Prediction of free imatinib concentrations based on total plasma concentrations in patients with gastrointestinal stromal tumours.

AIM: Total imatinib concentrations are currently measured for the therapeutic drug monitoring of imatinib, whereas only free drug equilibrates with cells for pharmacological action. Due to technical and cost limitations, routine measurement of free concentrations is generally not performed. In this study, free and total imatinib concentrations were measured to establish a model allowing the confident prediction of imatinib free concentrations based on total concentrations and plasma proteins measurements. METHODS: One hundred and fifty total and free plasma concentrations of imatinib were measured in 49 patients with gastrointestinal stromal tumours. A population pharmacokinetic model was built up to characterize mean total and free concentrations with inter-patient and intrapatient variability, while taking into account α1 -acid glycoprotein (AGP) and human serum albumin (HSA) concentrations, in addition to other demographic and environmental covariates. RESULTS: A one compartment model with first order absorption was used to characterize total and free imatinib concentrations. Only AGP influenced imatinib total clearance. Imatinib free concentrations were best predicted using a non-linear binding model to AGP, with a dissociation constant Kd of 319 ng ml(-1) , assuming a 1:1 molar binding ratio. The addition of HSA in the equation did not improve the prediction of imatinib unbound concentrations. CONCLUSION: Although free concentration monitoring is probably more appropriate than total concentrations, it requires an additional ultrafiltration step and sensitive analytical technology, not always available in clinical laboratories. The model proposed might represent a convenient approach to estimate imatinib free concentrations. However, therapeutic ranges for free imatinib concentrations remain to be established before it enters into routine practice.

Evaluation of clinician-operated sonography and fine-needle aspiration in the assessment of salivary gland tumours.

OBJECTIVES: To evaluate the combination of ultrasound (US) + fine-needle aspiration (FNA) in the assessment of salivary gland tumours in the hands of the otolaryngologist. DESIGN: A retrospective review of case notes was performed. SETTING: Two university teaching hospitals in Switzerland. PARTICIPANTS: One hundred and three patients with a total of 106 focal masses of the salivary glands were included. Clinician-operated US + FNA were the first line of investigation for these lesions. All patients underwent surgical excision of the lesion, which allowed for confirmation of diagnosis by histopathology in 104 lesions and by laboratory testing in two lesions. MAIN OUTCOME MEASURES: Primary--diagnostic accuracy in identifying true salivary gland neoplasms and detecting malignancy. Secondary--predicting an approximate and specific diagnosis in these tumours. RESULTS: The combination of US + FNA achieved a diagnostic accuracy of 99% in identifying and differentiating true salivary gland neoplasms from tumour-like lesions. In detecting malignancy, this combination permitted an accuracy of 98%. An approximate diagnosis was possible in 89%, and a specific diagnosis in 69% of our patients. CONCLUSIONS: Due to economic factors and a high diagnostic accuracy, the combination of US + FNA represents the investigation method of choice for most salivary gland tumours. We suggest that the otolaryngologist be employed in carrying out these procedures, as is already the rule in other medical specialties, while computed tomography and magnetic resonance imaging should be reserved to those few lesions, which cannot be delineated completely by sonography.

Pitfalls in colour photography of choroidal tumours.

Colour imaging of fundus tumours has been transformed by the development of digital and confocal scanning laser photography. These advances provide numerous benefits, such as panoramic images, increased contrast, non-contact wide-angle imaging, non-mydriatic photography, and simultaneous angiography. False tumour colour representation can, however, cause serious diagnostic errors. Large choroidal tumours can be totally invisible on angiography. Pseudogrowth can occur because of artefacts caused by different methods of fundus illumination, movement of reference blood vessels, and flattening of Bruch's membrane and sclera when tumour regression occurs. Awareness of these pitfalls should prevent the clinician from misdiagnosing tumours and wrongfully concluding that a tumour has grown.

Replicating adenoviruses targeting tumours with constitutive activation of the Wnt signalling pathway

Abstract Activation of the Wnt pathway through mutation of the adenomatous polyposis coli and 13-catenin genes is a hallmark of colon cancer. These mutations lead to constitutive activation of transcription from promoters containing binding sites for Tcf/LEF transcription factors. Tumour-selective replicating oncolytic viruses are promising agents for cancer therapy. They can in principle spread throughout a tumour mass until all the cancerous cells are killed, and clinical trials have shown that they are safe except at very high doses. Adenoviruses are interesting candidates for virotherapy because their biology is well understood and their small genome can be rapidly mutated. Adenoviruses with Tcf binding sites in the E2 early promoter replicate selectively in cells with an active Wnt pathway. Although these viruses can replicate in a broad panel of colon cancer cell lines, some colorectal cancer cells are only semi-permissive for Tcf-virus replication. The aim of my thesis was to increase the safety and the efficacy of Tcf-viruses for colon cancer virotherapy. I replaced the endogenous ElA viral promoter by four Tcf binding sites and showed that transcription from the mutant promoter was specifically activated by the Wnt pathway. A virus with Tcf binding sites in the ElA and E4 promoters was more selective for the Wnt pathway than the former Tcf-E2 viruses. Moreover, insertion of Tcf binding sites into all early promoters further increased viral selectivity, but reduced viral activity. I showed that Tcf-dependent transcription was inhibited by the interaction between ElA and p300, but deletion of the p300-binding site of ElA generally led to viral attenuation. In the semi-permissive cell lines, replication of Tcf-viruses remained lower than that of the wild-type virus. The E2 promoter was the most sensitive to the cell type, but I was unable to improve its activity by targeted mutagenesis. To increase the toxicity of the Tcf-E1A/E4 virus, I decided to express a suicide gene, yeast cytosine deaminase (yCD), late during infection. This enzyme converts the prodrug 5-FC to the cytotoxic agent 5-FU. yCD was expressed in a DNA replication-dependent manner and increased viral toxicity in presence of 5-FC. Tcf-ElA and yCD adenoviruses are potentially useful vectors for the treatment of liver metastases from colorectal tumours. Résumé Dans la quasi-totalité des cancers du côlon, la voie Wnt est activée par des mutations dans les gènes codant pour APC ou pour la (3-caténine. Ces mutations activent de façon constitutive la transcription de promoteurs contenant des sites de liaison pour les facteurs de transcription Tcf/LEF. Les virus réplicatifs spécifiques aux tumeurs sont des agents prometteurs pour la thérapie cancéreuse. En principe, ces vecteurs peuvent se propager dans une masse tumorale jusqu'à destruction de toutes les cellules cancéreuses, et des études cliniques ont démontré que de tels vecteurs n'étaient pas toxiques, sauf à de très hautes doses. Les adénovirus sont des candidats intéressants pour la thérapie virale car leur biologie est bien définie et leur petit génome peut être rapidement modifié. Des adénovirus comportant des sites de liaison à Tcf dans leur promoteur précoce E2 se répliquent sélectivement dans les cellules qui possèdent une voie Wnt active. Ces virus sont capables de se répliquer dans un grand nombre de cellules cancéreuses du côlon, bien que certaines de ces cellules ne soient que semi-permissives pour la réplication des virus Tcf. Le but de ma thèse était d'augmenter la sécurité et l'efficacité des virus Tcf. Le promoteur viral endogène ElA a été remplacé par quatre sites de liaison à Tcf, ce qui a rendu son activation dépendante de la voie Wnt. Un virus comportant des sites de liaison pour Tcf dans les promoteurs ElA et E4 était plus sélectif pour la voie Wnt que les précédents virus Tcf-E2, et un virus comportant des sites Tcf dans tous les promoteurs précoces était encore plus sélectif, mais moins actif. J'ai montré que l'interaction entre ElA et p300 inhibait la transcription dépendante de Tcf, mais la délétion du domaine concerné dans ElA a eu pour effet d'atténuer les virus. Dans les cellules semi-permissives, la réplication des virus Tcf était toujours plus basse que celle du virus sauvage. J'ai identifié le promoteur E2 comme étant le plus sensible au type cellulaire, mais n'ai pas pu augmenter son activité par mutagenèse. Pour augmenter la toxicité du virus Tcf-E1A/E4, j'ai décidé d'exprimer un gène suicide, la cytosine déaminase (yCD), pendant la phase tardive de l'infection. Cette enzyme transforme la procirogue 5-FC en l'agent cytotoxique 5-FU. yCD était exprimée après réplication de l'ADN viral et augmentait la toxicité virale en présence de 5-FC. Les virus Tcf-ElA et yCD sont des vecteurs potentiellement utiles pour le traitement des métastases hépatiques de cancers colorectaux.

Dysregulated activation of the CXCR4/CXCL12 Axisand its role in the malignant phenotype of neural crest-derived neuroblastoma tumours

Résumé: Le neuroblastome (NB) est un néoplasme dévastateur de la petite enfance, pour lequel il n'existe pas encore de traitement efficace. Les chimiokines et leurs récepteurs ont été impliqués dans la croissance des tumeurs et la formation de métastases, et en particulier, il a été rapporté que l'axe CXCR4/CXCL12 dirigeait le guidage, ainsi que l'invasion des cellules cancéreuses vers des organes spécifiques. Notre étude avait pour objectif d'analyser le rôle de CxCR4 exogène dans le comportement malin du NB, en étudiant la croissance des cellules tumorales, leur capacité de survie, de migration et d'invasion in vitro et en validant ces résultats grâce à un modèle orthotopique murin de la progression tumorale du NB in vivo. La surexpression de CXCR4 dans les cellules faiblement métastatiques IGR-NB8 n'exprimant pas CXCR4, a augmenté la mobilité des cellules vers CXCL12 in vitro. De plus, les cellules surexprimant CXCR4 ont été moins affectées par la privation de sérum que les cellules contrôles. Le volume des tumeurs chez les animaux greffés de manière orthotopique avec les cellules NB8-CXCR4-C3 était significativement plus élevé que celui des tumeurs issues des cellules contrôles NB8-E6 au moment du sacrifice des animaux. Cependant, aucune induction des métastases n'a été observée. La lignée cellulaire IGR-N91, aux propriétés invasives et métastatiques in vivo, exprime constitutivement des quantités modérées de CXCR4. La surexpression du récepteur dans cette lignée a accéléré la croissance tumorale in vivo, mais n'a pas augmenté pas l'occurrence des métastases. Les cellules IGR-N91, dans lesquelles l'expression de CXCR4 a été éteinte, suite à l'introduction de shRNA stable contre CXCR4, a présenté une croissance cellulaire plus lente, in vitro et in vivo. Afin d'identifier les gènes et les voies de signalisation impliqués dans les effets dépendants de CXCR4-CXCL12 dans le NB, des analyses du profil d'expression des gènes ont été effectuées sur les lignées cellulaires transfectées ou non (contrôle). Trois clones contrôles ont été comparés à 3 clones surexprimant CXCR4 pour chacune des lignées (IGR-NB8 et IGR-N91). Les analyses biostatiques ont identifié 10 gènes induits, dont CXCR4, et 31 gènes réprimés, communs entre tous les clones surexprimant CXCR4. Ces observations démontrent que la surexpression de CXCR4 dans le NB stimule la croissance, la survie et la migration chémotactique des cellules tumorales, mais est insuffisante pour induire ou augmenter leurs capacités invasives et métastatiques. Les voies de signalisation activées suite à la surexpression de CXCR4 et identifiées à travers le profil global de l'expression des gènes pourraient être des cibles intéressantes pour le développement de drogues capables d'inhiber la croissance tumorale. Abstact: Neuroblastoma (NB) is a devastating childhood neoplasm for which there is not yet an efficient treatment. Chemokines and their receptors have been involved in tumour growth and metastasis, and in particular the CXCR4/CXCL12 axis has been reported to mediate organ-specific cancer cells homing and invasion. The purpose of the study was to investigate the role of ectopic CXCR4 in the malignant behaviour of NB by studying tumour cell growth, survival, migration, and invasion in vitro and by validating these results using a murine orthotopic model of NB tumour progression in vivo. CXCR4 overexpression in the low metastatic, CXCR4-negative IGR-NB8 cells resulted in CXCL12-mediated chemotaxis in vitro. Furthermore, CXCR4 overexpressing cells were less affected by serum deprivation than mock-transduced cells. In vivo studies revealed that, at sacrifice, volumes of tumours developing in mice with orthotopically implanted NB8-CXCR4-C3 cells, were significantly increased compared to NB8-E6 control tumours. However, no induction of metastases was observed. The in vivo invasive and metastatic cell line IGR-N91 cell line constitutively expresses moderate levels of CXCR4. Overexpression of CXCR4 enhanced in vivo tumour growth but did not increase the occurrence of metastases. IGR-N91 cells where CXCR4 has been knocked-down by stable shRNA grew slower in vitro and in vivo. To identify genes and pathways involved in the CXCR4/CXCL12-mediated effects in NB expression, profiles analyses (Affymetrix) were performed on transduced and control cell lines. Three mock-transduced clones were compared to three CXCR4 overexpressing clones of either cell line IGR-NB8 and IGR-N91. Biostatistical analysis identified 10 commonly upregulated genes (including CXCR4) and 31 downregulated genes common to all CXCR4 overexpressing clones. These observations demonstrate that overexpression of CXCR4 in NB stimulates tumour cell growth, survival, and chemotactic migration but is not sufficient to induce or enhance invasive and metastatic capacities. Activated pathways upon CXCR4 overexpression, identified through global gene expression profiling may be interesting targets for drugs inhibiting tumour growth.

A novel gene expression signature in peripheral blood mononuclear cells for early detection of colorectal cancer.

BACKGROUND: Early detection and treatment of colorectal adenomatous polyps (AP) and colorectal cancer (CRC) is associated with decreased mortality for CRC. However, accurate, non-invasive and compliant tests to screen for AP and early stages of CRC are not yet available. A blood-based screening test is highly attractive due to limited invasiveness and high acceptance rate among patients. AIM: To demonstrate whether gene expression signatures in the peripheral blood mononuclear cells (PBMC) were able to detect the presence of AP and early stages CRC. METHODS: A total of 85 PBMC samples derived from colonoscopy-verified subjects without lesion (controls) (n = 41), with AP (n = 21) or with CRC (n = 23) were used as training sets. A 42-gene panel for CRC and AP discrimination, including genes identified by Digital Gene Expression-tag profiling of PBMC, and genes previously characterised and reported in the literature, was validated on the training set by qPCR. Logistic regression analysis followed by bootstrap validation determined CRC- and AP-specific classifiers, which discriminate patients with CRC and AP from controls. RESULTS: The CRC and AP classifiers were able to detect CRC with a sensitivity of 78% and AP with a sensitivity of 46% respectively. Both classifiers had a specificity of 92% with very low false-positive detection when applied on subjects with inflammatory bowel disease (n = 23) or tumours other than CRC (n = 14). CONCLUSION: This pilot study demonstrates the potential of developing a minimally invasive, accurate test to screen patients at average risk for colorectal cancer, based on gene expression analysis of peripheral blood mononuclear cells obtained from a simple blood sample.

Cancers de l'adulte jeune [Overview on cancer in young adults]

To make a diagnostic of cancer in a young adult (15-30 years of age) has important physical, psychological and social implications. The most frequent cancers seen at this age are cancer of the thyroid, testicular germ cell tumours, 'melanoma, Hodgkin's lymphoma, non-Hodgkin lymphoma, leukaemia, cerebral tumours and sarcomas. Even if the prognostic of most of these cancers is excellent, treatments are difficult and often associated with long-term side effects. A multidisciplinary approach of these patients is essential. A long-term follow-up by a general practicioner or an oncologist is indispensable.

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.

Menarche, menopause, and breast cancer risk: individual participant meta-analysis, including 118 964 women with breast cancer from 117 epidemiological studies.

BACKGROUND: Menarche and menopause mark the onset and cessation, respectively, of ovarian activity associated with reproduction, and affect breast cancer risk. Our aim was to assess the strengths of their effects and determine whether they depend on characteristics of the tumours or the affected women. METHODS: Individual data from 117 epidemiological studies, including 118 964 women with invasive breast cancer and 306 091 without the disease, none of whom had used menopausal hormone therapy, were included in the analyses. We calculated adjusted relative risks (RRs) associated with menarche and menopause for breast cancer overall, and by tumour histology and by oestrogen receptor expression. FINDINGS: Breast cancer risk increased by a factor of 1·050 (95% CI 1·044-1·057; p<0·0001) for every year younger at menarche, and independently by a smaller amount (1·029, 1·025-1·032; p<0·0001), for every year older at menopause. Premenopausal women had a greater risk of breast cancer than postmenopausal women of an identical age (RR at age 45-54 years 1·43, 1·33-1·52, p<0·001). All three of these associations were attenuated by increasing adiposity among postmenopausal women, but did not vary materially by women's year of birth, ethnic origin, childbearing history, smoking, alcohol consumption, or hormonal contraceptive use. All three associations were stronger for lobular than for ductal tumours (p<0·006 for each comparison). The effect of menopause in women of an identical age and trends by age at menopause were stronger for oestrogen receptor-positive disease than for oestrogen receptor-negative disease (p<0·01 for both comparisons). INTERPRETATION: The effects of menarche and menopause on breast cancer risk might not be acting merely by lengthening women's total number of reproductive years. Endogenous ovarian hormones are more relevant for oestrogen receptor-positive disease than for oestrogen receptor-negative disease and for lobular than for ductal tumours. FUNDING: Cancer Research UK.

Evaluation of tumour vascularisation in two rat sarcoma models for studying isolated lung perfusion. Injection route determines the origin of tumour vessels.

Isolated cytostatic lung perfusion (ILP) is an attractive technique allowing delivery of a high-dose of cytostatic agents to the lungs while limiting systemic toxicity. In developing a rat model of ILP, we have analysed the effect of the route of tumour cell injection on the source of tumour vessels. Pulmonary sarcomas were established by injecting a sarcoma cell suspension either by the intravenous (i.v.) route or directly into the lung parenchyma. Ink perfusion through either pulmonary artery (PA) or bronchial arteries (BA) was performed and the characteristics of the tumour deposits defined. i.v. and direct injection methods induced pulmonary sarcoma nodules, with similar histological features. The intraparenchymal injection of tumour cells resulted in more reliable and reproducible tumour growth and was associated with a longer survival of the animals. i.v. injected tumours developed a PA-derived vascular tree whereas directly injected tumours developed a BA-derived vasculature.

Contrast-enhanced ultrasound after devascularisation of neuroendocrine liver metastases: functional and morphological evaluation.

OBJECTIVE: To evaluate morphological and perfusion changes in liver metastases of neuroendocrine tumours by contrast-enhanced ultrasound (CEUS) after transarterial embolisation with bead block (TAE) or trans-arterial chemoembolisation with doxorubicin-eluting beads (DEB-TACE). METHODS: In this retrospective study, seven patients underwent TAE, and ten underwent DEB-TACE using beads of the same size. At 1 day before embolisation, 2 days, 1 month and 3 months after the procedure, a destruction-replenishment study using CEUS was performed with a microbubble-enhancing contrast material on a reference tumour. Relative blood flow (rBF) and relative blood volume (rBV) were obtained from the ratio of values obtained in the tumour and in adjacent liver parenchyma. Morphological parameters such as the tumour's major diameter and the viable tumour's major diameter were also measured. A parameter combining functional and morphological data, the tumour vitality index (TVI), was studied. The Wilcoxon rank-sum test and Fisher's test were used to compare treatment groups. RESULTS: At 3 months rBF, rBV and TVI were significantly lower (P = 0.005, P = 0.04 and P = 0.03) for the group with doxorubicin. No difference in morphological parameters was found throughout the follow-up. CONCLUSIONS: One parameter, TVI, could evaluate the morphological and functional response to treatments.

Induction in transgenic mice of HLA-A2.1-restricted cytotoxic T cells specific for a peptide sequence from a mutated p21ras protein.

Cytotoxic T cells (CTL) recognize short peptides that are derived from the proteolysis of endogenous cellular proteins and presented on the cell surface as a complex with MHC class I molecules. CTL can recognize single amino acid substitutions in proteins, including those involved in malignant transformation. The mutated sequence of an oncogene may be presented on the cell surface as a peptide, and thus represents a potential target antigen for tumour therapy. The p21ras gene is mutated in a wide variety of tumours and since the transforming mutations result in amino acid substitutions at positions 12, 13 and 61 of the protein, a limited number of ras peptides could potentially be used in the treatment of a wide variety of malignancies. A common substitution is Val for Gly at position 12 of p21ras. In this study, we show that the peptide sequence from position 5 to position 14 with Val at position 12-ras p5-14 (Val-12)-has a motif which allows it to bind to HLA-A2.1. HLA-A2.1-restricted ras p5-14 (Val-12)-specific CTL were induced in mice transgenic for both HLA-A2.1 and human beta2-microglobulin after in vivo priming with the peptide. The murine CTL could recognize the ras p5-14 (Val-12) peptide when they were presented on both murine and human target cells bearing HLA-A2.1. No cross-reactivity was observed with the native peptide ras p5-14 (Gly-12), and this peptide was not immunogenic in HLA-A2.1 transgenic mice. This represents an interesting model for the study of an HLA-restricted CD8 cytotoxic T cell response to a defined tumour antigen in vivo.