PTEN deficiency promotes macrophage infiltration and hypersensitivity of prostate cancer to IAP antagonist/radiation combination therapy

PTEN loss is prognostic for patient relapse post-radiotherapy in prostate cancer (CaP). Infiltration of tumor-associated macrophages (TAMs) is associated with reduced disease-free survival following radical prostatectomy. However, the association between PTEN loss, TAM infiltration and radiotherapy response of CaP cells remains to be evaluated. Immunohistochemical and molecular analysis of surgically-resected Gleason 7 tumors confirmed that PTEN loss correlated with increased CXCL8 expression and macrophage infiltration. However PTEN status had no discernable correlation with expression of other inflammatory markers by CaP cells, including TNF-α. In vitro, exposure to conditioned media harvested from irradiated PTEN null CaP cells induced chemotaxis of macrophage-like THP-1 cells, a response partially attenuated by CXCL8 inhibition. Co-culture with THP-1 cells resulted in a modest reduction in the radio-sensitivity of DU145 cells. Cytokine profiling revealed constitutive secretion of TNF-α from CaP cells irrespective of PTEN status and IR-induced TNF-α secretion from THP-1 cells. THP-1-derived TNF-α increased NFκB pro-survival activity and elevated expression of anti-apoptotic proteins including cellular inhibitor of apoptosis protein-1 (cIAP-1) in CaP cells, which could be attenuated by pre-treatment with a TNF-α neutralizing antibody. Treatment with a novel IAP antagonist, AT-IAP, decreased basal and TNF-α-induced cIAP-1 expression in CaP cells, switched TNF-α signaling from pro-survival to pro-apoptotic and increased radiation sensitivity of CaP cells in co-culture with THP-1 cells. We conclude that targeting cIAP-1 can overcome apoptosis resistance of CaP cells and is an ideal approach to exploit high TNF-α signals within the TAM-rich microenvironment of PTEN-deficient CaP cells to enhance response to radiotherapy.

MErCuRIC1: A Phase I study of MEK1/2 inhibitor PD-0325901 with cMET inhibitor crizotinib in RASMT and RASWT (with aberrant c-MET) metastatic colorectal cancer (mCRC) patients.

Background: RAS is mutated (RASMT) in ~55% of mCRC, and phase III studies have shown that patients harbouring RAS mutations do not benefit from anti-EGFR MoAbs. In addition, ~50% of RAS Wild Type (RASWT) will not benefit from the addition of an EGFR MoAb to standard chemotherapy. Hence, novel treatment strategies are urgently needed for RASMT and > 50% of RASWT mCRC patients. c-MET is overexpressed in ~50-60%, amplified in ~2-3% and mutated in ~3-5% of mCRC. Recent preclinical studies have shown that c-MET is an important mediator of resistance to MEK inhibitors (i) in RASMT mCRC, and that combined MEKi/METi resulted in synergistic reduction in tumour growth in RASMT xenograft models (1). A number of recent studies have highlighted the role of c-MET in mediating primary/secondary resistance to anti-EGFR MoAbs in mCRC, suggesting that patient with RASWT tumours with aberrant c-MET (RASWT/c-MET+) may benefit from anti-c-MET targeted therapies (2). These preclinical data supported the further clinical evaluation of combined MEKi/METi treatment in RASMT and RASWT CRC patients with aberrant c-MET signalling (overexpression, amplification or mutation; RASWT/c-MET+). Methods: MErCuRIC1 is a phase I combination study of METi crizotinib with MEKi PD-0325901. The dose escalation phase, utilizing a rolling six design, recruits 12-24 patients with advanced solid tumours and aims to assess safety/toxicity of combination, recommended phase II (RPII) dose, pharmacokinetics (PK) and pharmacodynamics (PD) (pERK1/2 in PBMC and tumour; soluble c-MET). In the dose expansion phase an additional 30-42 RASMT and RASWT/c-MET mCRC patients with biopsiable disease will be treated at the RPII dose to further evaluate safety, PK, PD and treatment response. In the dose expansion phase additional biopsy and blood samples will be obtained to define mechanisms of response/resistance to crizotinib/PD-0325901 therapy. Enrolment into the dose escalation phase began in December 2014 with cohort 1 still ongoing. EudraCT registry number: 2014-000463-40. (1) Van Schaeybroeck S et al. Cell Reports 2014;7(6):1940-55; (2) Bardelli A et al. Cancer Discov 2013;3(6):658-73. Clinical trial information: 2014-000463-40.

The prognostic and therapeutic value of EpHA2 in early colorectal cancer (CRC).

Background: EpHA2 is a 130 kD transmembrane glycoprotein belonging to ephrin receptor subfamily and involved in angiogenesis/tumour neovascularisation. High EpHA2 mRNA level has recently been implicated in cetuximab resistance. Previously, we found high EpHA2 levels in a panel of invasive colorectal cancer (CRC) cells, which was associated with high levels of stem-cell marker CD44. Our aim was to investigate the prognostic value of EpHA2 and subsequently correlate expression levels to known clinico-pathological variables in early stage CRC. Methods: Tissue samples from 509 CRC patients were analysed. EpHA2 expression was measured using IHC. Kaplan-Meier graphs were used. Univariate and multivariate analyses employed Cox Proportional Hazards Ratio (HR) method. A backward selection method (Akaike’s information criterion) was used to determine a refined multivariate model. Results: EpHA2 was highly expressed in CRC adenocarcinoma compared to matched normal colon tissue. In support of our preclinical invasive models, strong correlation was found between EpHA2 expression and CD44 and Lgr5 staining (p<0.001). In addition, high EpHA2 expression significantly correlated with vascular invasion (p=0.03).HR for OS for stage II/III patients with high EpHA2 expression was 1.69 (95%CI: 1.164-2.439; p=0.003). When stage II/III was broken down into individual stages, there was significant correlation between high EpHA2 expression and poor 5-years OS in stage II patients (HR: 2.18; 95%CI: 1.28-3.71; p=0.005).HR in the stage III group showed a trend to statistical significance (HR: 1.48; 95%CI=0.87-2.51; p=0.05). In both univariate and multivariate analyses of stage II patients, high EpHA2 expression was the only significant factor and was retained in the final multivariate model. Higher levels of EpHA2 were noted in our RAS and BRAF mutant CRC cells, and silencing EpHA2 resulted in significant decreases in migration/invasion in parental and invasive CRC sublines. Correlation between KRAS/NRAS/BRAFmutational status and EpHA2 expression in clinical samples is ongoing. Conclusions: Taken together, our study is the first to indicate that EpHA2 expression is a predictor of poor clinical outcome and a potential novel target in early stage CRC.

Quantification of HER2 heterogeneity in breast cancer-implications for identification of sub-dominant clones for personalised treatment

Breast cancer is a heterogeneous disease, at both an inter- and intra-tumoural level. Appreciating heterogeneity through the application of biomarkers and molecular signatures adds complexity to tumour taxonomy but is key to personalising diagnosis, treatment and prognosis. The extent to which heterogeneity exists, and its interpretation remains a challenge to pathologists. Using HER2 as an exemplar, we have developed a simple reproducible heterogeneity index. Cell-to-cell HER2 heterogeneity was extensive in a proportion of both reported 'amplified' and 'non-amplified' cases. The highest levels of heterogeneity objectively identified occurred in borderline categories and higher ratio non-amplified cases. A case with particularly striking heterogeneity was analysed further with an array of biomarkers in order to assign a molecular diagnosis. Broad biological complexity was evident. In essence, interpretation, depending on the area of tumour sampled, could have been one of three distinct phenotypes, each of which would infer different therapeutic interventions. Therefore, we recommend that heterogeneity is assessed and taken into account when determining treatment options.

Stratified analysis reveals chemokine-like factor (CKLF) as a potential prognostic marker in the MSI-immune consensus molecular subtype CMS1 of colorectal cancer

The Colorectal Cancer (CRC) Subtyping Consortium (CRCSC) recently published four consensus molecular subtypes (CMS’s) representing the underlying biology in CRC. The Microsatellite Instable (MSI) immune group, CMS1, has a favorable prognosis in early stage disease, but paradoxically has the worst prognosis following relapse, suggesting the presence of factors enabling neoplastic cells to circumvent this immune response. To identify the genes influencing subsequent poor prognosis in CMS1, we analyzed this subtype, centered on risk of relapse.
In a cohort of early stage colon cancer (n=460), we examined, in silico, changes in gene expression within the CMS1 subtype and demonstrated for the first time the favorable prognostic value of chemokine-like factor (CKLF) gene expression in the adjuvant disease setting [HR=0.18, CI=0.04-0.89]. In addition, using transcription profiles originating from cell sorted CRC tumors, we delineated the source of CKLF transcription within the colorectal tumor microenvironment to the leukocyte component of these tumors. Further to this, we confirmed that CKLF gene expression is confined to distinct immune subsets in whole blood samples and primary cell lines, highlighting CKLF as a potential immune cell-derived factor promoting tumor immune-surveillance of nascent neoplastic cells, particularly in CMS1 tumors. Building on the recently reported CRCSC data, we provide compelling evidence that leukocyte-infiltrate derived CKLF expression is a candidate biomarker of favorable prognosis, specifically in MSI-immune stage II/III disease.

Embracing an integromic approach to tissue biomarker research in cancer: Perspectives and lessons learned

Modern approaches to biomedical research and diagnostics targeted towards precision medicine are generating ‘big data’ across a range of high-throughput experimental and analytical platforms. Integrative analysis of this rich clinical, pathological, molecular and imaging data represents one of the greatest bottlenecks in biomarker discovery research in cancer and other diseases. Following on from the publication of our successful framework for multimodal data amalgamation and integrative analysis, Pathology Integromics in Cancer (PICan), this article will explore the essential elements of assembling an integromics framework from a more detailed perspective. PICan, built around a relational database storing curated multimodal data, is the research tool sitting at the heart of our interdisciplinary efforts to streamline biomarker discovery and validation. While recognizing that every institution has a unique set of priorities and challenges, we will use our experiences with PICan as a case study and starting point, rationalizing the design choices we made within the context of our local infrastructure and specific needs, but also highlighting alternative approaches that may better suit other programmes of research and discovery. Along the way, we stress that integromics is not just a set of tools, but rather a cohesive paradigm for how modern bioinformatics can be enhanced. Successful implementation of an integromics framework is a collaborative team effort that is built with an eye to the future and greatly accelerates the processes of biomarker discovery, validation and translation into clinical practice.

Challenging the cancer molecular stratification dogma: Intratumoral heterogeneity undermines consensus molecular subtypes and potential diagnostic value in colorectal cancer

Purpose:
A number of independent gene expression profiling studies have identified transcriptional subtypes in colorectal cancer (CRC) with potential diagnostic utility, culminating in publication of a CRC Consensus Molecular Subtype classification. The worst prognostic subtype has been defined by genes associated with stem-like biology. Recently, it has been shown that the majority of genes associated with this poor prognostic group are stromal-derived. We investigated the potential for tumor misclassification into multiple diagnostic subgroups based on tumoral region sampled.

Experimental Design:
We performed multi-region tissue RNA extraction/transcriptomic analysis using Colorectal Specific Arrays on invasive front, central tumor and lymph node regions selected from tissue samples from 25 CRC patients.

Results:
We identified a consensus 30 gene list which represents the intratumoral heterogeneity within a cohort of primary CRC tumors. Using a series of online datasets, we showed that this gene list displays prognostic potential (HR=2.914 (CI 0.9286-9.162) in stage II/III CRC patients, but in addition we demonstrated that these genes are stromal derived, challenging the assumption that poor prognosis tumors with stem-like biology have undergone a widespread Epithelial Mesenchymal Transition (EMT). Most importantly, we showed that patients can be simultaneously classified into multiple diagnostically relevant subgroups based purely on the tumoral region analysed.

Conclusions:
Gene expression profiles derived from the non-malignant stromal region can influence assignment of CRC transcriptional subtypes, questioning the current molecular classification dogma and highlighting the need to consider pathology sampling region and degree of stromal infiltration when employing transcription-based classifiers to underpin clinical decision-making in CRC.

Immune-derived PD-L1 gene expression defines a subgroup of stage II/III colorectal cancer patients with favorable prognosis that may be harmed by adjuvant chemotherapy

A recent phase 2 study of metastatic colorectal carcinoma (CRC) patients showed that mismatch repair gene status was predictive of clinical response to PD-1-targeting immune checkpoint blockade. Further examination revealed strong correlation between PD-L1 protein expression and microsatellite instability (MSI) in stage IV CRC, suggesting that the amount of PD-L1 protein expression could identify late stage patients who may benefit from immunotherapy. To assess whether the clinical associations between PD-L1 gene expression and MSI identified in metastatic CRC are also present in stage II/III CRC, we used in silico analysis to elucidate the cell types expressing the PD-L1 gene. We found a significant association of PD-L1 gene expression with MSI in early stage CRC (P < 0.001) and show that unlike in non-CRC tumors, PD-L1 is derived predominantly from the immune infiltrate. We demonstrate that PD-L1 gene expression has positive prognostic value in the adjuvant disease setting (PD-L1low v PD-L1high HR = 9.09; CI, 2.11-39.10). PD-L1 gene expression had predictive value, as patients with high PD-L1 expression appear to be harmed by standard-of-care treatment (HR = 4.95; CI,1.10-22.35). Building on the promising results from the metastatic CRC PD-1-targeting trial, we provide compelling evidence that PD-L1high/MSI/immunehigh stage II/III CRC patients should not receive standard chemotherapy. This conclusion supports the rationale to clinically evaluate this patient subgroup for PD-1 blockade treatment.

Estudio de Factibilidad para la creación de una empresa dedicada a la Producción y Comercialización de Yogur de Uvilla bajo en Calorias, ubicada e el Cantón Cayambe con Mercado objetivo la Ciudad de Quito

En el mundo, las empresas y países que han incursionado en la industria láctea se han especializado en la elaboración de algún producto específico. Dentro de los principales productos derivados de leche podemos mencionar: leche fluida o fresca, leche en polvo, quesos y otros productos lácteos. Según la Organización de las Naciones Unidas para la Agricultura y la Alimentación (FAO) la producción mundial de leche entera en el 2008 alcanzó un volumen de 693,2 millones de toneladas equivalentes en leche, lo que representa un crecimiento de 2,5% respecto al 2007.1

Análsis de Factibilidad para la creación de un Hotel en la Parroquia Tababela sector del Nuevo Areopuerto de la Cidad de Quito.

El Turismo es una fuente importante de ingresos para la economía nacional, la forma cómo vendemos los productos o los destinos y la calidad de los mismos crean un nicho de mercado de turismo nacional y extranjero. Produce divisas siendo una valiosa contribución en la economía de países en vías de desarrollo como es el nuestro. El Turismo genera empleo local tanto directamente en el Sector Turístico como en el de Apoyo y en la ordenación de los recursos. Además esta actividad genera una serie de servicios y por ende una gran gama de ocupación en el sector donde se trabaja, creando un rubro más de ingresos. El turismo en el Ecuador en los últimos años ha tenido un importante crecimiento, es muy claro observar el incremento de turistas para la serranía, la costa, el oriente y las Islas Galápagos. Hoy en día existe un incremento en relación al turismo receptivo dada la importancia que ha adquirido la ciudad de Quito como centro cultural y como un lugar elegido por los turistas como centro o base para el aprendizaje del español o inclusive para el traslado a los diferentes puntos del país. Quito es una de las ciudades de más alto crecimiento en América Latina. Al ser la capital de la República, es el centro político y administrativo del país, en torno al cual gira toda una gama de actividades políticas, económicas, sociales y culturales.

Estabilidade de sistemas comutados com reset parcial

A presente tese é dedicada ao estudo da estabilidade de sistemas definidos por famílias finitas de sistemas lineares invariantes e por regras de comutação que coordenam a comutação entre eles. Assumimos que, em cada instante de tempo onde ocorre comutação, a trajectória do estado do sistema possa sofrer um "salto" desencadeado pela aplicação de um reset. Estes sistemas são, neste trabalho, designados por sistemas comutados com reset. Os resets podem ser de dois tipos - totais ou parciais, dependendo se a totalidade ou apenas uma parte das componentes do estado está disponível para reset. Neste sentido, distinguimos sistemas comutados com reset (total) e sistemas comutados com reset parcial. Analisamos a estabilidade dos dois tipos de sistemas comutados referidos à luz da teoria de Lyapunov e sob duas perspectivas; por um lado determinamos sob que condições um sistema comutado com reset é estável e por outro, identificamos resets que, quando aplicados, asseguram a estabilidade do sistema. Neste último ponto, a escolha dos resets adequados a aplicar pode por si só revelar-se insuficiente para obter estabilidade, especialmente se apenas parte das componentes do estado estiver disponível para reset (caso de reset parcial).

A study of singular integral operators with shift

Nesta tese, consideram-se operadores integrais singulares com a acção extra de um operador de deslocacamento de Carleman e com coeficientes em diferentes classes de funções essencialmente limitadas. Nomeadamente, funções contínuas por troços, funções quase-periódicas e funções possuíndo factorização generalizada. Nos casos dos operadores integrais singulares com deslocamento dado pelo operador de reflexão ou pelo operador de salto no círculo unitário complexo, obtêm-se critérios para a propriedade de Fredholm. Para os coeficientes contínuos, uma fórmula do índice de Fredholm é apresentada. Estes resultados são consequência das relações de equivalência explícitas entre aqueles operadores e alguns operadores adicionais, tais como o operador integral singular, operadores de Toeplitz e operadores de Toeplitz mais Hankel. Além disso, as relações de equivalência permitem-nos obter um critério de invertibilidade e fórmulas para os inversos laterais dos operadores iniciais com coeficientes factorizáveis. Adicionalmente, aplicamos técnicas de análise numérica, tais como métodos de colocação de polinómios, para o estudo da dimensão do núcleo dos dois tipos de operadores integrais singulares com coeficientes contínuos por troços. Esta abordagem permite também a computação do inverso no sentido Moore-Penrose dos operadores principais. Para operadores integrais singulares com operadores de deslocamento do tipo Carleman preservando a orientação e com funções contínuas como coeficientes, são obtidos limites superiores da dimensão do núcleo. Tal é implementado utilizando algumas estimativas e com a ajuda de relações (explícitas) de equivalência entre operadores. Focamos ainda a nossa atenção na resolução e nas soluções de uma classe de equações integrais singulares com deslocamento que não pode ser reduzida a um problema de valor de fronteira binomial. De forma a atingir os objectivos propostos, foram utilizadas projecções complementares e identidades entre operadores. Desta forma, as equações em estudo são associadas a sistemas de equações integrais singulares. Estes sistemas são depois analisados utilizando um problema de valor de fronteira de Riemann. Este procedimento tem como consequência a construção das soluções das equações iniciais a partir das soluções de problemas de valor de fronteira de Riemann. Motivados por uma grande diversidade de aplicações, estendemos a definição de operador integral de Cauchy para espaços de Lebesgue sobre grupos topológicos. Assim, são investigadas as condições de invertibilidade dos operadores integrais neste contexto.

Novel critical phenomena in optimization driven processes on networks

The work presented in this Ph.D thesis was developed in the context of complex network theory, from a statistical physics standpoint. We examine two distinct problems in this research field, taking a special interest in their respective critical properties. In both cases, the emergence of criticality is driven by a local optimization dynamics. Firstly, a recently introduced class of percolation problems that attracted a significant amount of attention from the scientific community, and was quickly followed up by an abundance of other works. Percolation transitions were believed to be continuous, until, recently, an 'explosive' percolation problem was reported to undergo a discontinuous transition, in [93]. The system's evolution is driven by a metropolis-like algorithm, apparently producing a discontinuous jump on the giant component's size at the percolation threshold. This finding was subsequently supported by number of other experimental studies [96, 97, 98, 99, 100, 101]. However, in [1] we have proved that the explosive percolation transition is actually continuous. The discontinuity which was observed in the evolution of the giant component's relative size is explained by the unusual smallness of the corresponding critical exponent, combined with the finiteness of the systems considered in experiments. Therefore, the size of the jump vanishes as the system's size goes to infinity. Additionally, we provide the complete theoretical description of the critical properties for a generalized version of the explosive percolation model [2], as well as a method [3] for a precise calculation of percolation's critical properties from numerical data (useful when exact results are not available). Secondly, we study a network flow optimization model, where the dynamics consists of consecutive mergings and splittings of currents flowing in the network. The current conservation constraint does not impose any particular criterion for the split of current among channels outgoing nodes, allowing us to introduce an asymmetrical rule, observed in several real systems. We solved analytically the dynamic equations describing this model in the high and low current regimes. The solutions found are compared with numerical results, for the two regimes, showing an excellent agreement. Surprisingly, in the low current regime, this model exhibits some features usually associated with continuous phase transitions.

Route planning in wireless sensor networks for data gathering purposes

Dissertação de Mestrado, Engenharia Informática, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2015