgroups were defined. Functional structure and individual proteins prognostic value were evaluated and correlated with clinicopathologic parameters. 1,453 proteins were quantified, leading to two MIUC molecular subgroups. A protein-based functional structure was defined, including several nodes with specific biological activity. The functional structure showed differences between subtypes in metabolism, focal adhesion, RNA and splicing nodes. Focal adhesion node has prognostic value in the whole population. A 6-protein prognostic signature, associated with higher risk of relapse (5 year DFS 70% versus 20%) was defined. Additionally, we identified two MIUC subtypes groups. Prognostic information provided by pathologic characteristics is not enough to understand MIUC behavior. Proteomics analysis may enhance our understanding of prognostic and classification. These findings can lead to improving diagnosis and treatment selection in these patients.

Functional proteomics outlines the complexity of breast cancer molecular subtypes.

Breast cancer is a heterogeneous disease comprising a variety of entities with various genetic backgrounds. Estrogen receptor-positive, human epidermal growth factor receptor 2-negative tumors typically have a favorable outcome; however, some patients eventually relapse, which suggests some heterogeneity within this category. In the present study, we used proteomics and miRNA profiling techniques to characterize a set of 102 either estrogen receptor-positive (ER+)/progesterone receptor-positive (PR+) or triple-negative formalin-fixed, paraffin-embedded breast tumors. Protein expression-based probabilistic graphical models and flux balance analyses revealed that some ER+/PR+ samples had a protein expression profile similar to that of triple-negative samples and had a clinical outcome similar to those with triple-negative disease. This probabilistic graphical model-based classification had prognostic value in patients with luminal A breast cancer. This prognostic information was independent of that provided by standard genomic tests for breast cancer, such as MammaPrint, OncoType Dx and the 8-gene Score.

Combined Label-Free Quantitative Proteomics and microRNA Expression Analysis of Breast Cancer Unravel Molecular Differences with Clinical Implications.

Better knowledge of the biology of breast cancer has allowed the use of new targeted therapies, leading to improved outcome. High-throughput technologies allow deepening into the molecular architecture of breast cancer, integrating different levels of information, which is important if it helps in making clinical decisions. microRNA (miRNA) and protein expression profiles were obtained from 71 estrogen receptor-positive (ER(+)) and 25 triple-negative breast cancer (TNBC) samples. RNA and proteins obtained from formalin-fixed, paraffin-embedded tumors were analyzed by RT-qPCR and LC/MS-MS, respectively. We applied probabilistic graphical models representing complex biologic systems as networks, confirming that ER(+) and TNBC subtypes are distinct biologic entities. The integration of miRNA and protein expression data unravels molecular processes that can be related to differences in the genesis and clinical evolution of these types of breast cancer. Our results confirm that TNBC has a unique metabolic profile that may be exploited for therapeutic intervention.

Exploring correlations in gene expression microarray data for maximum predictive-minimum redundancy biomarker selection and classification.

An important issue in the analysis of gene expression microarray data is concerned with the extraction of valuable genetic interactions from high dimensional data sets containing gene expression levels collected for a small sample of assays. Past and ongoing research efforts have been focused on biomarker selection for phenotype classification. Usually, many genes convey useless information for classifying the outcome and should be removed from the analysis; on the other hand, some of them may be highly correlated, which reveals the presence of redundant expressed information. In this paper we propose a method for the selection of highly predictive genes having a low redundancy in their expression levels. The predictive accuracy of the selection is assessed by means of Classification and Regression Trees (CART) models which enable assessment of the performance of the selected genes for classifying the outcome variable and will also uncover complex genetic interactions. The method is illustrated throughout the paper using a public domain colon cancer gene expression data set.