PREPARATION AND CHARACTERIZATION OF COVALENTLY LINKED ENZYME-ANTIBODY CONJUGATES AND THEIR USE IN MEASURING MINUTE QUANTITIES OF PROTEIN ANTIGENS

The discovery and characterization of oncofetal proteins have led to significant advances in early cancer diagnosis and therapeutic monitoring of patients undergoing cancer chemotherapy. These tumor-associated antigens are presently measured by sensitive, specific immunoassay techniques based on the detection of minute amounts of labeled antigen or antibody incorporated into immune complexes, which must be isolated from free antigen and antibody.^ Since there are several disadvantages with using radioisotopes, the most common immunolabel, one major objective was to prepare covalently coupled enzyme-antibody conjugates and evaluate their use as a practical alternative to radiolabeled immune reagents. An improved technique for the production of enzyme-antibody conjugates was developed that involves oxidizing the carbohydrate moieties on a glycoprotein enzyme, then introducing antibody in the presence of polyethylene glycol (PEG). Covalent enzyme-antibody conjugates involving alkaline phosphatase and amyloglucosidase were produced and characterized.^ In order to increase the sensitivity of detecting the amyloglucosidase-antibody conjugate, an enzyme cycling assay was developed that measures glucose, the product of maltose cleavage by amyloglucosidase, in the picomole range. The increased sensitivity obtained by combined usage of the amyloglucosidase-antibody conjugate and enzyme cycling assay was then compared to that of conventional enzyme immunoassay (EIA).^ For immune complex isolation, polystyrene tubes and protein A-bearing Staphylococcus aureus were evaluated as solid phase matrices, upon which antibodies can be immobilized. A sandwich-type EIA, using antibody-coated S. aureus, was developed that measures human albumin (HSA) in the nanogram range. The assay, using an alkaline phosphatase-anti-HSA conjugate, was applied to the determination of HSA in human urine and evaluated extensively for its clinical applicability.^ Finally, in view of the clinical significance of alpha-fetoprotein (AFP) as an oncofetal antigen and the difficulty with its purification for use as an immunogen and assay standard, a chemical purification protocol was developed that resulted in a high yield of immunochemically pure AFP. ^

Cerebral High-grade Oligodendroglioma with Sarcomatous Transdifferentiation ("Oligosarcoma") in a Boxer Dog.

A 9-year-old Boxer dog was referred to the Veterinary Teaching Hospital of the University of Bern for a history of chronic neck pain and gait problems, which rapidly progressed to a non-ambulatory status. Magnetic resonance imaging (MRI) examination of the head revealed a large intra-axial space-occupying lesion that was divided in two portions interconnected by a thin isthmus at the level of the cerebellar tentorium. Histopathology revealed a biphasic malignant neoplasm composed of neuroepithelial and mesenchymal elements. The former displayed characteristics of conventional anaplastic oligodendroglioma involving brisk mitotic activity and glomeruloid microvascular proliferation on a background of a fibrillary round cells with "honeycomb-like" perinuclear vacuolation. Conversely, the sarcomatous moiety exhibited haphazard fascicles of spindle cells amidst an intricate mesh of pericellular basal lamina and broad bands of collagen. Both tumor cell populations immunoreacted for Olig-2 and – to a lesser extent – GFAP. In addition, the sarcomatous areas focally expressed vimentin, muscular actin, and smooth muscle actin. "Oligosarcoma" - an exquisitely uncommon pattern of oligodendroglial malignancy in humans - has not previously been reported to affect dogs, although oligodendroglioma is a common CNS tumor in this species. Whether canine oligosarcoma shares with its human counterpart not only morphological aspects, but also molecular signatures, clinical behavior and responsiveness to therapy merits further investigation. In humans, oligodendroglial differentiation tends to confer significant clinical advantage with respect to prognosis and adjuvant treatment options. The awareness of such hallmarks and the investigation of their impact on prognosis are crucial for improved therapeutical strategies in dogs.

Prevalence and prognostic significance of TMPRSS2-ERG gene fusion in lymph node positive prostate cancers.

BACKGROUND TMPRSS2-ERG gene fusion is the most frequent genetic alteration in prostate cancer. However, information about its distribution in lymph node positive prostate cancers and the prognostic significance in these advanced tumors is unknown. METHODS Gene fusion status was determined by fluorescence in situ hybridization on a tissue-microarray constructed from 119 hormone-naïve nodal positive, surgically treated prostate cancers containing samples from the primary tumors and corresponding lymph node metastases. Data were correlated with various tumor features (Gleason score, stage, cancer volume, nodal tumor burden) and biochemical recurrence-free, disease-specific, and overall survival. RESULTS TMPRSS2-ERG fusion was detected in 43.5% of the primary tumors. Conversely, only 29.9% of the metastasizing components showed the fusion. Concordance in TMPRSS2-ERG status between primary tumors and metastases was 70.9% (Kappa 0.39); 20.9% and 8.1% of the patients showed the mutation solely in their primary tumors and metastases, respectively. TMPRSS2-ERG fusion was not correlated with specific histopathological tumor features but predicted favorable biochemical recurrence-free, disease-specific and overall survival independently when present in the primary tumor (P < 0.05 each). CONCLUSION TMPRSS2-ERG fusion is more frequent in primary prostate cancer than in corresponding metastases suggesting no selection of fusion-positive cells in the metastatic process. The gene fusion in primary tumors independently predicts favorable outcome.

NFkappaB and STAT binding elements participate in regulation of MUC1 expression in normal and transformed mammary epithelial cells

The MUC1 gene encodes a transmembrane mucin glycoprotein that is overexpressed in several cancers of epithelial origin, including those of breast, pancreas, lung, ovary, and colon. Functions of MUC1 include protection of mucosal epithelium, modulation of cellular adhesion, and signal transduction. Aberrantly increased expression of MUC1 in cancer cells promotes tumor progression through adaptation of these functions. Some regulatory elements participating in MUC1 transcription have been described, but the mechanisms responsible for overexpression are largely unknown. A region of MUC1 5′ flanking sequence containing two conserved potential cytokine response elements, an NFκB site at −589/−580 and a STAT binding element (SBE) at −503/−495, has been implicated in high level expression in breast and pancreatic cancer cell lines. Persistent stimulation by proinflammatory cytokines may contribute to increased MUC1 transcription by tumor cells. ^ T47D breast cancer cells and normal human mammary epithelial cells (HMEC) were used to determine the roles of the κB site and SBE in basal and stimulated expression of MUC1. Treatment of T47D cells and HMEC with interferon-γ (IFNγ) alone enhanced MUC1 expression at the level of transcription, and the effect of IFNγ was further stimulated by tumor necrosis factor-α (TNFα). MUC1 responsiveness to these cytokines was modest in T47D cells but clearly evident in HMEC. Transient transfection of T47D cells with mutant MUC1 promoter constructs revealed that the κB site at −589/−580 and the SBE at −503/−495 and were required for cooperative stimulation by TNFα and IFNγ. Electrophoretic mobility shift assays (EMSA) revealed that the synergy was mediated not by cooperative binding of transcription factors but by the independent actions of STAT1α and NFκB p65 on their respective binding sites. Independent mutations in the κB site and SBE abrogated cytokine responsiveness and reduced basal MUC1 promoter activity by 45–50%. However, only the κB site appeared to be constitutively activated in T47D cells, in part by NFκB p65. These findings implicate two cytokine response elements in the 5 ′ flanking region of MUC1, specifically a κB site and a STAT binding element, in overexpression of MUC1 in breast cancer cells. ^

Quantitative comparison of late effects following photon versus proton external-beam radiation therapies: Toward an evidence-based approach to selecting a treatment modality

Radiation therapy has been used as an effective treatment for malignancies in pediatric patients. However, in many cases, the side effects of radiation diminish these patients’ quality of life. In order to develop strategies to minimize radiogenic complications, one must first quantitatively estimate pediatric patients’ relative risk for radiogenic late effects, which has not become feasible till recently because of the calculational complexity. The goals of this work were to calculate the dose delivered to tissues and organs in pediatric patients during contemporary photon and proton radiotherapies; to estimate the corresponding risk of radiogenic second cancer and cardiac toxicity based on the calculated doses and on dose-risk models from the literature; to test for the statistical significance of the difference between predicted risks after photon versus proton radiotherapies; and to provide a prototype of an evidence-based approach to selecting treatment modalities for pediatric patients, taking second cancer and cardiac toxicity into account. The results showed that proton therapy confers a lower predicted risk of radiogenic second cancer, and lower risks of radiogenic cardiac toxicities, compared to photon therapy. An uncertainty analysis revealed that the qualitative findings of this study are insensitive to changes in a wide variety of host and treatment related factors.

GENETIC ANALYSIS OF THE HIPPO PATHWAY IN MOUSE LIVER

Cancer therapy and tumor treatment remain unsolved puzzles. Genetic screening for tumor suppressor genes in Drosophila revealed the Hippo-signaling pathway as a kinase cascade consisting of five core components. Disrupting the pathway by deleting the main component genes breaks the balance of cell proliferation and apoptosis and results in epithelial tissue tumorigenesis. The pathway is therefore believed to be a tumor suppressor pathway. However, a corresponding role in mammals is yet to be determined. Our lab began to investigate the tumor suppression function of the potent mammalian Hippo pathway by putting floxed alleles into the mouse genome flanking the functional-domain-expressing exons in each component (Mst1, Mst2, Sav1, Lats1 and Lats2). These mice were then crossed with different cre-mouse lines to generate conditional knockout mice. Results indicate a ubiquitous tumor suppression function of these components, predominantly in the liver. A further liver specific analysis of the deletion mutation of these components, as well as the Yap/Taz double deletion mutation, reveals essential roles of the Hippo pathway in regulating hepatic quiescence and embryonic liver development. One of the key cellular mechanisms for the Hippo pathway’s involvement in these liver biological events is likely its cell cycle regulation function. Our work will help to develop potential therapeutic approaches for liver cancer.

Overexpression of DNA polymerase β in cell results in a mutator phenotype and a decreased sensitivity to anticancer drugs

DNA polymerase β (pol β) is the most error prone of all known eukaryotic DNA polymerases tested in vitro. Here, we show that cells overexpressing pol β cDNA have acquired a spontaneous mutator phenotype. By measuring the appearance of mutational events using three independent assays, we found that genetic instability increased in the cell lines that overexpressed pol β. In addition, these cells displayed a decreased sensitivity to cancer chemotherapeutic, bifunctional, DNA-damaging agents such as cisplatin, melphalan, and mechlorethamine, resulting in enhanced mutagenesis compared with control cells. By using cell-free extracts and modified DNA substrates, we present data in support of error-prone translesion replication as one of the key determinants of tolerance phenotype. These results have implications for the potential role of pol β overexpression in cancer predisposition and tumor progression during chemotherapy.

Loss of ovarian function promotes angiogenesis in human ovarian carcinoma

We show here that elevated levels of gonadotropins (luteinizing hormone and follicle stimulating hormone), as found in menopause or after ovariectomy, promote growth of human ovarian carcinoma by induction of tumor angiogenesis. Human epithelial ovarian cancer tumors progressed faster in ovariectomized mice. This induced growth could be attributed to the elevated levels of gonadotropins associated with loss of ovarian function because direct administration of gonadotropins also was effective in promoting tumor progression in vivo. On the other hand, gonadotropins had no direct effect on the proliferation of human ovarian cancer cells in vitro. Using MRI, we demonstrated that ovariectomy significantly (P < 0.02) induces neovascularization of human ovarian carcinoma spheroids implanted in nude mice. Moreover, conditioned medium of gonadotropin-treated human ovarian carcinoma cells showed increased mitogenic activity to bovine endothelial cells, and this activity could be blocked by neutralizing antibodies against luteinizing hormone and against vascular endothelial growth factor. Accordingly, gonadotropin stimulation resulted in a dose-dependent-induced expression of vascular endothelial growth factor in monolayer culture as well as in the outer proliferating cells of human ovarian cancer spheroids. These results demonstrate the significance of the elevated levels of gonadotropins, as found in menopause and in all ovarian cancer patients, on the progression of ovarian cancer and could explain the protective effect of estrogen replacement therapy. Based on these results, we suggest that hormonal therapy aimed at lowering the circulating levels of gonadotropins may possibly prolong remission in ovarian cancer by extending tumor dormancy.

Interações moleculares no mecanismo de ação da galectina-4 humana

A galectina-4 humana (HGal-4), pertencente à família das galectinas, possui dois domínios de reconhecimento de carboidratos (CRDs) com alta afinidade para β-galactosídeos e se encontra amplamente distribuída em células normais e neoplásicas de diferentes organismos. Suas funções snglobam uma grande variedade de eventos celulares, tais como processos inflamatórios, neoplásicos, progressão tumoral e metástase. Entretanto, muitas perguntas sobre suas interações com diferentes carboidratos, a especificidade destas interações e o papel específico das galectinas permanecem ainda sem resposta. No presente trabalho, propomos a investigação das interações galectina-glicano da galectina-4 humana e de seus domínios CRDs independentes (CRD-I e CRD-II) através de um conjunto de métodos biofísicos. Através do método de dicroísmo circular (CD), usando várias regiões espectrais, e fluorescência fomos capazes de entender mudanças ocorrentes na estrutura secundária e terciária das protéinas quando da interação com lactose/sacarose. Estes dados, juntamente com testes de hemaglutinação, mostraram que a glectina-4 e os CRDs respondem de forma distinta à ligação com açúcar. Por diferentes técnicas (fluorescência, ITC e MST) determinamos as constantes de dissociação para os domínios CRDs (Kd ~0,5 mM) e para HGal-4 e, de forma qualitativa, os valores obtidos indicaram possíveis estados oligoméricos dessas proteínas. A investigação da interação proteína-membrana da HGal-4 foi feita, primeiramente, com miméticos de membranas e monitorada pela técnica de RPE em crescente complexidade de composição de tais miméticos, indo desde composições mais simples, passando por lipid rafts na presença de diferentes glicolipídeos (GM1, LPS) e chegando-se à interação com células tumorais (U87MG, T98G e HT-29). Tais experimentos mostraram que galectina-4 reconhece e se liga naqueles modelos onde existem glicanos complexos na superfície. Investigamos também a participação de HGal-4 endógena e exógena no tratamento quimioterápico de células tumorais e verificamos um papel importante de HGal-4 para células HT-29. Finalizando esta tese, apresentamos o trabalho realizado em um ano de estágio na University of Oxford, durante o qual, investigamos a estrutura da região C-terminal de um receptor da família GPCR, qual seja o receptor de neurotensina NTS1. Aqui, mais uma vez, foi empregada a técnica de RPE que aliada à produção/marcação de mutantes do receptor, permitiu determinar que a hélice H8 se estabiliza quando em proteolipossomos.

Efeitos do treinamento de força no músculo esquelético em ratos com caquexia induzida pelo câncer

A ausência de terapias eficazes para a caquexia permanece como um problema central para o tratamento do câncer no mundo. Em contrapartida, o treinamento de força (i.e. também conhecido como treinamento resistido) tem sido amplamente utilizado como uma estratégia não farmacológica anticatabólica, prevenindo a perda da massa e da função da musculatura esquelética. Entretanto, o papel terapêutico do treinamento de força na caquexia do câncer permanece apenas especulativo. Portanto, nesse estudo avaliamos se o treinamento de força poderia atenuar a perda da massa e da função da musculatura esquelética em um severo modelo de caquexia do câncer em ratos. Para isso, ratos machos da linhagem Wistar foram randomizados em quatro grupos experimentais: 1) ratos sedentários injetados com solução salina na medula óssea (Controle); 2) ratos injetados com solução salina na medula óssea e submetidos ao treinamento de força (Controle + T); 3) ratos sedentários injetados com células do tumor Walker 256 na medula óssea (Tumor); e 4) ratos injetados com células do tumor Walker 256 na medula óssea e submetidos ao treinamento de força (Tumor + T). Foram avaliados a massa e a área de secção transversa da musculatura esquelética, marcadores de disfunção metabólica e do turnover proteico, a função da musculatura esquelética in vivo e ex vivo, o consumo alimentar, o crescimento tumoral e a sobrevida dos grupos experimentais com tumor. O grupo Tumor apresentou atrofia muscular após quinze dias da injeção das células tumorais como pode ser observado pela redução na massa dos músculos Plantaris (- 20,5%) e EDL (-20%). A atrofia no músculo EDL foi confirmada por análises histológicas, demonstrando uma redução de 43,8% na área de secção transversa. Embora o treinamento de força tenha aumentado o conteúdo proteico da lactato desidrogenase e revertido totalmente o conteúdo da forma fosforilada de 4EBP-1 (i.e. repressor da transcrição de mRNA), ele não atuou na morfologia da musculatura esquelética nos animais com tumor. Além disso, o treinamento de força não atenuou a perda de função da musculatura esquelética, a anorexia, o crescimento tumoral ou a taxa de mortalidade. Contudo, a força muscular, avaliada pelo teste de 1RM, apresentou uma correlação negativa com a sobrevida dos animais (p = 0,02), sugerindo que a perda de força prediz a mortalidade nesse modelo experimental de caquexia do câncer. Em suma, a injeção de células do tumor Walker 256 na medula óssea induz caquexia do câncer em ratos. O treinamento de força não foi eficaz em atenuar a perda de massa e função da musculatura esquelética nesse modelo. Entretanto, a força muscular prediz a sobrevida dos animais, sugerindo que novos estudos são necessários para elucidar o possível efeito terapêutico do treinamento de força para atenuar a caquexia do câncer e a progressão tumoral

Cáncer gástrico en pacientes de Méderi – Hospital Universitario Mayor entre los años 2011 - 2014

Introducción: El cáncer gástrico es uno de los más frecuentes a nivel mundial y Colombia se sitúa entre los países de mayor incidencia en este tipo de patología. Objetivo: Describir las características epidemiológicas, clínicas, el tratamiento administrado y los desenlaces inmediatos de los pacientes con diagnóstico de cáncer gástrico atendidos en el Hospital Universitario Mayor de Bogotá entre los años 2011 y 2014. Metodología: Se realizó un estudio observacional descriptivo con diagnóstico de cáncer gástrico. Se realizaron análisis univariados por medio de proporciones para las variables cualitativas y medidas de tendencia central para las variables cuantitativas según la distribución. Resultados: Un total de 189 pacientes fueron analizados. El dolor fue el síntoma más frecuente en los pacientes (30.7%) y el principal signo encontrado fue una masa palpable en abdomen (9,5%). Los pacientes fueron sometidos a diferentes abordajes terapéuticos, la mayoría recibieron manejo paliativo no quirúrgico (52.9%) y la opción quirúrgica más usada en los pacientes fue la gastrectomía total (20.6%), y la subtotal (16,4) seguidas de quimioterapia y/o radiación perioperatoria. Los pacientes que sobrevivieron a los 2 años fueron 7,4% del total. Conclusiones: El registro de los pacientes con cáncer gástrico es bueno en el Méderi-Hospital Universitario Mayor es bueno y permite caracterizar los pacientes, la presentación de la patología y los resultados del tratamiento que concuerdan con los presentados en contextos similares en la literatura.

Peptidomimetic ligands for tuning integrin-mediated signalling: rational design, synthesis and theranostic applications

Integrins are α/β-heterodimeric transmembrane adhesion receptors that mediate cell-cell and cell-ECM interactions. Integrins are bidirectional signalling receptors that respond to external signals (“outside-in” signalling) and in parallel, transduce internal signals to the matrix (“inside-out” signalling), to regulate vital cellular functions including migration, survival, growth and differentiation. Therefore, dysregulation of these tightly regulated processes often results in uncontrolled integrin activation and abnormal tissue expression that is responsible for many diseases. Because of their important roles in physiological and pathological events, they represent a validated target for therapeutic and diagnostic purposes. The aim of the present Thesis was focused on the development of peptidic ligands for α4β1 and αvβ3 integrin subtypes, involved in inflammatory responses (leukocytes recruitment and extravasation) and cancer progression (angiogenesis, tumor growth, metastasis), respectively. Following the peptidomimetic strategy, we designed and synthesized a small library of linear and cyclic hybrid α/β-peptidomimetics based on the phenylureido-LDV scaffolds for the treatment of chronic inflammatory autoimmune diseases. In order to implement a fast and non-invasive diagnostic method for monitoring the course of the inflammatory processes, a flat glass-surface of dye-loaded Zeolite L-crystal nanoparticles was coated with bioactive α4β1-peptidomimetics to detect specific integrin-expressing cells as biomarkers of inflammatory diseases. Targeted drug delivery has been considered a promising alternative to overcome the pharmacokinetic limitations of conventional anticancer drugs. Thus, a novel Small-Molecule Drug Conjugate was synthesized by connecting the highly cytotoxic Cryptophycin to the tumor-targeting RGDfK-peptide through a protease-cleavable linker. Finally, in view to making the peptide synthesis more sustainable and greener, we developed an alternative method for peptide bonds formation employing solvent-free mechanochemistry and ultra-mild minimal solvent-grinding conditions in common, inexpensive laboratory equipment. To this purpose, standard amino acids, coupling agents and organic-green solvents were used in the presence of nanocrystalline hydroxyapatite as a reusable, bio-compatible inorganic basic catalyst.

Ontological representation of tumor-node-metastasis classification and an ontology-driven classifier: a study on colorectal cancer

Dissertação de mestrado integrado em Engenharia Biomédica (área de especialização em Informática Médica)

Phase II study of weekly plitidepsin as second-line therapy for small cell lung cancer.

OBJECTIVE: To evaluate the antitumor activity and safety profile of plitidepsin administered as a 1h weekly intravenous (i.v.) infusion of 3.2mg/m(2) to patients with small cell lung cancer (SCLC) who relapsed or progressed after one line of chemotherapy. PATIENTS AND METHODS: This was a multicenter, open-label, single-arm, exploratory, phase II clinical trial. Treatment lasted until disease progression, unacceptable toxicity, patient refusal or treatment delay for >2 weeks. Objective response rate (primary efficacy endpoint) was evaluated according to response evaluation criteria in solid tumors (RECIST). The rate of stable disease (SD) lasting for at least 6 months and time-to-event variables were secondary endpoints of efficacy. Toxicity was assessed using National Cancer Institute Common Toxicity Criteria (NCI-CTC) version 2.0. RESULTS: Twenty pretreated SCLC patients (median age, 60 years) with extensive (n=13) or limited-stage disease (n=7) received a total of 24 treatment cycles (median, one cycle per patient; range, 1-2). Objective tumor responses were not observed and only one of the 17 evaluable patients had SD. With a median follow-up of 11.8 months, the progression-free survival and the median overall survival were 1.3 months and 4.8 months, respectively. The most troubling or common toxicities were fatigue, muscle weakness, lymphopenia, anemia (no patients showed neutropenia), and asymptomatic, non-cumulative increase of transaminases levels and alkaline phosphatase. CONCLUSION: This clinical trial shows that a cycle of 1h weekly i.v. infusion of plitidepsin (3.2mg/m(2)) was generally well tolerated other than fatigue and muscle weakness in patients with pretreated SCLC. One patient died due to multi-organ failure. The absence of antitumor activity found here precludes further studies of this plitidepsin schedule as second-line single-agent treatment of SCLC.

The results of surgery, with or without radiotherapy, for primary spinal myxopapillary ependymoma: a retrospective study from the rare cancer network.

PURPOSE: The aim of this study was to assess the outcome of patients with primary spinal myxopapillary ependymoma (MPE). MATERIALS AND METHODS: Data from a series of 85 (35 females, 50 males) patients with spinal MPE were collected in this retrospective multicenter study. Thirty-eight (45%) underwent surgery only and 47 (55%) received postoperative radiotherapy (RT). Median administered radiation dose was 50.4 Gy (range, 22.2-59.4). Median follow-up of the surviving patients was 60.0 months (range, 0.2-316.6). RESULTS: The 5-year progression-free survival (PFS) was 50.4% and 74.8% for surgery only and surgery with postoperative low- (<50.4 Gy) or high-dose (>or=50.4 Gy) RT, respectively. Treatment failure was observed in 24 (28%) patients. Fifteen patients presented treatment failure at the primary site only, whereas 2 and 1 patients presented with brain and distant spinal failure only. Three and 2 patients with local failure presented with concomitant spinal distant seeding and brain failure, respectively. One patient failed simultaneously in the brain and spine. Age greater than 36 years (p = 0.01), absence of neurologic symptoms at diagnosis (p = 0.01), tumor size >or=25 mm (p = 0.04), and postoperative high-dose RT (p = 0.05) were variables predictive of improved PFS on univariate analysis. In multivariate analysis, only postoperative high-dose RT was independent predictors of PFS (p = 0.04). CONCLUSIONS: The observed pattern of failure was mainly local, but one fifth of the patients presented with a concomitant spinal or brain component. Postoperative high-dose RT appears to significantly reduce the rate of tumor progression.