Thèses sur le sujet « Neoplasie solide »

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Dissertacao (Mestrado)
IPEN/D
Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP

Background: The JAK-STAT pathway is a cellular signaling pathway, which acts normally in humans and animals in the control of multiple important functions. Dysregulation of this pathway has been identified in human cancers, as well as a limited number of veterinary cancers. Objectives: The aims of this study were to identify the presence and tentative activity of components of the JAK-STAT pathway in selected canine tumors. Methods: Formalin-fixed, paraffin-embedded samples from mast cell tumors (MCT), hemangiosarcomas (HSA), thyroid carcinomas, and apocrine gland anal sac adenocarcinomas (AGASACA) were obtained from the Diagnostic Histopathology Laboratory at the Virginia Maryland College of Veterinary Medicine. Immunohistochemistry was performed to evaluate protein levels of JAK1, phospho-JAK1, JAK2, phospho-JAK2, STAT3, and phospho-STAT3. Signalment, treatment information, and survival information was obtained from the medical record for each case. Results: Tumor samples were scored for percent positive neoplastic cells. Positive staining was seen for all antibodies in all tumor types, with expression of JAK1, STAT3, and pSTAT3 being highest overall for all tumor types. Significant associations were seen between JAK1 and survival time in MCT (p = 0.03), pJAK1 and survival time in HSA (p = 0.009) and MCT (p = 0.04), and pSTAT3 and metastasis in MCT (p = 0.0008). Conclusions: The finding of positive staining for the components of the JAK-STAT pathway in the tumor samples evaluated indicates presence and tentative activity of this pathway in the studied cancers. Further study of JAK1, pJAK1, and pSTAT3 should be pursued to evaluate their potential as therapeutic targets.
MS

Cancer is one of the commonest conditions among patients admitted to Intensive Care Units (ICU). However, little is known about how it affects likelihood of ICU admission and subsequent clinical progress. What literature does exist is often not generalisable to current UK practice. The aims of the studies presented in this thesis are to determine the features that are associated with ICU admission in patients with solid tumours; to describe how the solid tumour population in ICU differs from the ICU population without cancer; how this impacts upon survival; and finally, to describe the long-term outcomes of solid tumour patients that have survived ICU and those features associated with mortality. I undertook a detailed systematic review of the international literature relating to survival following ICU admission for patients with solid tumours. This revealed a paucity of high quality studies and led to recommendations for improving the conduct and reporting of future research in this field. Using retrospective cohorts from prospectively collected databases, variables relating to patients in the West of Scotland diagnosed with a cancer between 1st January 2000 and 31st December 2009 were analysed. The rate of ICU admission within two years following cancer incidence was investigated, and the factors associated with admission described. The Scottish Intensive Care Society Audit Group (SICSAG) database was used to detail information pertaining to critical illness and to provide data on patients without an underlying tumour that were admitted to ICU during the same study period. Three cohorts were defined: patients with a solid tumour that were admitted to ICU, patients with a solid tumour that were not admitted to ICU, and ICU patients without a cancer diagnosis. One in twenty patients diagnosed with a solid tumour (5.2%) were admitted to ICU with the majority receiving organ support during their ICU stay. ICU admission tended to occur soon after cancer diagnosis and was therefore likely related to the cancer diagnosis or its treatment. The rate of ICU admission was greatest for bowel malignancies (16.5% of colorectal cancer patients) and for those tumours that require peri-operative ICU support for tumour resection surgery such as head and neck cancers (12.8%), stomach cancer (11.3%) and oesophageal cancer (10.2%). When compared with the ICU population without cancer, patients with solid tumours tended to be older (median age 68 years vs. 59 years, respectively), with a higher proportion of elective hospitalisations (52.7% vs. 10.0%) and were predominantly admitted to ICU with a surgical illness (89.3% vs. 55.0%). Surgical ICU admissions have a favourable ICU and hospital mortality if they have an underlying cancer diagnosis compared with surgical ICU patients without cancer (hospital mortality 22.9% vs. 28.1%, respectively). A potential explanation for this would be a higher proportion of level 2 admissions, lower utilisation of multi-organ support and an opportunity for pre-operative optimisation within the cancer group. ICU cancer patients admitted with a medical diagnosis have poorer short-term survival than those without cancer (hospital mortality 49.1% vs. 41.7%, respectively) and this difference is even more pronounced in those that received organ support (62.5% vs. 46.2%). In patients that survive an admission to ICU the presence of cancer has the largest impact upon mortality risk in the longer-term with a risk of death over three times greater than in the population of ICU survivors without cancer. Long-term survival varies considerably by underlying tumour type with four-year survival varying from 10.0% in patients with hepatocellular carcinoma to 73.3% in patients with testicular cancer. Cancer-related factors such as tumour stage have an important role in determining mortality risk in the longer term for survivors of ICU with cancer. In patients with colorectal cancer that had survived an ICU admission the risk of death after six-months was significantly higher in patients with Dukes D stage vs. Dukes A (HR 8.66). The work presented in this thesis systematically reviews and summarises the current published outcomes of patients with solid tumours admitted to ICU, demonstrates that among the solid tumour population ICU admission is common and shows that short-term outcomes vary significantly by features associated with both the critical illness and the underlying tumour type. In patients that survive an ICU admission the presence, type and stage of cancer is important for determining on-going mortality risk. This information may be used when clinicians are discussing potential outcomes following admission to critical care with cancer patients. Future studies should focus on the administration of treatments for cancer after critical illness and whether they differ from those received by those patients without an ICU admission. Prospective studies are required to describe the pre-ICU deteriorations in physiology in cancer patients with critical illness including those considered, but not admitted, to ICU. Outcomes for this latter group are unknown and given the high burden of illness severity documented in ICUs within the UK, these studies may identify a group of patients for whom critical care would be beneficial but is not currently provided.

Abstract This study aimed to assess the value of B-mode, nonenhanced and enhanced power Doppler ultrasonography (US), fine-needle aspiration biopsy (FNAB) and magnetic resonance (MR) imaging as adjunctive tools in breast diagnostics. The findings were compared to histology. The mammograms and US images of 84 palpable breast lesions were retrospectively reviewed, 63 of them also blindly. The cytologic reports of 57 lesions were reviewed. Eighty-one (96%) of all the 84 lesions, and 52 of the 53 cancers were visible as a local abnormality at US. The sensitivity, specificity, and overall accuracy of FNAB cytology was 92%, 83%, and 88%, respectively. There were no false negative malignancies in the three modalities combined. Sixty-five lesions not unequivocally benign at mammography were examined with B-mode, unenhanced and enhanced power Doppler US. Vascularity was also analyzed quantitatively. The sensitivity, specificity, and overall accuracy of the morphologic evaluation was 100%, 10%, and 57%, respectively. Rounded lesions were more vascular than spiculated lesions, but vascular assessment was only helpful when supporting a benign morphology. Forty breast lesions were examined with dynamic MR imaging and power Doppler US by obtaining time-signal intensity curves, which were analyzed morphologically and quantitatively. The shape of the MR curve acchieved 90% accuracy in differentiating between benign and malignant lesions. It enabled also differentiation between fibroadenomas and malignancies. The accuracy of the US curve was 38%. Quantitatively, statistically significant differences were found using all the MR variables, except between fibroadenomas and malignancies. Using the US variables, no significant difference was found. Twenty-eight patients (34 breasts) were examined by dynamic low-field and high-field MR imaging. The images were analyzed separately by two radiologists paying attention to lesion morphology and enhancement kinetics. In 27 breasts, results were compared to biopsy. Kappa statistics was used to compare the performance between the MR-scanners and readers. The sensitivity was 100% and 100%, the specificity 82% and 73%, and the accuracy 93% and 89% at low and high field, respectively. The inter-MR-scanner kappa value was 0.77 (substantial agreement), while the inter-observer kappa value was 0.86 and 0.81 at low and high field, respectively (almost perfect agreement).

Le developpement de la cytogenetique moleculaire, et en particulier celui des techniques d'hybridation in situ fluorescente (fish), a permis d'analyser plus finement l'instabilite chromosomique dans les cellules tumorales. Nous avons mis en place une nouvelle technique de fish appelee hybridation genomique comparative (cgh). La cgh permet de visualiser l'ensemble des desequilibres genomiques (amplifications et deletions) presents dans un echantillon tumoral. Bien que consideree comme une technique revolutionnaire, la cgh presente neanmoins l'inconvenient de ne detecter que les anomalies chromosomiques de grande taille. Apres avoir defini ses limites, nous l'avons utilisee lors d'une etude retrospective de 100 tumeurs des tissus mous, un groupe de cancer heterogene. Ces travaux ont montre l'implication specifique de certaines regions du genome dans la progression de ces tumeurs. L'etude de la predisposition au cancer constitue un bon moyen pour identifier les genes necessaires au maintien de l'integrite du genome. Dans ce cadre, nous avons participe au clonage positionnel du gene predisposant a la neoplasie endocrinienne multiple de type i (men1), un syndrome caracterise par une instabilite chromosomique constitutionnelle et des tumeurs affectant principalement les glandes endocrines. Ce travail a ete effectue au sein d'un consortium europeen. Notre contribution a consiste en l'etablissement d'une carte physique et genetique fiable de la region candidate minimale, localisee en 11q13. Le gene identifie code pour une proteine de 610 acides amines, denommee menin, ne presentant aucune homologie avec des proteines connues. Les mutations, dispersees sur toute la sequence, ne donnent pas indications sur des domaines fonctionnels importants. Afin d'elucider la fonction du gene men1, nous avons identifie son homologue murin et etudie l'instabilite chromosomique constitutionnelle dans des cellules issues de patients men1 et de leurs proches.

The PhD research work was performed at the Pediatric Hemato-Oncology Department of Padua University, one of the major AIEOP (Associazione Italiana di Emato-Oncologia Pediatrica) Centres and the coordinating centre of the Soft Tissue Sarcoma Committee (STSC) protocol EpSSG 2005 for pediatric soft tissue sarcomas. The primary goal of the PhD was analyzing new therapeutic strategies for resistant solid tumors children. The work moved from data analysis on adolescents with rhabdomyosarcoma (RMS) and resistant RMS patients treated with a new second line topotecan (T)/carboplatin (C) based regimen, through a specific training in clinical trials management to obtain the necessary expertise to write down a phase II trial for resistant RMS patients. In the last decades in many types of cancer the survival rates are reported to be less favorable in adolescents compared with younger children. To investigate whether this is true for adolescents with RMS, the results obtained in patients enrolled in protocols run by the Italian STSC were analyzed. Our study concluded that the higher prevalence of unfavorable tumor characteristics among adolescents seems to explain their worse outcome, the limited number of adolescents enrolled in STSC studied is worrisome and cooperation with adult oncologists needs to be improved. Another bad result is about prognosis of children with metastatic or resistant RMS: there is a strong need to find new strategies to improve the outcome of these patients. T and C are known to have activity against a variety of pediatric tumors so a T/C based chemotherapy has been proposed as second line CT for children relapsed after being treated in the STSC protocols. Our study shows that the T/C combination is tolerable in heavily pretreated patients; the response rate (RR) is somewhat lower when compared to other combinations tested in phase II studies but it’s of interest for the population with alveolar subtype. Clinical trials are one of the most important tools to explore new therapeutic approaches; for this reason the PhD involved a specific training in clinical trials management through participation to courses ad hoc, creation of a team committed to new drugs environment, participation to international phase II and III trials. During the last part of PhD the efforts were coordinated to write a phase II trial on treatment of leptomeningeal dissemination by RMS. Neoplastic meningitis is a devasting complication of both solid and hematologic tumors and despite treatment the median survival duration is in the range of 8-16 weeks. Among available therapeutic approaches intrathecal (IT) chemotherapy is one of the most widely used even if no impact on survival has been demonstrated. Few anticancer agents are used in this setting, therefore it’s essential to develop new IT agents with novel mechanism of action. Topotecan showed interesting results, then we designed a phase II study with IT topotecan in patients with RMS and EPNET tumors and leptomeningeal spread.
L’attività di ricerca del dottorato si è svolta press il Dipartimento di Pediatria dell’Università di Padova; l’ unità di Oncoematologia pediatrica è uno dei maggiori centri AIEOP (Associazione Italiana di Emato-Oncologia Pediatrica) ed è il centro coordinatore per il protocollo EpSSG 2005 del comitato sarcomi tessuti molli per la cura dei sarcomi. L’obiettivo del dottorato era analizzare nuove strategie terapeutiche in bambini con tumori solidi recidivi o resistenti. Il lavoro si è svolto passando per l’analisi di dati su una popolazione di pazienti adolescenti affetti da rabdomiosarcoma (RMS), l’analisi di dati su pazienti con RMS resistente trattati con un regime basato su topotecan/carboplatino, una formazione specifica sui trials clinici per giungere all’obiettivo della stesura di un protocollo di fase II su RMS resistenti. Negli ultimi anni è emerso come in molte neoplasie gli adolescenti vadano peggio rispetto ai bambini; per capire se ciò è vero anche per i pazienti adolescenti con RMS, sono stati analizzati i risultati ottenuti dai pazienti con RMS trattati con i protocolli del STSC. Lo studio concludeva che l’outcome peggiore è giustificato per gli adolescenti da una maggior incidenza di caratteristiche prognostiche sfavorevoli, che il tasso di arruolamento nei protocolli è insoddisfacente e pertanto urge una migliore collaborazione con gli oncologi dell’adulto. La prognosi dei pazienti con RMS metastatico o resistente resta negativa a tutt’oggi: cè una forte esigenza di identificare nuove strategie per migliorare la sopravvivenza di questi pazienti. Topotecan e Carboplatino sono farmaci con nota efficacia in vari tumori pediatrici pertanto la loro combinazione è stata proposta come terapia di seconda linea nei bambini che ricadono dopo trattamento secondo i protocolli del STSC. Il nostro studio ha dimostrato che la combinazione è ben tollerata, che i tassi di risposta sono leggermente inferiori rispetto ad altri regimi precedentemente studiati ma interessanti in particolare per l’istotipo alveolare. I trials clinici sono uno degli strumenti più validi per esplorare nuove strategie terapeutiche; per questo buona parte dell’attività di dottorato è stata dedicata a una formazione specifica nella gestione dei trials clinici, attraverso la partecipazione a corsi, la formazione di un gruppo dedicato ai nuovi farmaci, la partecipazione a trials nazionali e internazionali di fase II e III. Durante l’ultima parte del Dottorato gli sforzi sono stati coordinati alla stesura di un protocollo di fase II per il trattamento delle meningosi da RMS/PNET. La meningite neoplastica è una complicanza devastante di neoplasie sia solide che ematologiche; indipendentemente dal trattamento la sopravvivenza si aggira sulle 8-16 settimane. Tra i vari trattamenti la chemioterapia intratecale (CT IT) è molto usata nonostante non vi sia dimostrazione che impatti sulla sopravvivenza. Vi sono pochi chemioterapici ad uso IT disponibili, quindi è necessario trovarne altri. Topotecan sembra promettente in tal senso, pertanto abbiamo disegnato un protocollo di fase II sull’uso di topotecan IT in bambini e adolescenti affetti da RMS/PNET con disseminazione leptomeningea.

Orientador: Luiz Carlos Zeferino
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
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Resumo: Introdução: Os carcinomas luminais de mama apresentam resposta variável à quimioterapia neoadjuvante. A identificação precoce das pacientes que se beneficiarão desse tratamento é importante para se evitar tratamento desnecessário e suas decorrentes toxicidades. O objetivo desse estudo é identificar preditores de resposta à quimioterapia neoadjuvante no carcinoma luminal de mama. Metodologia: Nós incluímos pacientes com carcinoma luminal de mama tratadas com quimioterapia pré-operatória em nosso serviço, no anos de 2013 e 2014. Dados demográficos, clínicos e patológicos foram coletados. Análises de imunoistoquímica para receptores de estrogênio e progesterona, HER2 e Ki67 foram realizadas em amostras obtidas antes do tratamento. A quimioterapia foi composta por esquema baseado em antraciclinas e taxanos. Resposta patológica completa (RPC) foi definida como ausência de doença invasora nas peças cirúrgicas da mama e da axila. Resultados: Foram incluídas cento e vinte pacientes, totalizando 131 tumores. As taxas de RPC foram de 6,11% na amostra geral, 5,26% para tumores luminais B-like (HER2-negativos) e 12,9% para luminais B-like (HER2-positivos). Nenhum preditor independente de RPC foi identificado. Cinquenta e dois tumores (39,7%) apresentaram resposta clínica completa, e grau histológico III (OR=9,05; IC95% 1,07-7,98; p=0,04), status pré-menopausal (OR=9,05; IC95% 2,01-40,87; p=0,004), e índice de Ki67 ? 20% (OR=2,71; IC95% 1,04-6,99; p=0,04) foram preditores independentes desse desfecho. A análise da curva ROC mostrou que o índice de Ki67 pré-tratamento tem baixo desempenho na identificação de pacientes que apresentarão resposta clínica completa (área sob a curva=0,660; IC95% 0,563-0,758). Pacientes com resposta clínica completa apresentaram maior chance de serem submetidas a cirurgia conservadora. Conclusões: A taxa de RPC é baixa entre os carcinomas luminais de mama submetidos a quimioterapia neoadjuvante. No entanto, parâmetros clinico-patológicos podem ajudar a identificar pacientes que apresentarão resposta clínica completa
Abstract: Purpose: Luminal breast cancers show a variable response to neoadjuvant chemotherapy. The early determination of those patients who will benefit from this treatment is important to avoid overtreatment and the resulting toxicity. The purpose of this study is to identify predictors of response to neoadjuvant chemotherapy in luminal breast cancer. Methods: We included patients with luminal breast cancer treated with neoadjuvant chemotherapy at the institution in 2013 and 2014. Demographic, clinical, and pathological data were collected. Immunohistochemistry analyses of estrogen and progesteron receptors, HER2 and Ki67 were conducted in samples obtained before treatment. Chemotherapy consisted in anthracycline/taxane-based scheme. Pathologic complete response was defined as the absence of invasive disease in the breast and axilla surgical specimens. Results: One hundred and twenty patients, totaling 131 tumors were included. Pathologic complete response rates were 6.11% in the overall sample, 5.26% for luminal B-like (HER2-negative) tumors and 12.9% for luminal B-like (HER2-positive) tumors. None independent predictor of pathologic complete response was identified. Fifty-two tumors (39,7%) showed complete clinical response, and histologic grade III (OR=2.92; 95% CI 1.07-7.98; p = 0.04), premenopausal status (OR=9.05, 95% CI 2.01-40.87; p = 0.004), Ki67 index ? 20% (OR=2.71, 95% CI 1.04 to 6,99; p = 0.04) were independent predictors for this outcome. ROC curve analysis showed that Ki67 index has a poor performance in identifying patients who will present complete clinical response (area under the curve=0.660, 95% CI 0.563-0.758). Patients with complete clinical response were more likely to be submitted to conservative surgery. Conclusions: Pathologic complete response rate is low among luminal tumors undergoing neoadjuvant chemotherapy. However, clinicopathological parameters can help identify patients who will present complete clinical response
Mestrado
Oncologia Ginecológica e Mamária
Mestre em Ciências da Saúde

Introdução. O receptor epidérmico humano 2 (Her-2) e a topoisomerase-IIα (T2A) são dois marcadores biológicos importantes, ambos tendo um valor prognóstico e preditivo potencial em pacientes com tumores sólidos. A amplificação dos genes Her- 2 e T2A são eventos independentes, embora o último seja mais frequente em tumores com amplificação do Her-2 (34-90%), do que em tumores sem amplificação do Her-2 (5-10%). Existe uma melhor correlação entre amplificação e superexpressão do Her-2 no câncer de mama (CM) do que em outros tumores. No entanto, no CM, a correlação entre amplificação e superexpressão da T2A tem sido inconsistente, e existe uma carência de tais dados em outros tipos de tumores. A expressão da proteína T2A tem mostrado uma boa correlação com o índice de proliferação tumoral, particularmente no CM. Objetivos. Artigo 1: Sintetizar o conhecimento atual sobre a importância dos marcadores Her-2 e T2A nos tumores sólidos. Artigo 2: Investigar a prevalência de amplificação e superexpressão do Her-2 e da T2A, a correlação entre estas variáveis e a correlação entre as variáveis e estágio clínico, em amostras de câncer de ovário (CO) fixadas em parafina. Artigo 3: Investigar a prevalência de amplificação da T2A, assim como a correlação entre esta variável e a expressão da proteína T2A e do marcador de proliferação celular Ki-67, em amostras de CM fixadas em parafina, mostrando uma amplificação do Her-2. Métodos. Artigo 1: Os dados foram identificados através de busca em bases de dados eletrônicas (medline), livros de resumos de congressos e referências de artigos de revisão e originais. Artigo 2: 73 amostras de CO foram testadas para amplificação e superexpressão do Her-2 e T2A, por hibridização in situ fluorescente (FISH) e imuno-histoquímica (IHC), respectivamente. Artigo 3: 103 amostras de CM, com amplificação do Her-2, foram testadas para amplificação do gene T2A (por FISH) e superexpressão das proteínas T2A e Ki-67 (por IHC). Resultados. Artigo 2: Com base nos pontos de corte >1.5 e >2 (relação cópias/CEP17), as taxas de amplificação do Her-2 foram 15/64(23.4%) e 8/64(12.5%), versus 16/64(25%) e 5/64(7.8 %) para a T2A. Encontramos somente 3/72(4.2%) casos de superexpressão do Her-2(3+), contra 15/70(21.4%) para a T2A (marcagem em >10% das células). Foi observada uma modesta correlação entre amplificação e superexpressão da T2A (p= 0.01) e uma forte correlação entre amplificação da T2A e do Her-2, quando analisados como variáveis contínuas (p<0.001). A amplificação da T2A correlacionou-se com estágio FIGO avançado (p= 0.02). Artigo 3: Uma amplificação do gene T2A foi observada em 36.9%(38/103) dos casos. Os níveis de amplificação do Her-2 (número de cópias) não se correlacionaram com a amplificação da T2A. A porcentagem média de células positivas para a T2A (por IHC) foi de 5% e 10%, para casos T2A não-amplificados e amplificados, respectivamente. Uma correlação fraca, mas ainda significativa, foi observada entre amplificação do gene T2A e porcentagem de células T2A-positivas por IHC (Spearman=0.23, p=0.02); a correlação entre estas duas variáveis foi mais forte em tumores Ki-67 positivos. Conclusões. Artigo 2 : A avaliação da amplificação e da superexpressão do Her-2 e da T2A, por FISH e IHC, respectivamente, é realizável em amostras de CO. Foi observada uma boa correlação entre a amplificação dos genes Her-2 e T2A, mas a correlação entre amplificação do gene e superexpressão da proteína foi fraca para ambos marcadores. As taxas de amplificação dos genes Her-2 e T2A são mais elevadas quando não é realizada correção para o número de cópias do CEP17. Parece existir uma boa correlação entre amplificação da T2A e estágio clínico avançado. Estudos adicionais serão necessários para determinar o melhor ponto de corte para estes marcadores. Artigo 3: Contrariamente ao Her-2, a amplificação do gene T2A não parece necessariamente levar à superexpressão da proteína no CM. Outros fatores, como o índice de proliferação celular, podem interferir na síntese da proteína T2A. Embora a maioria dos casos de aberrações do gene T2A ocorram em tumores Her-2 positivos, os níveis de amplificação do Her-2 não se correlacionaram com a amplificação do gene T2A.
Background. The human epidermal receptor 2 (Her-2) and topoisomerase-IIα (T2A) are two important biomarkers, with potential prognostic and predictive value in patients with solid tumours. Her-2 and T2A gene amplification are separate events, although the latter is more frequently seen in Her-2 amplified (34-90%) than in Her-2 non-amplified (5-10%) tumours. There is a better correlation between Her-2 amplification and protein overexpression in breast cancer (BC) than in other tumour types. Nevertheless, there is a doubtful correlation between T2A amplification and overexpression in BC, with virtually no data available in other tumour types. In BC, the expression of the T2A protein has shown a good correlation with tumour proliferation rate. Objectives. Article 1: To summarise the available literature on Her-2 and T2A in solid tumours. Article 2: To investigate the prevalence of Her-2 and T2A amplification and overexpression, the correlation between these variables and with clinical stage, in paraffin-embedded samples of ovarian cancer (OC). Article 3: To investigate the prevalence of T2A amplification, as well as the correlation between this variable and the expression of T2A protein and the proliferation marker Ki-67, in paraffinembedded samples of Her-2 amplified BC. Methods. Article 1: The data were identified through search in electronic databases (medline), abstract books and references from review and original articles. Article 2: 73 samples of OC were tested for Her-2 and T2A amplification and overexpression, by fluorescence in situ hybridisation (FISH) and immunohistochemistry (IHC), respectively. Article 3: 103 samples of Her-2 amplified BC were tested for T2A amplification (by FISH) and overexpression (by IHC), and Ki-67 expression (by IHC). Results. Article 2: Based on cut-offs of ≥1.5 and ≥2 (ratio copies/CEP17), amplification rates for Her-2 were 15/64(23.4%) and 8/64(12.5%) versus 16/64(25%) and 5/64(7.8%) for T2A. We found only 3/72(4.2%) cases of Her-2 overexpression(3+) versus 15/70(21.4%) for T2A (staining in >10% of the cells). There was a modest correlation between T2A amplification and overexpression (p=0.01) and a strong correlation between T2A and Her-2 amplification when these markers were analysed as continuous variables (p<0.001). T2A amplification significantly correlated with advanced FIGO stage (p=0.02). Article 3: T2A gene amplification was observed in 36.9%(38/103) of the Her-2 amplified samples. Her-2 amplification level (i.e. copy number) was not predictive of T2A amplification. The median percentage of T2A positive cells for T2A non-amplified and amplified cases were 5% and 10%, respectively. A weak but still significant correlation was observed between T2A gene amplification level and percentage of positively stained cells (Spearman=0.23, p=0.02), the observed correlation being higher in patients with positive staining for Ki-67. Conclusions. Article 2: The assessment of Her-2 and T2A amplification and overexpression by FISH and IHC, respectively, is feasible in OC samples. There was a good correlation between Her-2 and T2A gene amplification, but the correlation between gene amplification and protein overexpression was poor for both markers. Amplification rates were higher in the absence of correction for the number of copies of the CEP17. Finally, we found a good correlation between T2A amplification and advanced disease stage. Further studies should aim to determine the optimal cut-offs for these markers. Article 3: Contrary to Her-2, T2A gene amplification does not always lead to protein overexpression in BC. Other factors, especially tumour proliferation rate, may interfere with the T2A protein status. Although the majority of the cases of T2A gene aberrations are seen in Her-2 positive tumours, the level of Her-2 amplification does not predict for T2A amplification.

A terapia fotodinâmica (Photodynamic Therapy - PDT) é um método de tratar neoplasias baseado na interação entre luz, oxigênio molecular e um agente fotossensibilizador. Após a administração do agente, o tumor é iluminado com luz visível, ativando-o e produzindo espécies reativas de oxigênio, altamente citotóxicas, que provocam morte celular e destruição tecidual. Com a destruição do tumor há ativação do sistema imune inato e o subsequente processo inflamatório determina a apresentação de antígenos tumorais aos linfócitos, promovendo uma resposta imunológica adaptativa contra o tecido tumoral. O presente trabalho visou estudar a PDT associando um laser de diodo como fonte de luz e o fotossensibilizante Azul de Metileno (AM) a 1%, avaliando a sua efetividade no tratamento do Tumor de Ehrlich (TE) em sua forma sólida e a resposta imunológica nos animais tratados. Em um primeiro estudo, avaliou-se macro e microscopicamente tumores tratados, determinando a capacidade do protocolo em induzir inflamação e destruição do tecido tumoral. No segundo estudo, a resposta imune foi estudada em camundongos desafiados com um segundo implante de células do tumor de Ehrlich. O primeiro implante tumoral foi tratado com a PDT ou a excisão cirúrgica, comparando-se com um grupo controle sem tratamento. Os parâmetros avaliados após 17 dias foram o crescimento tumoral (p>0,05), peso relativo dos órgãos linfóides [Baço (p<0,05) e Linfonodo poplíteo (p>0,05)], tamanho relativo do linfonodo (p<0,05), presença de metástase em linfonodo poplíteo (p>0,05), contagem de leucócitos sanguíneos (p>0,05) e análise morfométrica quantitativa do tumor secundário [determinação da fração volumétrica de células tumorais (p<0,05), infiltrado inflamatório (p<0,05), necrose (p>0,06) e porcentagem da área tumoral em necrose (p<0,05)]. A PDT com o AM foi capaz de induzir necrose do TE e inflamação, havendo diferenças da resposta imune sistêmica quando comparado aos animais tratados por meio de excisão cirúrgica do tumor de Ehrlich.
Photodynamic therapy (PDT) is a method of treating neoplasms based on the interaction between light, molecular oxygen and a photosensitizing agent. After administration of the photosensitizer, the tumor is illuminated with visible light, activating the agent and producing reactive oxygen species (ROS). This highly cytotoxic ROS cause cell death and tissue destruction. The activation of the innate immune system and the subsequent inflammation induces tumor antigen presentation to lymphocytes, promoting an adaptive immune response against the tumor cells. This work aimed to study the PDT using a diode laser as light source and Methylene Blue (MB) 1% as photosensitizer. It was accessed its effectiveness in treating Ehrlich Solid tumor (ET) and the immune response produced in treated animals. First the treated tumors were evaluated macroscopically and microscopically, determining the ability of the protocol to induce inflammation and tumor tissue destruction. In a second study, the immune response was studied in mice challenged with a second tumor cell implant. The primary tumor was treated with PDT or surgical excision, comparing with a control group without treatment. The parameters evaluated after 17 days were tumor growth (p> 0.05), relative weight of lymphoid organs [spleen (p <0.05) and popliteal lymph node (p> 0.05)], the relative size of the lymph node (p <0, 05), metastasis at lymph node (p>0,05), blood leukocyte count (p> 0.05) and quantitative morphometric analysis of secondary tumor [determining the volume fraction of tumor cells (p <0.05), inflammatory infiltrate (p <0.05), necrosis (p> 0.06) and tumor necrosis area (p <0.05)]. PDT with MB was able to induce necrosis of the ET and inflammation, with differences in the immune response when compared to animals treated surgically to remove the Ehrlich tumor in its solid form.

Karcinom pluća predstavlja vodeći uzrok smrtnosti među svim malignim obolenjima, a uprkos dostignućima u dijagnostici i terapiji u poslednjih 30 godina nije došlo do bitnog poboljšanja izuzetno niske ukupne stope petogodišnjeg preživljavanja kod ovih pacijenata. U momentu otkrivanja bolesti preko trećine svih novootkrivenih slučajeva nalazi se u IV stadijumu bolesti. Precizno i adekvatno praćenje odgovora tumora na terapijski tretman kod obolelih od karcinoma pluća u IV stadijumu kao i što ranije utvrđivanje progresije bolesti, odnosno neefikasnosti terapijskog tretmana kod ove grupe bolesnika od velikog je značaja, jer za ove pacijente hemioterapija predstavlja jedinu terapijsku opciju. Postojeće konvencionalne metode jednodimenzionalnih merenja i procena tumorskog odgovora na terapijski tretman prema RECIST kriterijumima ne koriste sve prednosti CT dijagnostike i oslanjaju se na subjektivnost manuelnih merenja. Primena naprednih radioloških tehnika, poput volumetrije, mogu doprineti razvoju imidžing praćenja terapijskog odgovora tumora kod pacijenata sa karcinomom pluća. Cilj istraživanja je evaluacija primene jednodimenzionalnog i volumetrijskog merenja u proceni terapijskog odgovora karcinoma pluća višeslojnom kompjuterizovanom tomografijom. Metodologija: Istraživanjem po tipu prospektivne studije obuhvaćeno je 100 pacijenata obolelih od karcinoma pluća u IV stadijumu bolesti u vreme otkrivanja, koji su ispitivani u periodu od 2013. do 2016. godine u Institutu za plućne bolesti Vojvodine u Sremskoj Kamenici i koji su ispunili kriterijume za ulazak u studiju. Kod svih pacijenata obuhvaćenih istraživanjem, dva radiologa nezavisno su evaluirali sve CT preglede grudnog koša, izvršili jednodimenzionalna manuelna i volumetrijska merenja odabranih target lezija što je omogućilo utvrđivanje unutarčitačke i međučitačke varijabilnosti i slaganja rezultata merenja target lezija primenom ispitivanih metoda. Na osnovu rezutata izvršenih merenja urađena je kategorizacija terapijskog odgovora tumora primenom konvencionalnih RECIST kriterijuma kao i primenom modifikovanog sistema kategorizacije za volumetrijsku evaluaciju terapijskog odgovora sa modifikovanim optimalnim graničnim vrednostima za klasifikovanje progresije bolesti i pozitivnog odgovora na terapiju, izračunatim na osnovu ispitivanog uzorka (model 3Dindividual). Urađeno je poređenje manuelne i volumetrijske procene terapijskog odgovora primenom razičitih ispitivanih sistema klasifikacije uz utvrđivanje stepena različite klasifikacije i analizu preživljavanja pacijenata do pojave progresije bolesti. Uticaj morfoloških karakteristika “target” lezija na rezultate volumetrijskog merenja određen je analizom odstupanja izmerenog volumena tumora u odnosu na aritmetičku sredinu između grupa lezija ispitivanih morfooških karakteristika. Rezultati: Primena volumetrijskog merenja, na ispitivanom uzorku, dovodi do niže stope varijabilnosti rezultata merenja dimenzija target lezija u odnosu na konvencionalnu manuelnu metodu merenja i u slučaju međučitačke varijabilnosti (0,9% vs 6,5%) i u pogledu unutarčitačke varijabilnosti (4,9% vs 0,9%). Volumetrijskom evaluacijom terapijskog odgovora tumora uz primenu modifikovanih graničnih vrednosti kategorizacije (model 3D-individual) postiže se značajno različita klasifikacija terapijskog odgovora u odnosu na primenu konvencionalnih RECIST kriterijuma. U slučaju volumetrijske evaluacije terapijskog odgovora, klasifikovanje pacijenata primenom novog sistema kategorizacije ”3D-individual” dovodi do različite klasifikacije u 22,2 % slučajeva u poređenju sa RECIST ekvivalent kriterijumima za volumetriju, uz održavanje jednako dobre predikcije PFS ova dva sistema. Rezultati istraživanja pokazali su da izgled ivica lezija i odnos lezije prema okolnim anatomskim strukturama imaju srednji uticaj na varijabilnost rezultata volumetrijskih merenja. Zaključak: Primena volumetrijskog merenja kao novog aspekta morfološke procene odgovora karcinoma pluća na primenjenu terapiju može unaprediti donošenje terapijskih odluka kako u lečenju individualnih bolesnika tako i u vođenju kliničkih istraživanja.
Lung cancer is the leading cause of mortality among all malignancies, and despite advances in diagnostics and therapy over the past 30 years, there has been no significant improvement in the extremely low overall rate of a five-year survival with these patients. At the time of the diagnosis, more than a third of all newly discovered cases are at the IV stage of the disease. Precise and adequate monitoring of the response of the tumor to therapeutic treatment with lung cancer patients in IV stage, as well as the early detection of progression of the disease or inefficiency of therapy in this group of patients is of great importance as chemotherapy is the only therapeutic option for these patients. The existing conventional methods of one-dimensional measurement and assessment of tumor response to therapeutic treatment according to RECIST criteria do not use all the advantages of CT diagnostics and rely on the subjectivity of manual measurements. Advanced radiological techniques, such as volumetry, can contribute to the development of the image monitoring of the therapeutic response of tumors in patients with lung cancer. The aim of this study is to evaluate the application of one-dimensional and volumetric measurement in the assessment of the therapeutic response to lung cancer with multslice computerized tomography. Methodology: A study per type of prospective study included 100 patients with lung cancer at the IV stage of the disease at the time of detection, which were tested in the period between 2013 and 2016 at the Institute of Pulmonary Diseases of Vojvodina in Sremska Kamenica and met the criteria for entering the study. With all patients involved in the study, two radiologists independently assessed all CT chest exams, performed one-dimensional manual and volumetric measurements of selected target lesions, which enabled the determination of intraobserver and interobserver variability and the agreement of the target lesion measurement results using the test method. Based on the results of the performed measurements, the categorization of the therapeutic response of the tumor with conventional RECIST criteria, as well as the application of a modified categorization system for volumetric assessment of the therapeutic response with modified optimal limit values for classification (progression of the disease and positive response to the therapy) was performed, calculated on the basis of the tested sample. Comparison of manual and volumetric estimates of the therapeutic response was made using various classification systems with the determination of the degree of difference in classification and analysis of survival of patients until the progression of the disease. The influence of morphological characteristics of target lesions on the results of volumetric measurement was determined by the analysis of the deviation of the measured tumor volume relative to the arithmetic mean between the groups of lesions of the examined morphological characteristics. Results: The application of volumetric measurements on the test sample leads to a lower rate of variability in the results of measuring the dimensions of the target lesions compared to the conventional manual measurement method, and in the case of interobserver variability (0.9% versus 6.5%) and in terms of intraobserver variability (4.9% to 0.9%). The volumetric assessment of the therapeutic response of the tumor using modified boundary categorization values (3Dindividual model) results in a significantly different classification of the therapeutic response in relation to the use of conventional RECIST criteria. In the case of volumetric assessment of the therapeutic response, the classification of patients using the new “3D-individual” categorization system leads to a misclassification in 22.2% of cases compared to RECIST equivalent to volumetric criteria, reflecting the equally good predictability of PFS in these two systems. The results of the study showed that the appearance of the lesion margins and relation to the surrounding anatomical structures influenced the variability of the results of volumetric measurements. Conclusion: The application of volumetric measurements as a new aspect of the morphological evaluation of lung cancer response to applied therapies can help in making therapeutic decisions both in the treatment of individual patients and in the conduct of clinical trials.

Thesis (PhD)--Stellenbosch University, 2012.
ENGLISH ABSTRACT: Introduction: The anthracyclines (ACs), daunorubicin (DNR) and doxorubicin (DXR) are two of the most effective drugs known for the treatment of systemic neoplasms and solid tumours. However, their clinical use is often hampered by their dosedependent cumulative cardiotoxicity, which leads to irreversible and fatal druginduced congestive heart failure. The mechanism by which ACs induces heart damage is not fully understood. Recent reports have indicated that DXR activates autophagy and ubiquitin proteasome-mediated degradation of specific transcription factors, however, no reports exists on the effect of ACs on the E3 ubiquitin ligases, MuRF-1 and MAFbx. The aim of the first part of the study was therefore to investigate the effect of DNR treatment on the protein and organelle degradation systems in the heart and to elucidate the signalling mechanisms involved. Although this model was ideal in allowing the investigation of the signalling pathways which are affected by DNR, it did not allow for further exploration or manipulation of signalling pathways that may be of potential benefit in this context. The in vitro model was therefore used to validate the hypothesis that increased autophagy alleviates AC-induced cardiotoxicity and delays the onset of cardiomyocyte death. The aims for the second part of the study were (i) to characterize the effect of DXR in H9C2 cells, (ii) to determine whether the induction/inhibition of autophagy in combination with DXR alleviates cytotoxicity and (iii) to investigate the influence of increased/decreased autophagy in combination with DXR on reactive oxygen species (ROS) production, mitochondrial function, endoplasmic reticulum (ER) stress and the ubiquitin proteasome pathway. In the final part of this study, an in vivo model was used to assess the potential benefit of autophagy in a novel GFP-LC-3 tumour bearing mouse model of acute DXR-induced cardiotoxicity. Material and Methods: Adult rats were divided into two groups where one group received six intraperitoneal injections of 2 mg/kg DNR on alternate days and the other group received saline injections as control. Hearts were excised and perfused on a working heart system the day after the last injection and freeze clamped for biochemical analysis. H9C2s were cultured and treated with Bafilomycin A1 (10 nM, inhibitor of autophagy) for 6 hrs, Rapamycin (50 μM, inducer of autophagy) for 24 hrs, DXR (3 μM) for 24 hrs or a combination of these drugs. Following treatment, cells were harvested and assessed for cell death, proteolytic activity and oxidative stress using western blotting, fluorescence microscopy and flow cytometry. In the final phase of the study, twenty-four female mice were injected at 8 weeks with a mouse breast cancer cell line (EO771) and after observation of tumour growth, animals were either treated with one injection (i.p.) of Rapamycin (4 mg/kg), two injections (i.p.) of DXR (10 mg/kg) or a combination of the two drugs. After the experimental protocol, mice were terminated and their hearts were rapidly excised. The hearts were divided cross-sectionally and utilized for biochemical and histological analyses. Results and Discussion: DNR treatment significantly attenuated myocardial function and increased apoptosis in the ex vivo heart model. DNR-induced cardiac cytotoxicity was associated with the upregulation of two E3 ubiquitin ligases, MuRF-1 and MAFbx as well as a significant increase in two markers of autophagy, beclin-1 and LC-3. These changes observed in the heart were also associated with attenuation of the PI3-kinase/Akt signalling pathway. The augmentation of autophagy with rapamycin before DXR treatment significantly reduced cell death in the in vitro model. Indeed, rapamycin treatment demonstrated to be a vital survival mechanism for acute DXR-induced cardiotoxicity as it decreased cellular ROS production, improved mitochondrial function and prevented nuclear translocation of DXR. Moreover, these changes in cardiomyocytes were also associated with a reduction in the ubiquitin-proteasome pathway (UPP). In the final part of this study, a novel tumour bearing GFP-LC3 mouse model was developed to confirm the results obtained in the in vitro study. It was demonstrated that acute DXR-induced cardiotoxicity resulted in increased apoptosis, the inhibition of autophagy and increased proteolysis via the UPP. These findings were associated with a reduction in body weight and cardiomyocyte cross-sectional area. The cardiotoxic effects of DXR were substantially reduced when autophagy was induced with rapamycin. Taken together, our data strongly indicates that it is possible to attenuate the cardiotoxic effects of doxorubicin in cancer patients by carefully controlling the levels of autophagy using rapamycin as adjuvant therapy.
AFRIKAANSE OPSOMMING: Inleiding: Die antrasikliene (AC’s), daunorubisien (DNR) en doksorubisien (DKS), is twee van die mees effektiewe AC wat bekend is vir die behandeling van sistemiese neoplasmas en soliede tumore. Hulle kliniese gebruik word egter deur dosis afhanklike kumulatiewe kardiotoksisiteit benadeel, wat tot onomkeerbare en dodelike kongestiewe hartversaking kan lei. Die meganisme waardeur AC’s hartversaking kan veroorsaak, word nog nie ten volle verstaan nie. Onlangse navorsing het aangetoon dat DKS autofagie en die ubikwitienproteosoom-bemiddelde degradasie van spesifieke transkripsie faktore aktiveer. Daar is egter geen literatuur wat die effek van AC’s op die E3-ubikwitienligases, MuRF-1 en MAFbx beskryfnie. Die doel van hierdie eerste afdeling van die studie is om die effek van DNR behandeling op die proteïen- en organel degradasie sisteme in die hart te ondersoek en om van die betrokke seinmeganismes te bepaal. Alhoewel hierdie model ideaal is om sommige seinweë wat deur DNR geaffekteer word, te ondersoek, kon seinoordragpaaie wat potensieël voordelig in hierdie konteks is, nie in bg. model gemanipuleer word nie. Die in vitro model is gebruik om die hipotese dat verhoogde outofagie AC-geïnduseerde kardiotoksisiteit verlaag en sodoende seldood verminder, te bevestig. Die doel van hierdie afdeling van die studie was: (i) om die effek van DKS op H9C2 selle te karakteriseer, (ii) om te bepaal of die induksie/inhibisie van outofagie in kombinasie met DKS kardiotoksisiteit verbeter (iii) om die invloed van verhoogde/verlaagde outofagie in kombinasie met DKS op reaktiwe suurstof species (ROS), mitokondriale funksie, endoplasmiese retikulum (ER) stress en die ubikwitienproteosoompad te ondersoek. In die finale deel van hierdie studie, is ‘n in vivo model gebruik om die moontlike voordelige effek van verhoogde outofagie in ‘n GFP-LC-3 tumor-draende muismodel met akute DKSgeïnduseerde kardiotoksisiteit, ondersoek. Materiaal en Metodes: Volwasse rotte is in twee groepe verdeel waar een groep ses intraperitoneale inspuitings van 2 mg/kg DNR op afwissellende dae ontvang het en die andergroep as ‘n kontrole, ‘n soutoplossing gekry het. Die harte is verwyder en geperfuseer op ‘n werkende hartsisteem een dag na die laaste inspuiting en gevriesklamp vir biochemiese analises. H9C2 selle is vir 6 uurgekweek en behandel met Bafilomisien A1 (10 nM, ‘n autofagie inhibitor), 24 uur met Rapamisien (50 μM, ‘n autofagie induseerder), 24 uur met DKS (3 μM) of ‘n kombinasie van hierdie middels. Na behandeling is selle ge-oes vir analises in seldood, proteolitiese aktiwiteit en oksidatiewe stress deur van westelike kladtegniek, fluoresensie mikroskopie en vloeisitometrie gebruik te maak. In die finale fase van hierdie studie is vier en twintig, agt weke oue wyfie muise ingespuit met ‘n muisborskankersellyn (E0771) en is tumorgroei waargeneem; die diere is of behandel met een rapamisien inspuiting (i.p) (4 mg/kg), of twee DKS inspuitings (i.p.) (10 mg/kg) of ‘n kombinasie van die twee middels. Na die eksperimentele protokol, is die muise van kant gemaak en hulle harte vinnig verwyder. Die harte is in twee verdeel en gebruik vir biochemiese- en histologiese analises. Resultate en Bespreking: DNR behandeling het kardiale funksie betekenisvol verswak en apoptose in die hart verhoog. DNR-geïnduseerde kardiotoksisiteit is geassosieer met die opregulering van E3-ligases, MuRF-1 en MAFbx en het ook ‘n betekenisvolle toename in twee outofagie merkers, beclin-1 en LC-3 veroorsaak. Hierdie veranderinge wat in die hart waargeneem is, is ook geassosieer met ‘n onderdrukking van die PI3-kinase/Akt seinweg. Die toename in outofagie met rapamisien voor DKS behandeling het seldood in die vorm van apoptose betekenisvol verlaag. Daarmee saam het verhoogde outofagie ‘n noodsaaklike oorlewings meganisme vir akute DKS-geïnduseerde kardiotoksisiteit gedemonstreer. Die rede hiervoor is dat dit ROS produksie verlaag het, mitokondriale funksie verbeter het en DKS translokasie vanuit die sitoplasma tot binne die nukleus verhoed het. Hierdie veranderinge in kardiomiosiete is ook met ‘n afname in die ubikwitienproteosoomseinweg (EPS) geassosieer. In die finale deel van hierdie studie, is ‘n nuwe tumor-draende muismodel ontwikkel om die resultate wat in die in vitro studie gekry is, te bevestig. Daar is bewys dat akute DKS-geïnduseerde kardiomiotoksisiteit aanleiding gegee het tot verhoogde apoptose, outofagie inhibisie en verhoogde proteolise via die EPS. Hierdie bevindinge is geassosieer met ‘n verlaging in liggaamsgewig en kardiomiosiet dwarssnit area. Die kardiotoksiese effekte van DKS is insiggewend verminder as autofagiege ïnduseer is met rapamisien. Om saam te vat: Ons data bevestig dat dit moontlik is om die kardiotoksiese effekte van DKS in kanker pasiënte te verminder deur outofagie vlakke te monitor en te kontroleer deur middel van rapamisien behandeling as bykomende terapie.

A administração intravenosa de solução salina hipertônica (HSS) induz alterações sistêmicas circulatórias como o aumento da pressão arterial e do volume circulante efetivo, além de ter efeitos locais sobre a microcirculação. No presente estudo foram analisados os efeitos produzidos pela administração de solução salina hipertônica 7,5% sobre a hemodinâmica do tumor através de estudos de imagem funcional e posteriormente, foi avaliado o seu potencial de otimizar a entrega de moléculas no tumor. A velocidade do sangue nos vasos tumorais estimada por Ultrassom Color Doppler foi aumentada após a injeção da HSS em comparação ao controle PBS em tumores de melanoma (B16F10 (p=0,019), SK-MEL-147 (p =0,028)) e de mama (4T1 (p=0,015)). Este mesmo efeito não foi observado nas artérias segmentarias do rim (p=0,476). Ultrassonografia com contraste por microbolhas (CEUS) foi realizada em xenoenxertos de tumor de melanoma (B16F10), carcinoma de cólon (HCT-116) e mama (MDA-MB-231), e como controle foi realizada imagem no rim e no músculo nos animais portadores destes tipos tumorais (n=3 por grupo). Após a injeção da HSS, o volume relativo de sangue foi aumentado nos tumores B16F10 (p=0,022) e HCT-116 (P = 0,039), mas o mesmo não foi observado com o tumor MDA-MB-231 (p=0,186). Além disso, não houve alterações nos tecidos normais (rim p = 0,957; músculo p = 0,104). Todos os testes estatísticos foram bicaudais. Quando a HSS foi utilizada como veículo para entrega de moléculas de baixo peso molecular como cisplatina e doxorrubicina no tratamento de tumores B16F10 e 4T1 respectivamente, não houve aumento da eficácia terapêutica, avaliada através do crescimento tumoral e peso dos tumores. O efeito da HSS sobre a retenção de macromoléculas nos tumores SK-Mel- 147 e 4T1, avaliado através de imagem por epifluorescência do contraste ótico IR-783, não foi suficientemente notório para rejeitar a hipótese nula. Assim, a HSS induz um aumento transitório na velocidade do sangue e do volume sanguíneo, de maneira relativamente seletiva para os tumores avaliados, com exceção do MDA-MB-231. Portanto, esta pode ser uma estratégia útil para aumentar a entrega de moléculas e otimizar tanto o efeito terapêutico, quanto o diagnóstico por imagem
Intravenous administration of Hypertonic saline solution (HSS) induces systemic circulatory changes including blood pressure rising, effective circulating volume increase as well as local effects on microvasculature. We analyzed the effects produced by Hypertonic Saline 7,5% administration on tumor hemodynamics through functional imaging studies as well as whether it enhances molecular delivery in tumor tissue when used as a vehicle. Blood velocity assessed by Color Doppler Ultrasound was increased after HSS injection compared to PBS in the following tumors: B16F10 (p=0,019), SKMEL- 147 (p=0,028) and 4T1 (p=0,015). No statistical difference was observed on the segmental kidney arteries (p=0,476). Dynamic Contrast enhanced ultrasound (CEUS) was done in B16F10, HCT-116 and MDA-MB-231 tumor xenografts, kidney and muscle tissues (n=3 per group). After HSS injection, relative blood volume was increased in B16F10 (p=0,022) and HCT-116 (p=0,039) but not on MDA-MB-231 (p=0,186). Changes on normal tissues were not statistically different (kidney p=0,957; muscle p=0,104). All statistical tests were two-sided. Administration of HSS as a vehicle for low molecular weight molecules cisplatin and doxorubicin in the treatment of B16F10 and 4T1 tumors respectively had no significant improvement of therapeutic efficacy, estimated by tumor growth and tumor weight measurements. Effect of HSS over retention of macromolecules in tumors SK-Mel-147 and 4T1, evaluated by epifluorescence imaging of the optical contrast IR- 783 was not large enough to reject the null hypothesis. HSS induces a transient increase in velocity of the blood as well as the blood volume that is relatively selective for the evaluated tumors with exception of MDA-MB-231. Data suggest that HSS administration might be a useful strategy to increase the delivery of molecules and optimize both therapy and diagnostic imaging

Negli ultimi anni si è assistito ad una notevole ricerca di marcatori di espressione genica nelle cellule tumorali finalizzata ad applicazioni diagnostiche e per monitorare le diverse terapie. Un target molto importante oggetto di diversi studi in vivo e in vitro è rappresentato dall’enzima telomerasi. In particolare, lo studio di espressione genica dei suoi componenti è apparso molto utile per rivelare cellule tumorali circolanti nel sangue periferico e per evidenziare quindi la presenza di micrometastasi. La presenza di metastasi scheletriche in pazienti oncologici è gravata da una serie di complicanze. I bisfosfonati sono una classe di farmaci con una potente attività di inibizione del riassorbimento osseo e vengono sempre più utilizzati nel trattamento di pazienti con metastasi ossee. Alcuni studi clinici hanno dimostrato che tali farmaci sono molto efficaci nel trattamento delle metastasi ossee di tumori prostatici e mammari. Recenti studi hanno inoltre rivelato che i bisfosfonati sono anche in grado di agire direttamente sulla cellula tumorale interferendo con la crescita e la sopravvivenza delle cellule metastatiche nell’osso. La presente tesi illustra i risultati relativi alla downregolazione dell’espressione dei geni della telomerasi utilizzando i bisfosfonati in vitro, su linee tumorali, ed in vivo, su pazienti neoplastici affetti da carcinoma prostatico e mammario. Relativamente agli studi in vitro, verranno presentati i dati di inibizione cellulare mediante test di vitalità sulle colture cellulari trattate con concentrazioni crescenti di diversi bisfosfonati e verrà analizzata la relativa espressione dei geni della telomerasi mediante la tecnica di RT Real Time PCR. Verranno inoltre valutati gli effetti dello zoledronato su linee prostatiche co-incubate con il geranylgeraniol che è noto ripristinare la via del mevalonato. Per quanto riguarda gli studi in vivo, saranno presentati i dati relativi all’espressione dei geni della telomerasi nei sieri dei pazienti oncologici trattati con lo zoledronato. La presente esperienza ha dimostrato che: • I geni della telomerasi rappresentano un target alternativo all’azione dei bisfosfonati sulle cellule tumorali; • Gli amino-bisfosfonati riducono la proliferazione nelle cellule tumorali mediante la down-regolazione dell’espressione dei geni della telomerasi; • I bisfosfonati agiscono sull’espressione genica in una maniera concentrazione-dipendente; • L’utilizzo dello zoledronato in vivo è in grado di abbassare i livelli di espressione dei geni della telomerasi; • L’utilizzo di tale target molecolare in vivo è un valido metodo per monitorare l’azione del trattamento con zoledronato in pazienti neoplastici.
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Objectives: Oggetto della nostra sperimentazione è stato dimostrare la fattibilità e l'efficacia dell'utilizzo degli ultrasuoni focalizzati ad alta intensità (HIFU High Intensity Focused Ultrasound) sia in ambito curativo che palliativo in pazienti affetti da neoplasie epatiche, pancreatiche, uterine e ossee. Methods: Da Ottobre 2011 a Ottobre 2014 presso un'unica unità di riferimento, si sono trattati con metodica HIFU 208 pazienti, per un totale di 243 lesioni, distinti in 154 pazienti trattati a scopo curativo e 54 a scopo palliativo. Ogni paziente è stato sottoposto ad un trattamento USgHIFU su una singola sessione, valutando come primary outcomes l'efficacia terapeutica nel lungo termine e la sopravvivenza totale e come secondary outcomes le complicanze nel breve e lungo termine e la qualità di vita. Results: In ambito di epatocarcinoma sono stati trattati 69 pazienti, 79 lesioni, rapporto M/F 54/15, con dimensioni medie 4,5 cm, tempo di trattamento medio 110 min, potenza di trattamento medio 376 Watt, tasso di complicanze totale del 10,1%, efficacia terapeutica primaria in 55 pazienti (79,7%), tasso di sopravvivenza complessivo a 12 mesi, 18 mesi e 36 mesi del 85,3%, 30,3% e 16,5% rispettivamente, tasso di sopravvivenza libero da malattia a 12 mesi, 18 mesi e 36 mesi del 39,8%, 18,4% e 0%, rispettivamente. In ambito di fibromi uterini sono state sottoposte a termoablazione 85 pazienti per un totale di 110 fibromi con un diametro medio di 4,3 cm ± 2 (2-10,5) ,volumi di 59,8 cm3 ± 80.2 (1,5- 398,4), tempo di trattamento medio 79 min, potenza di trattamento medio 306 Watt, tasso di complicanze totale del 14,2%, miglioramento dei sintomi nell' 85.8% con una totale scomparsa nel 60% dei casi e con una riduzione media dei volumi come percentuale del volume iniziale a 1, 3, 6, 12, 24 e 36 mesi del 20,5%, 27,6%, 31,8%, 47,7%, 48,1%, 48,9% rispettivamente. In ambito di carcinomi del pancreas, 41 pazienti sottoposti a HIFU, rapporto M/F 34/7, con dimensioni medie 4,2 cm (3-8,3), tempo di trattamento medio 147 min, potenza di trattamento 369 Watt, volume di tumore ablato nel 77,5% tra il 100-90%, nel 17,5% tra il 90%-50%, nel 5% entro il 50%, nessuna ablazione inefficace. VAS pretrattamento, a 1, 3, 6, 12 mesi post HIFU, 5,1 ± 0,9 (range 3-9), 2,3 ± 1,2 (range 0-5), 2,1 ± 1,1 (range 0-5), 2,2 ± 1,3 (range 0-5), 2,1 ± 0,9 (range 0-5), rispettivamente (P value < 0,001). Sopravvivenza totale a 6, 12 e 18 mesi del 54%, 29,7% e 11,4% rispettivamente. In ambito di metastasi ossee, sono stati trattati 13 pazienti, rapporto M/F 8/5, con dimensioni medie 36278 mm3 ± 1952,32 (range 2340-78000), tempo di trattamento medio 90 min, potenza di trattamento 320 Watt, tasso di complicanze totale del 23,07%, volume di tumore ablato nel 69,2% tra il 100-90%, nel 23,07% tra il 90%-50%, nel 7,73% entro il 50%, nessuna ablazione inefficace, VAS pretrattamento, a 1, 3, 6, 12 mesi post HIFU, 5,46 ± 1,71 (range 3-9), 2,2 ± 1,3 (range 0-4), 2,1 ± 0,9 (range 0-4), 2,2 ± 1,1 (range 0-4), 2,1 ± 1,0 (range 0-5), rispettivamente (P value < 0,001). Sopravvivenza totale a 1, 2 e 3 anni del 84,2%, 15,1% e 0% rispettivamente. Conclusions: la termoablazione con ultrasuoni focalizzati ad alta intensità si è confermata essere anche nella nostra esperienza una procedura sicura ed efficace, in termini di tasso di complicanze, di adeguatezza terapeutica, di qualità di vita e di sopravvivenza totale, sia in ambito curativo che palliativo.

O cancro do pâncreas é uma das neoplasias malignas mais letais, com uma taxa de sobrevida 1 ano após o diagnóstico de 24% e 5 anos após o diagnóstico de apenas 9%. Estes dados espelham, contudo, o comportamento do subtipo histológico mais prevalente - o adenocarcinoma ductal. Porém, nem todas as neoplasias malignas do pâncreas são adenocarcinomas e nem todas estão a associadas a um prognóstico tão reservado. A Neoplasia Pseudopapilar do Pâncreas é o exemplo disso: descrita pela Organização Mundial da Saúde como uma neoplasia maligna de baixo grau, é um tumor raro associado a um excelente prognóstico, mesmo na presença de metástases (taxa sobrevida aos 5 anos de 97%). Mais que uma neoplasia com um prognostico peculiarmente favorável, é uma neoplasia única em todas as suas componentes: na sua histogénese, epidemiologia, apresentação clínica, características imagiológicas, citológicas e imunihistoquímicas e no tratamento. Estas particularidades têm que estar bem presentes e consolidadas no raciocínio clínico de qualquer médico, para que estas neoplasias sejam devidamente reconhecidas e tratadas. Neste sentido, foi realizado este artigo de revisão que visa sumariar os mais relevantes tópicos relacionados com esta entidade clínica.
Pancreatic cancer is one of the most lethal malignant neoplasms, with a 1year survival rate after diagnosis of 24%, and a 5years survival rate of only 9%. While this illustrates the behavior of its main histologic type - ductal adenocarcinoma, there are other histologic subtypes of pancreatic cancer that can harbor excellent prognosis. Solid pseudopapillary neoplasm, described as a rare low grade malignant neoplasm by the World Health Organization, is the best example of that, having a 5year survival rate of about 97%, even in the presence of metastasis. Not only the prognosis, but everything about this entity is unique: its histogenesis, epidemiology, presentation, imaging characteristics, cytology features, immunohistochemical profile and treatment. This explains the urge to improve our understanding about this entity and so our ability to accurately recognize and manage it. Having this in mind, this article aims to summarize the most relevant topics regarding this entity.

O cancro do pâncreas é uma das neoplasias malignas mais letais, com uma taxa de sobrevida 1 ano após o diagnóstico de 24% e 5 anos após o diagnóstico de apenas 9%. Estes dados espelham, contudo, o comportamento do subtipo histológico mais prevalente - o adenocarcinoma ductal. Porém, nem todas as neoplasias malignas do pâncreas são adenocarcinomas e nem todas estão a associadas a um prognóstico tão reservado. A Neoplasia Pseudopapilar do Pâncreas é o exemplo disso: descrita pela Organização Mundial da Saúde como uma neoplasia maligna de baixo grau, é um tumor raro associado a um excelente prognóstico, mesmo na presença de metástases (taxa sobrevida aos 5 anos de 97%). Mais que uma neoplasia com um prognostico peculiarmente favorável, é uma neoplasia única em todas as suas componentes: na sua histogénese, epidemiologia, apresentação clínica, características imagiológicas, citológicas e imunihistoquímicas e no tratamento. Estas particularidades têm que estar bem presentes e consolidadas no raciocínio clínico de qualquer médico, para que estas neoplasias sejam devidamente reconhecidas e tratadas. Neste sentido, foi realizado este artigo de revisão que visa sumariar os mais relevantes tópicos relacionados com esta entidade clínica.
Pancreatic cancer is one of the most lethal malignant neoplasms, with a 1year survival rate after diagnosis of 24%, and a 5years survival rate of only 9%. While this illustrates the behavior of its main histologic type - ductal adenocarcinoma, there are other histologic subtypes of pancreatic cancer that can harbor excellent prognosis. Solid pseudopapillary neoplasm, described as a rare low grade malignant neoplasm by the World Health Organization, is the best example of that, having a 5year survival rate of about 97%, even in the presence of metastasis. Not only the prognosis, but everything about this entity is unique: its histogenesis, epidemiology, presentation, imaging characteristics, cytology features, immunohistochemical profile and treatment. This explains the urge to improve our understanding about this entity and so our ability to accurately recognize and manage it. Having this in mind, this article aims to summarize the most relevant topics regarding this entity.

Es ist schon lange bekannt, dass das Immunsystem eine wichtige Rolle in der Immunabwehr von malignen Tumoren spielt. In der vorliegenden Arbeit wurde das Verhalten der unten genannten Zellen, Rezeptoren und löslichem Protein MICA im peripheren Blut von Patienten mit soliden und hämatologischen Tumorerkrankungen sowie einer Kontrollgruppe mittels Durchflußzytometrie und ELISA-Verfahren untersucht. NK-Zellen ( u.a. CD 56+CD16-NK-Zellen, CD 56-CD16+ Nk-Zellen, CD56+CD16+NK-Zellen), NKT-Zellen und verschiedene T-Lymphozyten ( u.a. αβ-Lymphozyten, δγ-Lymphozyten, Vγ9Vδ1-Lymphozyetn, Vγ9Vδ2-Lymphozyten) sind auf direkten oder indirekten Weg über aktivierende Rezeptoren ( u.a. NKG2D, NKp44, NKp46, NKp33), inhibierende Rezeptoren ( u.a. p 58.1, p58.2, p70, NKG2A) und Chemokinrezeptoren ( u.a. CXCR1) an der Lyse / Apoptose von Tumorzellen über HLA-abhängige (MICA) und HLA-unabhängig Moleküle beteiligt. Es ließ sich zeigen, dass MICA genauso im Serum von hämatologischen Patienten wie auch bei soliden Tumorerkrankungen vorkommt, wobei vor allem Patienten mit fortgeschrittenen Stadien und Metastasierung hohe Werte aufwiesen. Es wurde eine Verminderung der Gesamtzahl an Lymphozyten und αβ-T-Lymphozyten bei hämatologischen und bei soliden Tumoren im peripheren Blut festgestellt. Weiterhin zeigte sich, wie bereits in der Literatur berichtet, bei hämatologischen Neoplasien ein signifikant erhöhter Wert an NK-Zellen und CD56-16+NK-Zellen sowie eine verminderte Expression von NKG2D auf NK-Zellen, auf αβ-T-Lymphozyten und auf γδ-T-Lymphozyten. Es ließ sich auch eine verminderte Expression von p58.1 und NKG2A auf γδ-Lymphozyten im Vergleich zur gesunden Kontrollgruppe nachweisen. Ein signifikanter Unterschied im Vergleich zu soliden Tumoren fand sich nicht, bis auf den oben genannten Lymphozytenanteil. Der Nachweis, dass erhöhte lösliche Serum MICA-Werte die Parameter wie KIRs und KARs sowie Chemokinrezetoren oder die Apopotose der Immunzellen bei hämatologischen oder bei soliden Tumorerkrankungen beeinflusst, konnte in dieser Arbeit nicht geführt werden. Die vorliegende Arbeit trägt dazu bei, die Immunabwehr gegenüber soliden und hämatologischen Tumorerkrankungen besser zu verstehen
Natural killer cells (NK), NKT cells, αβ-T cells and δγ-T cells play an important role in tumor defence. These cells eliminate tumor cells through killer inhibitory receptors (KIR) and stimulatory killer activating receptors (KAR), which can lyse target cells by binding to the major histocompatibility complex class I-related chain A (MICA) protein. The aim of this study is to analyze the reduction of these immune cells, the association between the levels of soluble MICA with KIR- and KAR-expressing on αβ cells, γδ cells and NK cells and their interaction with chemokine receptor CXCR1in the peripheral blood of patients with haematological and epithelial malignancies. ELISA and flow cytometric analysis were used in comparison to controls. Reduced numbers of αβ-T- cells and the presence of soluble MICA could be demonstrated in the serum of patients with both haematological and epithelial malignancies. Higher levels of soluble MICA were associated with advanced stages of disease and metastasation. Patient samples showed also lower numbers of NK cells and CD56-16+NK cells as well as a reduced expression of NKG2D on NK, αβ and γδ T cells. Confirming existing evidence, there was also a reduced expression of p58.1 and NKG2A on γδ cells in comparison to controls. No significant differences between solid and haematological malignancies were found, except for this type of lymphocytes. The study did not provide evidence that increased levels of soluble MICA influence KIRs and KARs, the chemokine receptors or the apoptosis of immune cells

Tese de Doutoramento em Genética Molecular Comparativa
As nanopartículas de lípidos sólidos (SLN, do inglês “solid lipid nanoparticles”) são transportadores coloidais de fármacos, constituídos por uma matriz de lípidos sólidos à temperatura corporal e à temperatura ambiente, estabilizados por agentes tensioactivos apropriados. No âmbito da presente tese, estes sistemas foram desenvolvidos com objectivo de administração de fármacos pouco solúveis em água, e para facilitar a administração direccionada a células de cancro. O objectivo deste trabalho consistiu em explorar o potencial das SLN no tratamento da invasão celular de cancro de mama, nomeadamente das células HER2/neu positivas. Foram desenvolvida partículas cuja composição consistiu no Imwitor 900K ou Compritol 888 ATO (como lípidos sólidos), no cetyltrimethylammonium bromide (CTAB) como lípido catiónico/tensioactivo e no Lutrol F68 ou Miranol C32 Ultra como agente tensioactivo. Como método de produção, foi utilizada a homogenização a alta pressão ou a alta velocidade, seguindo-se a optimização das respectivas formulações, as quais foram utilizadas para os estudos posteriores. As SLN foram caracterizadas em termos de distribuição de tamanho médio das partículas, cristalinidade de matriz lipídica sólida e estabilidade durante armazenamento. Foram obtidas SLN com tamanho médio das partículas entre 115 nm e 334 nm e d0.90 inferior a 1 μm. O estado sólido das mesmas foi confirmado por calorimetria diferencial de varrimento e por difracção de raios X. Todas as formulações apresentaram estabilidade adequada ao longo de 5 semanas, quer à temperatura ambiente, quer a 4 °C. Apesar da liofilização com o crioprotector trealose, a estabilidade das SLN não liofilizadas revelou-se bastante superior. A formulação designada como cSLN-C manteve-se estável durante um período mínimo de 12 semanas. As SLN são, em geral, consideradas como transportadores coloidais com baixa toxicidade. Mesmo assim, o efeito das SLN per si tem importância na interpretação da interacção de formulações que contém um fármaco ou um anticorpo com as células. As SLN desenvolvidas neste trabalho não apresentaram toxicidade na concentração de 0,01 mg/ml. Utilizando a concentração de 0,1 mg/ml a viabilidade celular diminuiu dependendo da linha celular utilizada e tempo de exposição. A dose 1,0 mg/ml foi tóxica nas linhas celulares seleccionadas para este trabalho. Dentro destas, MCF-7 (carcinoma de mama, receptor de estrogénio positivo, HER2-neu negativo) foram as mais susceptíveis aos danos causados pelas SLN, as BT-474 (carcinoma mamário, HER2-neu positivo), HepG2 (hepatocarcinoma) e Caco-2 (cólon adenocarcinoma) foram menos susceptíveis em ordem decrescente. A toxicidade das SLN foi causada por disrupção de integridade das membranas celulares. Danos em ácido deoxiribonucléico (ADN) foram detectados por ensaio cometa. Foram reportados poucos danos – quando comparado com controlo sem tratamento (não significativo nas concentrações não tóxicas). Também foram detectados danos em purinas, que não causaram quebras de ADN. Alguns sinais de stress oxidativo foram detectados em células HepG2: a fluorescência de diacetato de diclorofluoresceina (DCFDA) encontrou-se aumentada relativamente aos controlos sem tratamento e aos positivos, verificou-se um aumento da actividade da enzima superóxido dismutase e uma diminuição da actividade de glutationa reductase. Apesar destes sinais de existência de stress oxidativo, os lípidos membranares não foram afectados (determinação como substâncias reactivas ao ácido tiobarbitúrico, TBARS). Estes resultados estão em concordância com poucos danos detectados em ADN (relativamente ao controlo sem tratamento). Os danos causados por stress oxidativo podem ocorrer em células com capacidade antioxidante inferior à das células HepG2. A capacidade de indução de stress oxidativo pode, hipoteticamente, ser vantajosa em veiculação de fármacos quimioterapêuticos, cujo mecanismo de acção exige existência de radicais livres, e pode, parcialmente, contribuir para a melhoria de eficácia destes medicamentos, quando veiculadas em SLN in vitro e in vivo. A Curcumina foi seleccionada como fármaco-modelo com potencial actividade antineoplásica. A baixa solubilidade aquosa, instabilidade em pH alcalino e fotossensibilidade são propriedades que fazem da curcumina um fármaco ideal para a encapsulação em SLN. Contudo, a solubilidade em vários lípidos foi igual ou inferior a 1 %. A baixa solubilidade em lípidos influenciou a capacidade de carga. Em combinação com as limitações atribuídas à toxicidade das SLN, apenas pode ser administrada 10 μg/ml (27 μM) no máximo, uma dose que é insuficiente para observar os efeitos anticancerígenos da curcumina. Um anticorpo anti- HER2/neu foi colocado na superfície das SLN utilizando a interacção streptavidina-biotina. O efeito de complexo anticorpo-SLN foi governado pela toxicidade das próprias SLN. A conjugação com o anticorpo melhorou significativamente a internalização de complexos nas células de cancro mamário. O efeito foi mais marcado em células BT474, HER2/neu positivas. O tratamento com complexo SLN-anticorpo causou uma diminuição de viabilidade celular das linhas de cancro de mama superior ao efeito das partículas isoladas ou do anticorpo isolado.
Solid lipid nanoparticles (SLN) are colloidal carriers consisting of lipid cores that are solid at body and room temperature dispersed in aqueous phase and stabilized by suitable surfactant. They were developed to improve drug delivery of drugs that are poorly soluble in water and to enable targeted delivery to cancer cells. The aim of this work was to explore the potential of SLN in treatment of breast cancer cell invasion, namely HER2/neu positive breast cancer cells. A series of SLN composed of Imwitor 900K or Compritol 888 ATO as solid lipid, cetyltrimethylammonium bromide (CTAB) as cationic lipid/surfactant and Lutrol F68 or Miranol C32 Ultra as surfactants was developed. Optimized high shear homogenisation of high pressure homogenisation were used as preparation methods. SLN were characterized in terms of particle size distibution, lipid core crystalinity and storage stability. SLN with mean particle size between 115 nm and 334 nm and d0.50 below 0.5 μm were obtained; the crystalline state of lipid cores was confirmed by differential scanning calorimetry and X-ray diffraction. All SLN were stable for at least 4 weeks at room temperature and 4 °C, which was superior to stability of the same SLN freezedryed with trehalose. Compritol-composed SLN were stable over 12 weeks. SLN are in general considered as safe colloidal carriers, their effect on living cells however cannot be neglected when interpreting the studies of interaction of drug-loaded and/or targeted SLN with cells. SLN developed in this work were non-toxic to living cells at a dose 0.01 mg/ml; cell viability was reduced to various extent at 0.1 mg/ml depending on cell line and time of exposure and at 1.0 mg/ml the SLN were toxic to the selected cell lines. Among the used cell lines, MCF-7 cells (breast carcinoma, estrogen receptor positive, Her2/neu negative) were the most susceptible to our SLN, followed by BT-474 (breast carcinoma, HER2/neu positive), HepG2 (hepatocarcinoma) and Caco-2 (colorectal carcinoma) cells. Toxicity of SLN was caused mostly by disruption of membrane integrity. DNA damage was examined by comet assay and was detected in a limited extent, compared to untreated controls (not significant at non-toxic concentrations). Damage to purine bases that did not directly lead to DNA strand breaks was also detected. Some signs of oxidative stress was detected in HepG2 cells: dichlorofluorescein-diacetate (DCFDA) assay revealed increase in free radicals content compared to untreated and positive controls, activity of superoxid dismutase was found increased and activity of glutathion reductase was drastically decreased. Despite these signs of oxidative stress, membrane lipids were not affected – as determined by thiobarbituric acid reactive species (TBARS) determination. This finding is in line with only slightly increased DNA strand breaks (compared to untreated control). Damage caused by oxidative stress after SLN exposure may however occur in cells with lower antioxidant capacity than HepG2 cells. The capacity to induce oxidative stress can hypotethically be beneficial for delivery of chemotherapeutic drugs – that require some free radical increase for their action – and may partly explain many reports on SLN improving efficiency of chemotherapeutics in vitro and in vivo. Curcumin was selected as model drug with potential chemotherapeutic effect. Its low solubility, instability at alkaline pH and light make it an ideal candidate for encapsulation into SLN. Unfortunately, its solubility in solid lipids was limited to 1% (w/w) and to lipid mixtures containing either monoacylglycerides or polyethylenglycol. This affected the resulting drug loading, which together with limitations by SLN toxicity only enabled use of dose equal or lower than 10 μg/ml (27μM) of curcumin – i.e. doses lower than those at which anticancer effects were observed. An antiHER2/neu antibody was attached to SLN surface via streptavidin-biotin binding. The effect of targeted complex was influenced mostly by the toxicity of SLN alone, but at non-toxic dose of SLN a synergistic effect between SLN and the antibody was observed. The antibody improved significantly cell internalization into breast cancer cells, mostly in HER2/neu positive BT-474 cells but to some extent also in MCF-7 cells. Exposure to targeted SLN leads to cell viabilities lower than when exposed to antibody alone or SLN alone.

PURPOSE: This phase I dose-escalation study was undertaken to establish the maximum tolerated dose of the sequence-selective minor groove DNA binding agent SJG-136 in patients with advanced solid tumors. The study also investigated antitumor activity and provided pharmacokinetic and pharmacodynamic data. EXPERIMENTAL DESIGN: Sixteen patients were assigned sequentially to escalating doses of SJG-136 (15-240 microg/m(2)) given as a 10-minute i.v. infusion every 21 days. The dose was subsequently reduced in incremental steps to 45 microg/m(2) due to unexpected toxicity. RESULTS: The maximum tolerated dose of SJG-136 was 45 microg/m(2). The main drug-related adverse event was vascular leak syndrome (VLS) characterized by hypoalbuminemia, pleural effusions, ascites, and peripheral edema. Other unexpected adverse events included elevated liver function tests and fatigue. The VLS and liver toxicity had delayed onset and increased in severity with subsequent cycles. Disease stabilization was achieved for >6 weeks in 10 patients; in 2 patients this was maintained for >12 weeks. There was no evidence of DNA interstrand cross-linking in human blood lymphocytes with the use of the comet assay. Evidence of DNA interaction in lymphocytes and tumor cells was shown through a sensitive gamma-H2AX assay. SJG-136 had linear pharmacokinetics across the dose range tested. CONCLUSIONS: SJG-136 was associated with dose-limiting VLS and hepatotoxicity when administered by short injection every 21 days. DNA damage was noted, at all dose levels studied, in circulating lymphocytes. The etiology of the observed toxicities is unclear and is the subject of further preclinical research. Alternative clinical dosing strategies are being evaluated.