Practical identifiability analysis of a mechanistic model for the time to distant metastatic relapse and its application to renal cell carcinoma.

Distant metastasis-free survival (DMFS) curves are widely used in oncology. They are classically analyzed using the Kaplan-Meier estimator or agnostic statistical models from survival analysis. Here we report on a method to extract more information from DMFS curves using a mathematical model of primary tumor growth and metastatic dissemination. The model depends on two parameters, α and µ, respectively quantifying tumor growth and dissemination. We assumed these to be lognormally distributed in a patient population. We propose a method for identification of the parameters of these distributions based on least-squares minimization between the data and the simulated survival curve. We studied the practical identifiability of these parameters and found that including the percentage of patients with metastasis at diagnosis was critical to ensure robust estimation. We also studied the impact and identifiability of covariates and their coefficients in α and µ, either categorical or continuous, including various functional forms for the latter (threshold, linear or a combination of both). We found that both the functional form and the coefficients could be determined from DMFS curves. We then applied our model to a clinical dataset of metastatic relapse from kidney cancer with individual data of 105 patients. We show that the model was able to describe the data and illustrate our method to disentangle the impact of three covariates on DMFS: a categorical one (Führman grade) and two continuous ones (gene expressions of the macrophage mannose receptor 1 (MMR) and the G Protein-Coupled Receptor Class C Group 5 Member A (GPRC5a) gene). We found that all had an influence in metastasis dissemination (µ), but not on growth (α).

Experimental and computational modeling for signature and biomarker discovery of renal cell carcinoma progression.

BACKGROUND: Renal Cell Carcinoma (RCC) is difficult to treat with 5-year survival rate of 10% in metastatic patients. Main reasons of therapy failure are lack of validated biomarkers and scarce knowledge of the biological processes occurring during RCC progression. Thus, the investigation of mechanisms regulating RCC progression is fundamental to improve RCC therapy. METHODS: In order to identify molecular markers and gene processes involved in the steps of RCC progression, we generated several cell lines of higher aggressiveness by serially passaging mouse renal cancer RENCA cells in mice and, concomitantly, performed functional genomics analysis of the cells. Multiple cell lines depicting the major steps of tumor progression (including primary tumor growth, survival in the blood circulation and metastatic spread) were generated and analyzed by large-scale transcriptome, genome and methylome analyses. Furthermore, we performed clinical correlations of our datasets. Finally we conducted a computational analysis for predicting the time to relapse based on our molecular data. RESULTS: Through in vivo passaging, RENCA cells showed increased aggressiveness by reducing mice survival, enhancing primary tumor growth and lung metastases formation. In addition, transcriptome and methylome analyses showed distinct clustering of the cell lines without genomic variation. Distinct signatures of tumor aggressiveness were revealed and validated in different patient cohorts. In particular, we identified SAA2 and CFB as soluble prognostic and predictive biomarkers of the therapeutic response. Machine learning and mathematical modeling confirmed the importance of CFB and SAA2 together, which had the highest impact on distant metastasis-free survival. From these data sets, a computational model predicting tumor progression and relapse was developed and validated. These results are of great translational significance. CONCLUSION: A combination of experimental and mathematical modeling was able to generate meaningful data for the prediction of the clinical evolution of RCC.

Mitochondria transfer from tumor-activated stromal cells (TASC) to primary Glioblastoma cells.

The tumor microenvironment (TME) controls many aspects of cancer development but little is known about its effect in Glioblastoma (GBM), the main brain tumor in adults. Tumor-activated stromal cell (TASC) population, a component of TME in GBM, was induced in vitro by incubation of MSCs with culture media conditioned by primary cultures of GBM under 3D/organoid conditions. We observed mitochondrial transfer by Tunneling Nanotubes (TNT), extracellular vesicles (EV) and cannibalism from the TASC to GBM and analyzed its effect on both proliferation and survival. We created primary cultures of GBM or TASC in which we have eliminated mitochondrial DNA [Rho 0 (ρ0) cells]. We found that TASC, as described in other cancers, increased GBM proliferation and resistance to standard treatments (radiotherapy and chemotherapy). We analyzed the incorporation of purified mitochondria by ρ0 and ρ+ cells and a derived mathematical model taught us that ρ+ cells incorporate more rapidly pure mitochondria than ρ0 cells.

Transfer of Drug Resistance Characteristics Between Cancer Cell Subpopulations: A Study Using Simple Mathematical Models.

Resistance to chemotherapy is a major cause of cancer treatment failure. The processes of resistance induction and selection of resistant cells (due to the over-expression of the membrane transporter P-glycoprotein, P-gp) are well documented in the literature, and a number of mathematical models have been developed. However, another process of transfer of resistant characteristics is less well known and has received little attention in the mathematical literature. In this paper, we discuss the potential of simple mathematical models to describe the process of resistance transfer, specifically P-gp transfer, in mixtures of resistant and sensitive tumor cell populations. Two different biological hypotheses for P-gp transfer are explored: (1) exchange through physical cell-cell connections and (2) through microvessicles released to the culture medium. Two models are developed which fit very well the observed population growth dynamics. The potential and limitations of these simple "global" models to describe the aforementioned biological processes involved are discussed on the basis of the results obtained.

[Epidemiological panorama of venomous snake bites in the state of Yucatan, Mexico (2003-2012)]. / Panorama epidemiológico de las mordeduras por serpiente venenosa en el estado de Yucatán, México (2003-2012).

No information has been yet published on the epidemiological panorama of snakebite in the state of Yucatan. The aim of this study was to evaluate the geographic and temporal patterns of this problem in the state. Snakebite data was obtained from the Program of Zoonosis of the Health Services of Yucatan between 2003 and 2012. A total of 821 snakebite cases and an incidence of 41.9 accidents/100,000 inhabitants were recorded during this period. The annual average cases and incidence were 82.1 and 4.1 (bites/100,000 inhabitants), respectively. The highest number of snakebites occurred in 2005, while in 2003 the lowest number was recorded. Geographically, we observed a great disparity between municipalities, some of them reaching very high levels of incidence. This geographical variation may reflect the distribution and abundance of venomous snakes on one hand, and human population densities and their activities on the other. This study will help health authorities to know preliminarily the magnitude of snakebites in Yucatan and improving strategies to mitigate it.

An integrated system to assess marine extinctions.

More than 20 global marine extinctions and over 700 local extinctions have reportedly occurred during the past 500 years. However, available methods to determine how many of these species can be confidently declared true disappearances tend to be data-demanding, time-consuming, and not applicable to all taxonomic groups or scales of marine extinctions (global [G] and local [L]). We developed an integrated system to assess marine extinctions (ISAME) that can be applied to any taxonomic group at any geographic scale. We applied the ISAME method to 10 case studies to illustrate the possible ways in which the extinction status of marine species can be categorized as unverified, possibly extinct, or extinct. Of the 10 case studies we assessed, the ISAME method concludes that 6 should be categorized as unverified extinctions due to problems with species' identity and lack of reliable evidence supporting their disappearance (periwinkle-Littoraria flammea [G], houting-Coregonus oxyrinchus [G], long-spined urchin-Diadema antillarum [L], smalltooth sawfish-Pristis pectinata [L], and largetooth sawfish-P. pristis [L]). In contrast, ISAME classified the Guadalupe storm-petrel (Oceanodroma macrodactyla [G]) and the lost shark (Carcharhinus obsolerus [G]) as possibly extinct because the available evidence indicates that their extinction is plausible-while the largetooth sawfish [L] and Steller's sea cow (Hydrodamalis gigas [G]) were confirmed to be extinct. Determining whether a marine population or species is actually extinct or still extant is needed to guide conservation efforts and prevent further biodiversity losses.

A Simple 3D Cell Culture Method for Studying the Interactions between Human Mesenchymal Stromal/Stem Cells and Patients Derived Glioblastoma.

We have developed a 3D biosphere model using patient-derived cells (PDCs) from glioblastoma (GBM), the major form of primary brain tumors in adult, plus cancer-activated fibroblasts (CAFs), obtained by culturing mesenchymal stem cells with GBM conditioned media. The effect of MSC/CAFs on the proliferation, cell-cell interactions, and response to treatment of PDCs was evaluated. Proliferation in the presence of CAFs was statistically lower but the spheroids formed within the 3D-biosphere were larger. A treatment for 5 days with Temozolomide (TMZ) and irradiation, the standard therapy for GBM, had a marked effect on cell number in monocultures compared to co-cultures and influenced cancer stem cells composition, similar to that observed in GBM patients. Mathematical analyses of spheroids growth and morphology confirm the similarity with GBM patients. We, thus, provide a simple and reproducible method to obtain 3D cultures from patient-derived biopsies and co-cultures with MSC with a near 100% success. This method provides the basis for relevant in vitro functional models for a better comprehension of the role of tumor microenvironment and, for precision and/or personalized medicine, potentially to predict the response to treatments for each GBM patient.

Comparative efficacy of two primary care interventions to assist withdrawal from long term benzodiazepine use: a protocol for a clustered, randomized clinical trial.

BACKGROUND: Although benzodiazepines are effective, long-term use is not recommended because of potential adverse effects; the risks of tolerance and dependence; and an increased risk of hip fractures, motor vehicle accidents, and memory impairment. The estimated prevalence of long-term benzodiazepine use in the general population is about 2,2 to 2,6%, is higher in women and increases steadily with age. Interventions performed by General Practitioners may help patients to discontinue long-term benzodiazepine use. We have designed a trial to evaluate the effectiveness and safety of two brief general practitioner-provided interventions, based on gradual dose reduction, and will compare the effectiveness of these interventions with that of routine clinical practice. METHODS/DESIGN: In a three-arm cluster randomized controlled trial, general practitioners will be randomly allocated to: a) a group in which the first patient visit will feature a structured interview, followed by visits every 2-3 weeks to the end of dose reduction; b) a group in which the first patient visit will feature a structured interview plus delivery of written instructions to self-reduce benzodiazepine dose, or c) routine care. Using a computerized pharmaceutical prescription database, 495 patients, aged 18-80 years, taking benzodiazepine for at least 6 months, will be recruited in primary care health districts of three regions of Spain (the Balearic Islands, Catalonia, and Valencia). The primary outcome will be benzodiazepine use at 12 months. The secondary outcomes will include measurements of anxiety and depression symptoms, benzodiazepine dependence, quality of sleep, and alcohol consumption. DISCUSSION: Although some interventions have been shown to be effective in reducing benzodiazepine consumption by long-term users, the clinical relevance of such interventions is limited by their complexity. This randomized trial will compare the effectiveness and safety of two complex stepped care interventions with that of routine care in a study with sufficient statistical power to detect clinically relevant differences. TRIAL REGISTRATION: Current Controlled Trials: ISRCTN13024375.

Identification of a transient state during the acquisition of temozolomide resistance in glioblastoma.

Drug resistance limits the therapeutic efficacy in cancers and leads to tumor recurrence through ill-defined mechanisms. Glioblastoma (GBM) are the deadliest brain tumors in adults. GBM, at diagnosis or after treatment, are resistant to temozolomide (TMZ), the standard chemotherapy. To better understand the acquisition of this resistance, we performed a longitudinal study, using a combination of mathematical models, RNA sequencing, single cell analyses, functional and drug assays in a human glioma cell line (U251). After an initial response characterized by cell death induction, cells entered a transient state defined by slow growth, a distinct morphology and a shift of metabolism. Specific genes expression associated to this population revealed chromatin remodeling. Indeed, the histone deacetylase inhibitor trichostatin (TSA), specifically eliminated this population and thus prevented the appearance of fast growing TMZ-resistant cells. In conclusion, we have identified in glioblastoma a population with tolerant-like features, which could constitute a therapeutic target.

Interplay of Darwinian Selection, Lamarckian Induction and Microvesicle Transfer on Drug Resistance in Cancer.

Development of drug resistance in cancer has major implications for patients' outcome. It is related to processes involved in the decrease of drug efficacy, which are strongly influenced by intratumor heterogeneity and changes in the microenvironment. Heterogeneity arises, to a large extent, from genetic mutations analogously to Darwinian evolution, when selection of tumor cells results from the adaptation to the microenvironment, but could also emerge as a consequence of epigenetic mutations driven by stochastic events. An important exogenous source of alterations is the action of chemotherapeutic agents, which not only affects the signalling pathways but also the interactions among cells. In this work we provide experimental evidence from in vitro assays and put forward a mathematical kinetic transport model to describe the dynamics displayed by a system of non-small-cell lung carcinoma cells (NCI-H460) which, depending on the effect of a chemotherapeutic agent (doxorubicin), exhibits a complex interplay between Darwinian selection, Lamarckian induction and the nonlocal transfer of extracellular microvesicles. The role played by all of these processes to multidrug resistance in cancer is elucidated and quantified.

[Sperm parameters in 113 subjects after recent fatherhood did not correlate with WHO standards]. / Los valores espermáticos de 113 individuos con fertilidad reciente no mostraron correlación con los parámetros establecidos por la OMS.

OBJECTIVES: This is a descriptive prospective study of semen analysis in 113 men who were candidates for vasectomy as an option for fertility control after a recent event of fertility. METHODS: Each subject provided a semen sample for the analysis of volume, pH, spermatozoid concentration, mobility, and morphology. The results were compared with the reference values established by WHO. RESULTS: Different from what was expected, a great proportion of these fertile subjects, 54.9%, showed at least one altered parameter in comparison to minimal values established by WHO. Generally, median values for the whole group were normal: volume 2.4 ml, spermatozoid concentration measured in Makler and Neubauer chambers 89 and 91 millions/ml, respectively; sperm mobility (A + B) 56%; vitality 78%; and normal morphology 79%. CONCLUSIONS: In this group of fertile subjects we found a high probability to have altered semen parameters, which argues questions the real value and significance of sperm analysis in the diagnosis of a subfertile patient.