A disposable glass-based immunosensor for monitoring the cancer biomarker CEA in urine

NanoPT 2014 International Conference, in Portugal, on February 12-14. Poster presentation based on topic Nanobio/Nanomedicine

Sialyl Tn-Expressing Bladder Cancer Cells Induce a Tolerogenic Phenotype in Innate and Adaptive Immune Cells

Despite the wide acceptance that glycans are centrally implicated in immunity, exactly how they contribute to the tilt immune response remains poorly defined. In this study, we sought to evaluate the impact of the malignant phenotype-associated glycan, sialyl-Tn (STn) in the function of the key orchestrators of the immune response, the dendritic cells (DCs). In high grade bladder cancer tissue, the STn antigen is significantly overexpressed and correlated with the increased expression of ST6GALNAC1 sialyltransferase. Bladder cancer tissue presenting elevated expression of ST6GALNAC1 showed a correlation with increased expression of CD1a, a marker for bladder immature DCs and showed concomitant low levels of Th1-inducing cytokines IL-12 and TNF-α. In vitro, human DCs co-incubated with STn+ bladder cancer cells, had an immature phenotype (MHC-IIlow, CD80low and CD86low) and were unresponsive to further maturation stimuli. When contacting with STn+ cancer cells, DCs expressed significantly less IL-12 and TNF-α. Consistent with a tolerogenic DC profile, T cells that were primed by DCs pulsed with antigens derived from STn+ cancer cells were not activated and showed a FoxP3high IFN-γlow phenotype. Blockade of STn antigens and of STn+ glycoprotein, CD44 and MUC1, in STn+ cancer cells was able to lower the induction of tolerance and DCs become more mature. Overall, our data suggest that STn-expressing cancer cells impair DC maturation and endow DCs with a tolerogenic function, limiting their capacity to trigger protective anti-tumour T cell responses. STn antigens and, in particular, STn+ glycoproteins are potential targets for circumventing tumour-induced tolerogenic mechanisms.

Immunohistochemistry detection of putative miR-200c and miR-203 targets in breast cancer patients

Part of this thesis will be published in the following: Gomes, B.C., Santos, B. 2015. Methods for studying microRNAs expression and their targets in formalin-fixed, paraffin-embedded (FFPE) breast cancer tissues. In Methods in Molecular Biology: Cancer Drug Resistance (Rueff, J. & Rodrigues, A.S. eds), Springer Protocols.

Thomsen-Friedenreich antigens in bladder cancer: evaluation of their prognostic value

RESUMO: Introdução. O cancro de bexiga é uma patologia comum que representa o 6° e o 5° cancro mais incidente em Portugal e na Itália, respetivamente. Em mais de metade dos casos ocorre reincidência durante o primeiro ano, requerendo acompanhamento clínico ao longo da vida. A instilação intravesical de Bacillus Calmette-Guérin (BCG) (uma estirpe atenuada do Mycobacterium bovis) representa uma imunoterapia eficaz no combate ao cancro de bexiga, no entanto, muitos aspetos da interação de BCG com as células tumorais bem como com as células do sistema imunitário permanecem por desvendar. As células tumorais de bexiga expressam frequentemente as formas sialiladas dos antigénios de Thomsen-Friedenreich (TF), i.e., sialil-T (sT) e sialil-Tn (sTn). Contudo ainda se desconhece o significado da sua expressão na malignidade tumoral e se afeta a eficácia da terapêutica BCG. Objetivo do estudo. Investigar o papel dos antigénios sT e sTn no fenótipo maligno de células de cancro de bexiga bem como na resposta mediada pelo sistema imunitário à terapia com BCG. Metodologia. Para tal, foram utilizadas as linhas celulares de cancro da bexiga HT1376 e MCR, geneticamente modificadas por transdução com vetores codificantes para as sialiltransferases ST3GAL1 ou ST6GALNAC1, de forma a expressar homogeneamente os antigénios sT ou sTn respetivamente. Estes modelos celulares foram estudados após confronto com BCG. O nível de BCG internalizado foi avaliado por citometria de fluxo. O perfil global de expressão genética dos modelos celulares antes e após incubação com BCG foi analisado pela tecnologia de microarray. O perfil de citocinas secretadas pelos modelos celulares após incubação com BCG, bem como de macrófagos estimulados pelo secretoma de células de cancro de bexiga que por sua vez foram estimuladas previamente por BCG, foi estudado pelo sistema multiplex de “imuno-esferas”. Resultados. A análise do transcritoma dos modelos celulares revelou que grupos de genes envolvidos em funções específicas foram modulados em paralelo nos dois modelos celulares, após transdução, independentemente da sialiltransferase expressa. Ou seja, em células que expressavam a sialiltransferase ST3GAL1 ou ST6GALNAC1, os genes envolvidos na regulação da segregação cromossómica e na reparação do DNA foram consistentemente regulados negativamente. Genes descritos na literatura como marcadores para o cancro de bexiga foram também modulados. A incubação com BCG resultou numa tendência ao aumento da expressão de genes relevantes na preservação e estabilidade genómica e menor malignidade, no entanto, apenas em células que expressavam sT ou sTn. Entre as dez citocinas testadas, apenas a IL-6 e IL-8 foram expressas pelas linhas celulares de cancro da bexiga, com indução destas após estimulação com BCG, e principalmente em células que expressavam ST3GAL1 ou ST6GALNAC1. Em macrófagos, citocinas inflamatórias, tais como IL-1β, IL-6 e TNFα, e a citocina anti-inflamatória IL-10, foram induzidas apenas pelo secretoma de células de cancro da bexiga confrontadas com BCG, com maior relevância quando estas expressavam ST3GAL1 ou ST6GALNAC1, prevendo a estimulação de macrófagos semelhantes aos de tipo M1 e uma melhor resposta à terapia com BCG. Conclusões. O efeito geral da expressão destas sialiltransferases e dos produtos enzimáticos sT ou sTn nas células de cancro de bexiga conduz a um fenótipo de maior malignidade. Contudo, a maior avidez de estas na produção de citocinas inflamatórias após confronto com BCG, bem como a maior capacidade de estimulação de macrófagos, predirá uma resposta à terapia com BCG mais eficaz em tumores que expressem os antigénios de TF sialilados. Tais conclusões são totalmente concordantes com os nossos mais recentes dados clínicos obtidos em colaboração, que mostram que em doentes com cancro de bexiga que expressam sTn respondem melhor a terapia BCG. ----------ABSTRACT: Background. Bladder cancer is a common malignancy representing the 6th and the 5th most incident cancer in Portugal and in Italy, respectively. More than half of the cases relapse within one year, requiring though a lifelong follow-up. Intravesical instillation of Bacillus Calmette-Guérin (BCG) (an attenuated strain of Mycobacterium bovis) represents an effective immunotherapy of bladder cancer, although many aspects of the interaction of BCG with cancer cells and host immune cells remain obscure. Bladder cancer cells often express the sialylated forms of the Thomsen-Friedenreich (TF), i.e., sialil-T (sT) e sialil-Tn (sTn). However, it’s still unknown the sense of such expression in tumour malignancy and in the BCG therapy efficacy. Aim of the study. To investigate the role of the sT and sTn antigens on the malignant phenotype of bladder cancer cells and the immune mediated response to BCG therapy. Experimental. We have utilized populations of the bladder cancer cell lines HT1376 and MCR, genetically modified by transduction with the sialyltransferases ST3GAL1 or ST6GALNAC1 to express homogeneously sT or sTn antigens. The level of BCG internalized was assessed by flow cytometry. The whole gene expression profile of BCG-challenged or unchallenged bladder cancer cell lines was studied by microarray technology. The profile of cytokines secreted by BCG-challenged bladder cancer cells and that of macrophages challenged by the secretome of BCG-challenged bladder cancer cells was studied by multiplex immune-beads assay. Results. Transcriptome analysis of the sialyltransferase-transduced cells revealed that groups of genes involved in specific functions were regulated in parallel in the two cell lines, regardless the sialyltransferase expressed. Namely, in sialyltransferase-expressing cells, genes involved in the proper chromosomal segregation and in the DNA repair were consistently down-regulated, while genes reported in literature as markers for bladder cancer were modulated. BCG-challenging induced a tendency to up-regulation of the genes preserving genomic stability and reducing malignancy, but only in cells expressing either sT or sTn. Among the ten cytokines tested, only IL-6 and IL-8 were expressed by bladder cancer cell lines and up-regulated by BCG-challenging, mainly in sialyltransferases-expressing cells. In macrophages, inflammatory cytokines, such as IL-1β, IL-6 and TNFα, and the antinflammatory IL-10 were induced only by the secretome of BCG-challenged bladder cancer cells, particularly when expressing either sialyltransferase, predicting the stimulation of M1-like macrophages and a better response to BCG therapy. Conclusions. The general effect of the expression of the two sialyltransferases and their products in the bladder cancer cells is toward a more malignant phenotype. However, the stronger ability of sialyltransferase expressing cells to produce inflammatory cytokines upon BCG-challenging and to stimulate macrophages predicts a more effective response to BCG in tumours expressing the sialylated TF antigens. This is fully consistent with our recent clinical data obtained in collaboration, showing that patients with bladder cancer expressing sTn respond better to BCG therapy.

Study of glycosidic changes in lung cancer: potential effectors in hematogenous metastasis

RESUMO: O cancro do pulmão (LC), uma das principais causas de mortalidade relacionada com cancro em Portugal, pode levar à formação de metástases hematogénicas. A adesão das células tumorais ao endotélio é considerada um dos passos fundamentais envolvidos na metástase. Em células sanguíneas, esta adesão é mediada por ligandos de E-selectina (E-SL), glicoproteínas ou glicolípidos decorados principalmente com sialyl-Lewis x (sLex) e sialyl-Lewis a (sLea). Tem sido descrito a expressão destes antigénios em LC, contudo o seu papel funcional em permitir a adesão das células de LC ao endotélio é ainda pouco compreendido. Foram analisadas amostras emparelhadas normais e tumorais de pacientes com cancro de pulmão de não-pequenas células (NSCLC) e três linhas celulares de LC. Immunoblotting assays com anti-sLex/sLea e molécula quimérica de E-selectina demonstraram que tecidos tumorais de LC sobreexpressam significativamente E-SL e resultados de citometria de fluxo demonstraram uma expressão elevada de E-SL nas linhas celulares. Para compreender o mecanismo da sobreexpressão de E-SL em tecidos tumorais e linhas celulares de LC, foi analisada a expressão de genes envolvidos na biossíntese de E-SL, nomeadamente FUT3, FUT4, FUT6, FUT7, ST3GAL3, ST3GAL4, ST3GAL6, β4GALT1, GCNT1 e GALNT3. Observou-se a sobreexpressão das fucosiltransferases FUT3, FUT6 e FUT7 em tecidos tumorais de LC e FUT3 em linhas celulares de LC, sendo que neste último, esta expressão é correlacionada com um aumento da adesão das células de LC às selectinas endoteliais. Foi observado que uma baixa expressão de FUT4 em tecidos tumorais está associada com estadios menos avançados de NSCLC. Foram analisadas ainda proteínas decoradas com sLex/sLea, tendo-se identificado como E-SL o antigénio carcinoembrionário em NSCLC. Em resumo, esta tese contribuiu para uma melhor compreensão das alterações glicosídicas e moléculas que podem influenciar a progressão tumoral do LC, podendo permitir identificar futuramente novos biomarcadores de diagnóstico/prognóstico e potenciais alvos terapêuticos para o NSCLC.--------------------------ABSTRACT: Lung cancer (LC), one of the major causes of mortality related to cancer in Portugal, may lead to hematogenous metastasis. Adhesion of cancer cells to endothelium is considered one of the crucial steps involved in metastasis. In blood cells, this adhesion is initiated by endothelial selectin ligands (E-SL) that are glycoproteins or glycolipids decorated mostly with sialyl-Lewis x (sLex) and sialyl-Lewis a (sLea). While LC has been described as expressing these sialyl Lewis antigens, its functional role in allowing LC adhesion to endothelium is still poorly understood. We analyzed paired tumor and normal tissues samples from non-small cell lung cancer (NSCLC) patients and three LC cell lines. Immunoblotting assays with anti-sLex/sLea and E-selectin chimera demonstrated that LC tumor tissues significantly overexpress E-SL and flow cytometry results indicated that E-SL are also abundantly expressed in LC cell lines. To understand the mechanism behind the overexpression of E-SL in LC tissues and cell lines, we analyzed the expression of genes involved in its biosynthesis, namely FUT3, FUT4, FUT6, FUT7, ST3GAL3, ST3GAL4, ST3GAL6, β4GALT1, GCNT1 and GALNT3. It was observed the overexpression of fucosyltransferases FUT3, FUT6 and FUT7 in LC tumor tissues and FUT3 in LC cell lines, being this last one correlated with an increased reactivity of the LC cells to endothelial selectins. It was described that low expression of FUT4 in tumor tissues is correlated with early stages of NSCLC. We also analyzed scaffolds proteins of sLex/sLea and it was identified the carcinoembryonic antigen as an E-SL in NSCLC. In summary, this thesis contributed to a better understanding of the glycosidic changes and molecules that can influence tumor progression of LC, allowing identifying in the future new diagnosis/prognosis biomarkers and potential therapeutic targets for NSCLC.

P53 and Rb tumor suppressor gene alterations in gastric cancer

Inactivation of tumor suppressor genes has been frequently observed in gastric carcinogenesis. Our purpose was to study the involvement of p53, APC, DCC, and Rb genes in gastric carcinoma. METHOD: Loss of heterozygosity of the p53, APC, DCC and Rb genes was studied in 22 gastric cancer tissues using polymerase chain reaction; single-strand conformation polymorphism of the p53 gene exons 5-6 and exons 7-8 was studied using 35S-dATP, and p53 expression was detected using a histological immunoperoxidase method with an anti-p53 clone. RESULTS AND DISCUSSION: No loss of heterozygosity was observed in any of these tumor suppressor genes; homozygous deletion was detected in the Rb gene in 23% (3/13) of the cases of intestinal-type gastric carcinoma. Eighteen (81.8%) cases showed band mobility shifts in exons 5-6 and/or 7-8 of the p53 gene. The presence of the p53 protein was positive in gastric cancer cells in 14 cases (63.6%). Normal gastric mucosa showed negative staining for p53; thus, the immunoreactivity was likely to represent mutant forms. The correlation of band mobility shift and the immunoreactivity to anti-p53 was not significant (P = .90). There was no correlation of gene alterations with the disease severity. CONCLUSIONS: The inactivation of Rb and p53 genes is involved in gastric carcinogenesis in our environment. Loss of the Rb gene observed only in the intestinal-type gastric cancer should be further evaluated in association with Helicobacter pylori infection. The p53 gene was affected in both intestinal and diffuse histological types of gastric cancer.

MET is required for the recruitment of anti-tumoural neutrophils

Mutations or amplification of the MET proto-oncogene are involved in the pathogenesis of several tumours, which rely on the constitutive engagement of this pathway for their growth and survival. However, MET is expressed not only by cancer cells but also by tumour-associated stromal cells, although its precise role in this compartment is not well characterized. Here we show that MET is required for neutrophil chemoattraction and cytotoxicity in response to its ligand hepatocyte growth factor (HGF). Met deletion in mouse neutrophils enhances tumour growth and metastasis. This phenotype correlates with reduced neutrophil infiltration to both the primary tumour and metastatic sites. Similarly, Met is necessary for neutrophil transudation during colitis, skin rash or peritonitis. Mechanistically, Met is induced by tumour-derived tumour necrosis factor (TNF)-a or other inflammatory stimuli in both mouse and human neutrophils. This induction is instrumental for neutrophil transmigration across an activated endothelium and for inducible nitric oxide synthase production upon HGF stimulation. Consequently, HGF/MET-dependent nitric oxide release by neutrophils promotes cancer cell killing, which abates tumour growth and metastasis. After systemic administration of a MET kinase inhibitor, we prove that the therapeutic benefit of MET targeting in cancer cells is partly countered by the pro-tumoural effect arising from MET blockade in neutrophils. Our work identifies an unprecedented role of MET in neutrophils, suggests a potential 'Achilles' heel' of MET-targeted therapies in cancer, and supports the rationale for evaluating anti-MET drugs in certain inflammatory diseases.

Hypertension in Patients with Cancer

There is a known association between chemotherapy and radiotherapy for treatment of cancer patients and development or worsening of hypertension. The aim of this article is to review this association. A literature search was conducted for articles reporting this association on the databases PubMed, SciELO and LILACS between 1993 and 2013. There was a high coprevalence of hypertension and cancer, since both diseases share the same risk factors, such as sedentary lifestyle, obesity, smoking, unhealthy diet and alcohol abuse. The use of chemotherapy and adjuvant drugs effective in the treatment of cancer increased the survival rate of these patients and, consequently, increased the incidence of hypertension. We described the association between the use of angiogenesis inhibitors (bevacizumab, sorafenib and sunitinib), corticosteroids, erythropoietin and non-steroidal anti-inflammatory drugs with the development of hypertension. We also described the relationship between hypertension and carotid baroreceptor injury secondary to cervical radiotherapy. Morbidity and mortality increased in patients with cancer and hypertension without proper antihypertensive treatment. We concluded that there is need for early diagnosis, effective monitoring and treatment strategies for hypertension in cancer patients in order to reduce cardiovascular morbidity and mortality.

Development of a bifunctional CD1d-anti-HER2 scFv fusion molecule to redirect the iNKT cell-mediated immune response to the tumor site

Abstract : Invariant natural killer T lymphocytes (iNKT) are a unique subpopulation of T lymphocytes recognizing glycolipid antigens in the context of the MHC class I-like molecule CD1d. Upon activation with the high affinity ligand α-galactosylceramide (αGalCer), iNKT cells rapidly produce large amounts of the pro-inflammatory cytokine interferon gamma (IFN-γ) and potently activate cells of the innate and adaptive immune response, such as dendritic cells (DCs), NK and T cells. In this context, iNKT cells have been shown to efficiently mediate antitumor activity, and recent research has focused on the manipulation of these cells for antitumor therapies. However, a major drawback of αGalCer as a free drug is that a single injection of this ligand leads to a short-lived iNKT cell activation followed by a long-term anergy, limiting its therapeutic use. In contrast, we demonstrate here that when αGalCer is loaded on a recombinant soluble CD1d molecule (αGalCer/sCD1d), repeated injections lead to a sustained iNKT and NK cell activation associated with IFN-γ secretion as well as with DC maturation. Most importantly, when the αGalCer/sCD1d is fused to an anti-HER2 scFv antibody fragment, potent inhibition of experimental lung metastasis and established subcutaneous tumors is obtained when systemic treatment is started two to seven days after the injection of HER2-expressing B16 melanoma cells, whereas at this time free αGalCer has no effect. The antitumor activity of the sCD1d-anti-HER2 fusion protein is associated with HER2-specific tumor localization and accumulation of iNKT, NK and T cells at the tumor site. Importantly, active T cell immunization combined with the sCD1d-anti-HER2 treatment leads to the accumulation of antigen-specific CD8 T cells exclusively in HER2-expressing tumors, resulting in potent tumor inhibition. In conclusion, sustained activation and tumor targeting of iNKT cells by recombinant αGalCer/sCD1d molecules thus may promote a combined innate and adaptive immune response at the tumor site that may prove to be effective in cancer immunotherapy. RESUME : Les lymphocytes «invariant Natural Killer T » (iNKT) forment une sous-population particulière de lymphocytes T reconnaissant des antigènes glycolipidiques présentés sur la molécule non-polymorphique CD1d, analogue aux protéines du complexe majeur d'histocompatibilité de classe I. Après activation avec le ligand de haute affinité α-galactosylceramide (αGalCer), les cellules iNKT produisent des grandes quantités de la cytokine pro-inflammatoire interferon gamma (IFN-γ) et activent les cellules du système immunitaire inné et acquis, telles que les cellules dendritiques (DC), NK et T. En conséquence, on a montré que les cellules iNKT exercent des activités anti-tumorales et la recherche s'est intéressée à la manipulation de ces cellules pour développer des thérapies anti-tumorales. Néanmoins, le désavantage majeur de l'αGalCer, injecté seul, est qu'une seule dose de ce ligand aboutit à une activation des cellules iNKT de courte durée suivie par un état anergique prolongé, limitant l'utilisation thérapeutique de ce glycolipide. En revanche, l'étude présentée ici démontre que, si l'αGalCer est chargé sur des molécules récombinantes soluble CD1d (αGalCer/sCDld), des injections répétées aboutissent à une activation prolongée des cellules iNKT et NK associée avec la sécrétion d'IFN-γ et la maturation des cellules DC. Plus important, si on fusionne la molécule αGalCer/sCD1d avec un fragment single-chain (scFv) de l'anticorps anti-HER2, on observe une importante inhibition de métastases expérimentales aux poumons et de tumeurs sous-cutanées même lorsque le traitement systémique est commencé 2 à 7 jours après la greffe des cellules de mélanome B16 transfectées avec l'antigène HER2. Dans les mêmes conditions le traitement avec l'αGalCer seul est inefficace. L'activité anti-tumorale de la protéine sCDld-anti-HER2 est associée à son accumulation spécifique dans des tumeurs exprimant le HER2 ainsi qu'avec une accumulation des cellules iNKT, NK et T à la tumeur. De plus, une immunisation active combinée avec le traitement sCD1d-anti-HER2 aboutit à une accumulation des lymphocytes T CD8 spécifiques de l'antigène d'immunisation, ceci exclusivement dans des tumeurs qui expriment l'antigène HER2. Cette combinaison résulte dans une activité anti-tumeur accrue. En conclusion, l'activation prolongée des cellules iNKT redirigées à la tumeur par des molécules recombinantes αGalCer/sCDld conduit à l'activation de la réponse innée et adaptative au site tumoral, offrant une nouvelle stratégie prometteuse d'immunothérapie contre le cancer. RESUME POUR UN LARGE PUBLIC : Le cancer est une cause majeure de décès dans le monde. Sur un total de 58 millions de décès enregistrés au niveau mondial en 2005, 7,6 millions (soit 13%) étaient dus au cancer. Les principaux traitements de nombreux cancers sont la chirurgie, en association avec la radiothérapie et la chimiothérapie. Néanmoins, ces traitements nuisent aussi aux cellules normales de notre corps et parfois, ils ne suffisent pas pour éliminer définitivement une tumeur. L'immunothérapie est l'une des nouvelles approches pour la lutte contre le cancer et elle vise à exploiter la spécificité du système immunitaire qui peut distinguer des cellules normales et tumorales. Une cellule exprimant un marqueur tumoral (antigène) peut être reconnue par le système immunitaire humoral (anticorps) et/ou cellulaire, induisant une réponse spécifique contre la tumeur. L'immunothérapie peut s'appuyer alors sur la perfusion d'anticorps monoclonaux dirigés contre des antigènes tumoraux, par exemple les anticorps dirigés contre les protéines oncogéniques Her-2/neu dans le cancer du sein. Ces anticorps ont le grand avantage de spécifiquement se localiser à la tumeur et d'induire la lyse ou d'inhiber la prolifération des cellules tumorales exprimant l'antigène. Aujourd'hui, six anticorps monoclonaux non-conjugés sont approuvés en clinique. Cependant l'efficacité de ces anticorps contre des tumeurs solides reste limitée et les traitements sont souvent combinés avec de la chimiothérapie. L'immunothérapie spécifique peut également être cellulaire et exploiter par immunisation active le développement de lymphocytes T cytotoxiques (CTL) capables de détruire spécifiquement les cellules malignes. De telles «vaccinations »sont actuellement testées en clinique, mais jusqu'à présent elles n'ont pas abouti aux résultats satisfaisants. Pour obtenir une réponse lymphocytaire T cytotoxique antitumorale, la cellule T doit reconnaître un antigène associé à la tumeur, présenté sous forme de peptide dans un complexe majeur d'histocompatibilité de classe I (CHM I). Cependant les cellules tumorales sont peu efficace dans la présentation d'antigène, car souvent elles se caractérisent par une diminution ou une absence d'expression des molécules d'histocompatibilité de classe I, et expriment peu ou pas de molécules d'adhésion et de cytokines costimulatrices. C'est en partie pourquoi, malgré l'induction de fortes réponses CTL spécifiquement dirigés contre des antigènes tumoraux, les régressions tumorales obtenus grâce à ces vaccinations sont relativement rares. Les lymphocytes «invariant Natural Killer T » (iNKT) forment une sous-population particulière de lymphocytes T reconnaissant des antigènes glycolipidiques présentés sur la molécule non-polymorphique CD1d, analogue aux protéines CMH I. Après activation avec le ligand de haute affinité α-galactosylceramide (αGalCer), les cellules iNKT produisent des grandes quantités de la cytokine pro-inflammatoire interferon gamma (IFN-γ) et activent les cellules du système immunitaire inné et acquis, telles que les cellules dendritiques (DC), NK et T. En conséquence, on a montré que les cellules iNKT exercent des activités anti-tumorales et la recherche s'est intéressée à la manipulation de ces cellules pour développer des thérapies anti-tumorales. Néanmoins, le désavantage majeur de l'αGalCer, injecté seul, est qu'une seule dose de ce ligand aboutit à une activation des cellules iNKT de courte durée suivie par un état anergique prolongé, limitant l'utilisation thérapeutique de ce glycolipide. Notre groupe de recherche a donc eu l'idée de développer une nouvelle approche thérapeutique où la réponse immunitaire des cellules iNKT serait prolongée et redirigée vers la tumeur par des anticorps monoclonaux. Concrètement, nous avons produit des molécules récombinantes soluble CD1d (sCD1d) qui, si elles sont chargés avec l'αGalCer (αGalCer/sCDld), aboutissent à une activation prolongée des cellules iNKT et NK associée avec la sécrétion d'IFN-γ et la maturation des cellules DC. Plus important, si la molécule αGalCer/sCD1d est fusionnée avec un fragment single-chain (scFv) de l'anticorps anti-HER2, la réponse immunitaire est redirigée à la tumeur pour autant que les cellules cancéreuses expriment l'antigène HER2. Les molécules αGalCer/sCDld ainsi présentées activent les lymphocytes iNKT. Avec cette stratégie, on observe une importante inhibition de métastases expérimentales aux poumons et de tumeurs sous-cutanées, même lorsque le traitement systémique est commencé 2 à 7 jours après la greffe des cellules de mélanome B16 transfectées avec l'antigène HER2. Dans les mêmes conditions le traitement avec l'αGalCer seul est inefficace. L'activité anti-tumorale de la protéine sCDld-anti-HER2 est associée à son accumulation spécifique dans des tumeurs exprimant le HER2 ainsi qu'avec une accumulation des cellules iNKT, NK et T à la tumeur. En conclusion, l'activation prolongée des cellules iNKT redirigées à la tumeur par des molécules récombinantes αGalCer/sCD1d conduit à l'activation de la réponse innée et adaptative au site tumoral, offrant une nouvelle stratégie prometteuse d'immunothérapie contre le cancer.

The use of mannan antigen and anti-mannan antibodies in the diagnosis of invasive candidiasis: recommendations from the Third European Conference on Infections in Leukemia.

INTRODUCTION: Timely diagnosis of invasive candidiasis (IC) remains difficult as the clinical presentation is not specific and blood cultures lack sensitivity and need a long incubation time. Thus, non-culture-based methods for diagnosing IC have been developed. Mannan antigen (Mn) and anti-mannan antibodies (A-Mn) are present in patients with IC. On behalf of the Third European Conference on Infections in Leukemia, the performance of these tests was analysed and reviewed. METHODS: The literature was searched for studies using the commercially available sandwich enzyme-linked immunosorbent assays (Platelia™, Bio-Rad Laboratories, Marnes-la-Coquette, France) for detecting Mn and A-Mn in serum. The target condition of this review was IC defined according to 2008 European Organization for Research and Treatment of Cancer/Mycoses Study Group criteria. Sensitivity, specificity and diagnostic odds ratios (DOR) were calculated for Mn, A-Mn and combined Mn/A-Mn testing. RESULTS: Overall, 14 studies that comprised 453 patients and 767 controls were reviewed. The patient populations included in the studies were mainly haematological and cancer cases in seven studies and mainly intensive care unit and surgery cases in the other seven studies. All studies but one were retrospective in design. Mn sensitivity was 58% (95% confidence interval [CI], 53-62); specificity, 93% (95% CI, 91-94) and DOR, 18 (95% CI 12-28). A-Mn sensitivity was 59% (95% CI, 54-65); specificity, 83% (95% CI, 79-97) and DOR, 12 (95% CI 7-21). Combined Mn/A-Mn sensitivity was 83% (95% CI, 79-87); specificity, 86% (95% CI, 82-90) and DOR, 58 (95% CI 27-122). Significant heterogeneity of the studies was detected. The sensitivity of both Mn and A-Mn varied for different Candida species, and it was the highest for C. albicans, followed by C. glabrata and C. tropicalis. In 73% of 45 patients with candidemia, at least one of the serological tests was positive before the culture results, with mean time advantage being 6 days for Mn and 7 days for A-Mn. In 21 patients with hepatosplenic IC, 18 (86%) had Mn or A-Mn positive test results at a median of 16 days before radiological detection of liver or spleen lesions. CONCLUSIONS: Mn and A-Mn are useful for diagnosis of IC. The performance of combined Mn/A-Mn testing is superior to either Mn or A-Mn testing.

Neoadjuvant chemoradiotherapy with or without panitumumab in patients with wild-type KRAS, locally advanced rectal cancer (LARC): a randomized, multicenter, phase II trial SAKK 41/07.

BACKGROUND: We conducted a randomized, phase II, multicenter study to evaluate the anti-epidermal growth factor receptor (EGFR) mAb panitumumab (P) in combination with chemoradiotherapy (CRT) with standard-dose capecitabine as neoadjuvant treatment for wild-type KRAS locally advanced rectal cancer (LARC). PATIENTS AND METHODS: Patients with wild-type KRAS, T3-4 and/or N+ LARC were randomly assigned to receive CRT with or without P (6 mg/kg). The primary end-point was pathological near-complete or complete tumor response (pNC/CR), defined as grade 3 (pNCR) or 4 (pCR) histological regression by Dworak classification (DC). RESULTS: Forty of 68 patients were randomly assigned to P + CRT and 28 to CRT. pNC/CR was achieved in 21 patients (53%) treated with P + CRT [95% confidence interval (CI) 36%-69%] versus 9 patients (32%) treated with CRT alone (95% CI: 16%-52%). pCR was achieved in 4 (10%) and 5 (18%) patients, and pNCR in 17 (43%) and 4 (14%) patients. In immunohistochemical analysis, most DC 3 cells were not apoptotic. The most common grade ≥3 toxic effects in the P + CRT/CRT arm were diarrhea (10%/6%) and anastomotic leakage (15%/4%). CONCLUSIONS: The addition of panitumumab to neoadjuvant CRT in patients with KRAS wild-type LARC resulted in a high pNC/CR rate, mostly grade 3 DC. The results of both treatment arms exceeded prespecified thresholds. The addition of panitumumab increased toxicity.

Peptabody-EGF: a novel apoptosis inducer targeting ErbB1 receptor overexpressing cancer cells.

The epidermal growth factor receptor (EGFR) plays a central role in cell life by controlling processes such as growth or proliferation. This receptor is commonly overexpressed in a number of epithelial malignancies and its upregulation is often associated with an aggressive phenotype of the tumor. Thus, targeting of EGFR represents a very promising challenge in oncology, and antibodies raised against this receptor have been investigated as potential antitumor agents. Various putative mechanisms of action were proposed for such antibodies, including decreased proliferation, induction of apoptosis, stimulation of the immunological response against targeted cancer cells or combinations thereof. We report here the development of an alternative high affinity molecule that is directed against EGFR. Production of this pentameric protein, named peptabody-EGF, includes expression in a bacterial expression system and subsequent refolding and multimerization of peptabody monomers. The protein complex contains 5 human EGF ligand domains, which confer specific binding towards the extracellular portion of EGFR. Receptor binding of the peptabody-EGF had a strong antiproliferative effect on different cancer cell lines overexpressing EGFR. However, cells expressing constitutive levels of the target receptor were barely affected. Peptabody-EGF treated cancer cells exhibited typical characteristics of apoptosis, which was found to be induced within 30 min after the addition of the peptabody-EGF. In vitro experiments demonstrated a significantly higher binding activity for peptabody-EGF than for the therapeutic monoclonal EGFR antibody Mab-425. Furthermore, the antitumor action provoked by the peptabody-EGF was greatly superior than antibody mediated effects when tested on EGFR overexpressing cancer cell lines. These findings suggest a potential application of this high affinity molecule as a novel tool for anti-EGFR therapy.

Molecular pathology of colorectal cancer.

Colorectal cancer (CRC) is one of the most intensively studied cancer types, partly because of its high prevalence but also because of the existence of its precursor lesions, tubular or villous adenomas, and more recently (sessile) serrated adenomas, which can be detected endoscopically and removed. The morphological steps in the adenoma-carcinoma sequence have been elucidated at a molecular level, which has been facilitated by identification of the genes responsible for familial intestinal cancer. However, apart from early detection of familial forms of CRC and its use in genetic counseling, until recently such detailed molecular knowledge has had little impact on clinical management of the disease. This has dramatically changed in the last decade. With drugs specifically targeting the epidermal growth factor receptor (EGFR) having been shown effective in CRC, mechanisms responsible for resistance have been explored. The finding that KRAS mutated cancers do not respond to anti-EGFR treatment has had a profound impact on clinical management and on molecular diagnostics of CRC. Additional genetic tests for mutations in NRAS, BRAF and PIK3CA contribute to determining who to treat, and others will follow. New therapies effective in patients with advanced CRC are under investigation. Remaining burning questions for optimal management are which patients will relapse after resection of the primary tumor and which patients will respond to the standard 5FU-oxaliplatin adjuvant treatment regimen. Predictive tests to address these issues are eagerly awaited. New classifications of CRC, based on molecular parameters, are emerging, and we will be confronted with new subtypes of CRC, for which the definition is based on combinations of gene expression patterns, chromosomal alterations, gene mutations and epigenetic characteristics. This will be instrumental in designing new approaches for therapy but will also be translated into molecular diagnostics. Both will contribute to improved clinical management of CRC.

MMP-9 as predictive factor for response and progression free survival in breast cancer patients treated with bevacizumab and pegylated liposomal doxorubicin. A multicenter, single-arm phase II trial (SAKK24/06). On behalf of the Swiss Group for Clinical Cancer Research (SAKK).

The benefit of bevacizumab (Bv) has been shown in different tumors including colorectal cancer, renal cancer, pulmonary non-small cell cancer and also breast cancer. However to date, there is no established test evaluating the angiogenic status of a patient and monitoring the effects of anti-angiogenic treatments. Tumor angiogenesis is the result of a balance between multiple pro- and anti¬angiogenic molecules. There is very little published clinical data exploring the impact of the anti-angiogenic therapy on the different angiogenesis-related molecules and the potential role of these molecules as prognostic or predictive factors.