Surgical site infections in colon surgery: the patient, the procedure, the hospital, and the surgeon.

Objective: To determine the role of the surgeon in the occurrence of surgical site infection (SSI) following colon surgery, with respect to his or her adherence to guidelines and his or her experience.Design, Setting, and Patients: Prospective cohort study of 2393 patients who underwent colon surgery performed by 31 surgeons in 9 secondary and tertiary care public Swiss hospitals, recruited from a surveillance program for SSI between March 1, 1998, and December 31, 2008, and followed up for 1 month after their operation.Main Outcome Measures: Risk factors for SSI were identified in univariate and multivariate analyses that included the patients' and procedures' characteristics, the hospitals, and the surgeons as candidate covariates. Correlations were sought between surgeons' individual adjusted risks, their self-reported adherence to guidelines, and the delay since their board certification.Results: A total of 428 SSIs (17.9%) were identified, with hospital rates varying from 4.0% to 25.2% and individual surgeon rates varying from 3.7% to 36.1%. Features of the patients and procedures associated with SSI in univariate analyses were male sex, age, American Society of Anesthesiologists score, contamination class, operation duration, and emergency procedure. Correctly timed antibiotic prophylaxis and laparoscopic approach were protective. Multivariate analyses adjusting for these features and for the hospitals found 4 surgeons with higher risk of SSI (odds ratio [OR] = 2.37, 95% confidence interval [CI], 1.51-3.70; OR = 2.19, 95% CI, 1.41-3.39; OR = 2.15, 95% CI, 1.02-4.53; and OR = 1.97, 95% CI, 1.18-3.30) and 2 surgeons with lower risk of SSI (OR = 0.43, 95% CI, 0.19-0.94; and OR = 0.19, 95% CI, 0.04-0.81). No correlation was found between surgeons' individual adjusted risks and their adherence to guidelines or their experience.Conclusion: For reasons beyond adherence to guidelines or experience, the surgeon may constitute an independent risk factor for SSI after colon surgery.

Enhanced generation of specific tumor-reactive CTL in vitro by selected Melan-A/MART-1 immunodominant peptide analogues.

The Melan-A/MART-1 gene, which is expressed by normal melanocytes as well as by most fresh melanoma samples and melanoma cell lines, codes for Ags recognized by tumor-reactive CTL. HLA-A*0201-restricted Melan-A-specific CTL recognize primarily the Melan-A(27-35) (AAGIGILTV) and the Melan-A(26-35) (EAAGIGILTV) peptides. The sequences of these two peptides are not necessarily optimal as far as binding to HLA-A*0201 is concerned, since both lack one of the dominant anchor amino acid residues (leucine or methionine) at position 2. In this study we introduced single amino acid substitutions in either one of the two natural peptide sequences with the aim of improving peptide binding to HLA-A*0201 and/or recognition by specific CTL. Surprisingly, analogues of the Melan-A(27-35) peptide, which bound more efficiently than the natural nonapeptide to HLA-A*0201, were poorly recognized by tumor-reactive CTL. In contrast, among the Melan-A(26-35) peptide analogues tested, the peptide ELAGIGILTV was not only able to display stable binding to HLA-A2.1 but was also recognized more efficiently than the natural peptide by two short-term cultured tumor-infiltrated lymph node cell cultures as well as by five of five tumor-reactive CTL clones. Moreover, in vitro generation of tumor-reactive CTL by stimulation of PBMC from HLA-A*0201 melanoma patients with this particular peptide analogue was much more efficient than that observed with either one of the two natural peptides. These results suggest that the Melan-A(26-35) peptide analogue ELAGIGILTV may be more immunogenic than the natural peptides in HLA-A*0201 melanoma patients and should thus be considered as a candidate for future peptide-based vaccine trials.

Radioimmunotherapy of colorectal cancer liver metastases: combination with radiotherapy.

The expected therapeutic gain of a combined radioimmunotherapy (RIT) with conventional radiotherapy (RT) would be a synergy of tumor irradiation, provided that toxic, dose-limiting side effects concern different organs. We have shown in a model of subcutaneous human colon cancer transplants in nude mice that RIT with 131I-labeled anti-CEA antibody fragments combined with fractionated RT give an additive therapeutic effect without increase of side effects. A second study of different timing schedules of RIT and RT has shown that close association of both therapies without delay is more efficient than a therapy with a treatment-free interval of two weeks. In a new model of human colon cancer liver metastases in nude mice, early treatment with RIT and with RT has been curative, whereas therapies initiated later were less efficient, suggesting that the combined therapy is likely to be more efficient in an adjuvant situation after surgery. At the clinical level, six patients with limited liver metastatic disease from colorectal cancer were treated with RIT using 200 mCi 131I-labeled anti-CEA MAb F(ab')2 fragments combined with fractionated external beam RT of 20 Gy to the entire liver. As expected, spontaneously reversible bone marrow toxicity grade 3 to 4 and reversible liver toxicity grade 1 to 3 have been observed. By computerized tomography, three patients showed stable disease and one patient partial remission, whereas two patients had progressive disease. In conclusion, animal experiments have shown a clear advantage of combined RT and RIT, and the clinical study shows the feasibility of such a therapy in patients with colorectal cancer liver metastases.

Integrated Analysis of Molecular and Clinical Prognostic Factors in Stage II/III Colon Cancer.

Background The prognostic potential of individual clinical and molecular parameters in stage II/III colon cancer has been investigated, but a thorough multivariable assessment of their relative impact is missing. Methods Tumors from patients (N = 1404) in the PETACC3 adjuvant chemotherapy trial were examined for BRAF and KRAS mutations, microsatellite instability (MSI), chromosome 18q loss of heterozygosity (18qLOH), and SMAD4 expression. Their importance in predicting relapse-free survival (RFS) and overall survival (OS) was assessed by Kaplan-Meier analyses, Cox regression models, and recursive partitioning trees. All statistical tests were two-sided. Results MSI-high status and SMAD4 focal loss of expression were identified as independent prognostic factors with better RFS (hazard ratio [HR] of recurrence = 0.54, 95% CI = 0.37 to 0.81, P = .003) and OS (HR of death = 0.43, 95% CI = 0.27 to 0.70, P = .001) for MSI-high status and worse RFS (HR = 1.47, 95% CI = 1.19 to 1.81, P < .001) and OS (HR = 1.58, 95% CI = 1.23 to 2.01, P < .001) for SMAD4 loss. 18qLOH did not have any prognostic value in RFS or OS. Recursive partitioning identified refinements of TNM into new clinically interesting prognostic subgroups. Notably, T3N1 tumors with MSI-high status and retained SMAD4 expression had outcomes similar to stage II disease. Conclusions Concomitant assessment of molecular and clinical markers in multivariable analysis is essential to confirm or refute their independent prognostic value. Including molecular markers with independent prognostic value might allow more accurate prediction of prognosis than TNM staging alone.

Transcription factor PROX1 modifies Wnt signaling in colon cancer

PR0X1 est un facteur de transcription très conservé au cours de l'évolution. PROX1 joue un rôle essentiel dans de nombreuses étapes de l'embryogenèse, telles que le développement du système lymphatique ou la migration des hépatocytes. Récemment, il a été démontré que PROX1 contribue à la progression des tumeurs colorectales, en tant que gène cible de la voie de signalisation Wnt. En utilisant des approches de co- immunoprécipitation et de ligature de proximité, nous avons trouvé que PROX1 fait également partie du complexe transcriptionnel TCF/ß-catenin, à la fois dans les cellules humaines de cancer du colon et dans les cellules murines de l'épithélium de l'intestin, dans lesquelles la voie de signalisation Wnt est activée. Dans le but de comprendre le mécanisme d'action de PROX1, nous avons analysé le génome des cellules cancéreuses de colon à la recherche des sites de fixation de PROX1, TCF4 et ß-catenin. Nous avons ainsi pu montrer que TCF4, ß-catenin et PROX1 se fixent simultanément sur une sous- population d'amplificateurs génomiques, sur lesquels PROX1 agit comme répresseur. Ces résultats suggèrent que, spécifiquement dans le cadre du cancer du colon, PROX1 agit en tant que modificateur de la voie de transduction du signal Wnt/ß-catenin. De plus, nous proposons que ceci constitue un des mécanismes par lesquels la signalisation durable de Wnt, qui est observée dans la majorité des cancers du colon, transforme le programme génétique des progéniteurs intestinaux, initialement normal, en output spécifique de ce type de cancers, ce qui contribue plus tard à la croissance infinie de la tumeur, à son caractère invasif et à sa dissémination.

Antibody-indocyanin conjugates for immunophotodetection of human squamous cell carcinoma in nude mice.

We have recently shown that immunophotodetection of human colon carcinomas in nude mice and in patients is possible by using anti-carcinoembryonic antigen monoclonal antibodies (MAb) coupled to fluorescein. The most common clinical application of photodiagnosis has been for the detection of squamous cell carcinomas (SCC) in the upper respiratory tract, but the free dyes used have a poor tumor selectivity. We selected the known MAb E48 directed against SCC and coupled it to a fluorescent dye: indopentamethinecyanin (indocyanin). This dye has an advantage over fluorescein in that it emits a more penetrating fluorescent red signal at 667 nm after excitation with a laser ray of 640 nm. In vitro, an conjugate with an indocyanin:MAb molar ratio of 2, and an additional trace labeling with 125I, showed more than 80% of binding to cells from the SCC line A431. In vivo, when injected i.v. into nude mice bearing xenografts of the same carcinoma line, the MAb E48-(indocyanin)2 conjugate was almost as efficient as the unconjugated MAb E48 in terms of specific tumor localization: 15% of the injected dose per g of tumor at 24 h after injection and a tumor:overall normal tissue ratio of 6-8. There was no selective tumor localization of an irrelevant IgG1-(indocyanin)2 conjugate. Immunophotodetection of the s.c. SCC xenografts on mice given injections of 100 micrograms of MAb E48-(indocyanin), conjugate (representing 1 microgram of indocyanin) was performed at 24 h. Upon laser irradiation, clearly detectable red fluorescence from the indocyanin-MAb conjugate was observed specifically in the SCC xenografts across the mouse skin. In comparison, injection of 100 micrograms of a MAb E48 coupled to 2 micrograms of fluorescein gave a specific green fluorescence signal in the tumor xenografts, which was detectable, however, only after removing the mouse skin. Injection i.v. of a 15 times higher amount of free indocyanin (15 micrograms) gave a diffuse red fluorescence signal all over the mouse body with no definite increase in intensity in the tumor, indicating a lack of tumor selectivity of the free dye. The results demonstrate the possibility of broadening and improving the efficiency of tumor immunophotodiagnosis by coupling to a MAb directed against SCC, a fluorescent dye absorbing and emitting at higher wavelength than fluorescein, and thus having deeper tissue penetration and lower tissue autofluorescence. Such a demonstration opens the way to a new form of clinical immunophotodiagnosis and possibly to the development of a more specific approach to phototherapy of early bronchial carcinomas.

Non steroidal anti-inflammatory drugs and COX-2 inhibitors as anti-cancer therapeutics: hypes, hopes and reality.

Non-steroidal anti-inflammatory drugs (NSAIDs) and specific inhibitors of cyclooxygenase (COX)-2, are therapeutic groups widely used for the treatment of pain, inflammation and fever. There is growing experimental and clinical evidence indicating NSAIDs and COX-2 inhibitors also have anti-cancer activity. Epidemiological studies have shown that regular use of Aspirin and other NSAIDs reduces the risk of developing cancer, in particular of the colon. Molecular pathology studies have revealed that COX-2 is expressed by cancer cells and cells of the tumor stroma during tumor progression and in response to chemotherapy or radiotherapy. Experimental studies have demonstrated that COX-2 over expression promotes tumorigenesis, and that NSAIDs and COX-2 inhibitors suppress tumorigenesis and tumor progression. Clinical trials have shown that NSAIDs and COX-2 inhibitors suppress colon polyp formation and malignant progression in patients with familial adenomatous polyposis (FAP) syndrome. Recent advances in the understanding of the cellular and molecular mechanisms of the anti-cancer effects of NSAIDs and COX-2 inhibitors have demonstrated that these drugs target both tumor cells and the tumor vasculature. The therapeutic benefits of COX-2 inhibitors in the treatment of human cancer in combination with chemotherapy or radiotherapy are currently being tested in clinical trials. In this article we will review recent advances in the understanding of the anti-tumor mechanisms of these drugs and discuss their potential application in clinical oncology.

Selective accumulation of mature DC-Lamp+ dendritic cells in tumor sites is associated with efficient T-cell-mediated antitumor response and control of metastatic dissemination in melanoma.

The clinical relevance of dendritic cells (DCs) at the tumor site remains a matter of debate concerning their role in the generation of effective antitumor immunity in human cancers. We performed a comprehensive immunohistochemical analysis using a panel of DC-specific antibodies on regressing tumor lesions and sentinel lymph nodes (SLNs) in melanoma patients. Here we show in a case report involving spontaneous regression of metastatic melanoma that the accumulation of DC-Lamp+ DCs, clustered with tumor cells and lymphocytes, is associated with local expansion of antigen-specific memory effector CTLs. These findings were extended in a series of 19 melanoma-positive SLNs and demonstrated a significant correlation between the density of DC-Lamp+ DC infiltrates in SLNs with the absence of metastasis in downstream lymph nodes. This study, albeit performed in a limited series of patients, points to a pivotal role of mature DCs in the local expansion of efficient antitumor T-cell-mediated immune responses at the initial sites of metastasis and may have important implications regarding the prognosis, staging, and immunotherapy of melanoma patients.

Development and evaluation of antibody-MHC/peptide conjugates as a new form of cancer immunotherapy

Summary One of the major goals of cancer immunotherapy is the induction of a specific and effective antitumor cytotoxic T lymphocyte (CTL) response. However, the downregulation of Class I Major Histocompatibility Complexes (MHC) expression and the low level of tumor peptide presentation on tumor cell surface, ás well as the low immunogenicity of tumor specific antigens, limit the effectiveness of anti-tumor CTL responses. On the other hand, monoclonal antibodies, which bind with high affinity to tumor cell surface markers, are powerful tumor targeting tools. However, their capacity to .kill cancer cells is limited and mAb cancer treatments usually require the addition of different form of chemotherapy. The new cancer immunotherapy strategy described herein combines the advantage of the high tumor targeting capacity of monoclonal antibodies (mAb) with the powerful cytotoxicity of CD8 T lymphocytes directed against highly antigenic peptide-MHC complexes. Monoclonal antibody Fab fragments directed against a cell surface tumor associated antigen (TAA) are chemically coupled to soluble MHC class I complexes carrying a highly antigenic peptide. Antibody guided targeting and oligomerization of numerous antigenic class IMHC/peptide complexes on tumor cell surfaces can redirect the cytotoxicity of peptide-specific CD8 T cells towards target cancer cells. After the description of the production of murine anti-tumor xMHC/peptide conjugates in the first part of this thesis, the therapeutic potential of such conjugates were sequentially investigated in different syngeneic tumor mouse models. As a first proof of principle, transgenic OT-1 mice and later CEA transgenic C57BL/6 (B6) mice, adoptively transferred with OT-1 spleen cells and immunized with ovalbumin, were used as a model of high frequency of ova peptide specific T cells. In these mice, growth inhibition and regression of palpable colon carcinoma expressing CEA, were obtained by systemic injection of anti-CEA Fab/H-2Kb/ova peptide conjugates. Next, LCMV virus and influenza virus infection of B6 mice were used as viral models to redirect natural antiviral CTL responses to tumors via conjugates loaded with viral peptides. We showed that in mice infected with the LCMV virus, subcutaneous CEA-expressing tumor cells were inhibited by the H2Db/GP33 restricted anti-viral CTL response when preincubated before grafting with anti-CEA Fab-H-2Db/GP33 peptide conjugates. In mice infected with the influenza virus, lung metastases expressing the HER2 antigen were inhibited by the H-2Db/NP366 restricted CTLs response when preincubated before injection with anti-Her2 Fab-H-2Db/NP366 peptide conjugates. In the last chapter, the stability of the peptide in the anti-CEA Fab-H-2Db/GP33 conjugates was improved by the covalent photocross-link of the GP33 peptide in the H-2Db MHC groove. Thus, LCMV immune mice could reject CEA expressing tumors when treated with systemic injections of anti-CEA FabH-2Db/GP33 cross-linked conjugates. These results are encouraging for the potential application of this strategy in clinic. Such conjugates could be used alone in patients boosted by the relevant virus, or used in combination with existing T cell based ìmmunotherapy. Résumé Une des principales approches utilisées dans l'immunothérapie contre le cancer consiste en l'induction d'une réponse T cytotoxique (CTL) spécifiquement dirigée contre la tumeur. Cependant, le faible niveau d'expression des complexes majeurs d'histocompatibilité de classe I (CMH I) et de présentation des peptides tumoraux à la surface des cellules cancéreuses ainsi que la faible immunogenicité des antigens tumoraux, limitent l'efficacité de la réponse CTL. D'autre part,. l'injection d'anticorps monoclonaux (mAb), se liant avec une haute affinité aux marqueurs de surface des cellules tumorales, a fourni des résultats cliniques encourageant. Cependant l'efficacité de ces mAbs contre des tumeur solides reste limitée et necessite souvent l'addition de chimiotherapie. La nouvelle stratégie thérapeutique décrite dans ce travail associe le fort pouvoir de localisation des anticorps monoclonaux et le fort pouvoir cytotoxique des lymphocytes T CD8+. Des fragments Fab d'anticorps monoclonaux, dirigés contre des antigènes surexprimés à la surface de cellules tumorales, ont été chimiquement couplés à des CMH I solubles, portant un peptide fortement antigénique. Le ciblage et l'oligomérisation à la surface des cellules tumorales de nombreux CMH I présentant un peptide antigénique, va réorienter la cytotoxicité des cellules T CD8+ spécifiques du peptide présenté, vers les cellules tumorales cibles. Après une description de la production de conjugé anti-tumeur x CMH Upeptide dans la première partie de cette thèse, le potentiel thérapeutique de tels conjugés a été successivement étudiés in vivo dans différents modèles de tumeur syngénéiques. Tout d'abord, des souris OT-1 transgéniques, puis des souris C57BL/6 (B6) transférées avec des cellules de rate OT-1 puis immunisées avec l'ovalbumine, ont été employées comme modèle de haute fréquence de cellules T CD8+ spécifiques du peptide ova. Chez ces souris, l'inhibition de la croissance et la régression de nodules palpables de carcinomes exprimant l'antigène caccino embryonaire (ACE), ont été obtenues par l'injection systémique de conjugés anti-ACE Fab/H-2Kb/ova. Par la suite, l'infection de souris B6 par le virus LCMV et par le virus de la grippe, ont été utilisés comme modèles viraux pour redirigées des réponses anti-virales naturelles vers les tumeurs, en utilisant des conjugés chargés avec des peptides viraux. Nous avons montré que .chez les souris infectées par le LCMV, la croissance de carcinome sous-cutané est empêchée par la réponse anti-virale, spécifique du complexe H2Db/GP33, lorsque les cellules tumorales greffées sont pré-incubées avec des conjugés anti-CEA Fab-H-2Db/GP33. Dans le cas de souris infectées par le virus de la grippe, la métastatisation de mélanomes pulmonaires exprimant l'antigène HER-2 est inhibée par la réponse anti-virale spécifique du complexe H-2Db/NP366, après pré-incubation des cellules tumorales avec des conjugés anti-Her2 FabxH-2Db/NP366. Dans le dernier chapitre, la liaison covalente du peptide GP33 dans le complexe H-2Db a amélioré la stabilité des conjugés correspondants et a permis le traitement systémique de souris greffées avec des tumeurs exprimant l'ACE et infectées par le LCMV. L'ensemble de ces résultats sont encourageant pour l'application de cette strategie en clinique. De tels conjugués pourraient être employés seuls ou en combinaison avec des protocols d'immunisation peptidique anti-tumoral. Résumé pour un large public Dans les pays industrialisés, le cancer se situe au deuxième rang des causes de mortalité après les maladies cardiovasculaires. Les principaux traitement de nombreux cancers sont la chirurgie, en association avec la radiothérapie et la chimiothérapie. L'immunothérapie est l'une des nouvelles approches mises en oeuvre pour la lutte contre le cancer. Elle peut être humorale, et 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 proliferation des cellules tumorales exprimant l'antigène. Certains sont utilisés en clinique pour le traitement de lymphomes, de carcinomes de l'ovaire et du sein ou encore de carcinomes metastatiques du côlon. Cependant l'efficacité de ces anticorps contre des tumeurs solides reste limitée et les traitements exigent souvent d'être combiner avec de la chimiothérapie. L'immunothérapie spécifique peut également être cellulaire et reposer sur une démarche de type vaccinal, consistant à générer des lymphocytes T cytotoxiques (cytotoxic T lymphocytes :CTL) capables de détruire spécifiquement les cellules malignes. 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. Or les cellules tumorales ne presentent pas efficacement les peptides antigèniques, car elles se caractérisent par une diminution ou une absence d'expression des antigènes d'histocompatibilité de classe I, des molécules d'adhésion et des cytokines costimulatrices, et par une faible expression des antigènes associés aux tumeurs. C'est en partie pourquoi, malgré l'induction de fortes réponses CTL specifiquement dirigés contre des antigens tumoraux, les régressions tumorales obtenus grace à ces vaccinations sont relativement rares. Alors que chez les personnes atteintes du cancer on observe l'instauration d'une tolérance immunitaire vis-à-vis de la tumeur, à l'inverse, notre systeme immunitaire reste parfaitement capable de combattre des infection virales classiques, tels que la grippe, qui font aussi appel à une réponse T cytotoxique. Notre groupe de recherche a donc eu l'idee de développer une nouvelle approche thérapeutique où une réponse immunitaire anti-virale très efficace serait redirigée vers les tumeurs par des anticorps monoclonaux. Concrètement, nous avons chimiquement couplés des fragments d'anticorps monoclonaux dirigés contre des antigènes surexprimés à la surface de cellules tumorales, à des CMH I portant un peptide viral antigénique. Les cellules tumorales, ciblées par le fragment anticorps et couvertes d' antigènes viraux présentés par des molécules de CMH I, peuvent ainsi tromper les lymphocytes cytotoxiques anti-viraux qui vont détruire les cellules tumorales comme si elles étaient infectées par le virus. Suite à des résultats prometteurs obtenus in vitro avec différents conjugués anticorps-CMH humain de type HLA.A2/peptide Flu, le but du projet était de tester in vivo des conjugués anticorps-CMH I murins sur des modèles expérimentaux de souris. Tout d'abord, des souris transgéniques pour un recepteur T specifique du peptide ova, puis des transferts adoptifs de ces cellules T specifiques dans des souris immunocompétentes, ont été choisi comme modèle de haute fréquence des cellules T spécifiques, et ont permi de valider le principe de la strategie in vivo. Puis, deux modèles viraux ont été elaboré avec le virus LCMV et le virus Influenza, pour réorienter des réponses antivirales naturelles vers les tumeurs grâce à des conjugés chargés avec des peptides viraux. Nous avons montré la grande capacité de nos conjugués à rediriger des réponses cytotoxiques vers les tumeurs et inhiber la croissance de tumeurs syngénéiques sous cutanés et pulmonaires. Ces résultats d'inhibition tumorales obtenus dans des souris immunocompétentes, grâce à l'injection de conjugués anticorps xCMH/peptide et réorientant deux réponses antivirales différentes vers deux modèles tumoraux syngeneiques, sont encourageant pour l'application de cette nouvelle stratégie en clinique.

Functional avidity of tumor antigen-specific CTL recognition directly correlates with the stability of MHC/peptide multimer binding to TCR.

Avidity of Ag recognition by tumor-specific T cells is one of the main parameters that determines the potency of a tumor rejection Ag. In this study we show that the relative efficiency of staining of tumor Ag-specific T lymphocytes with the corresponding fluorescent MHC class I/peptide multimeric complexes can considerably vary with staining conditions and does not necessarily correlate with avidity of Ag recognition. Instead, we found a clear correlation between avidity of Ag recognition and the stability of MHC class I/peptide multimeric complexes interaction with TCR as measured in dissociation kinetic experiments. These findings are relevant for both identification and isolation of tumor-reactive CTL.

Myeloid cells contribute to tumor lymphangiogenesis.

The formation of new blood vessels (angiogenesis) and lymphatic vessels (lymphangiogenesis) promotes tumor outgrowth and metastasis. Previously, it has been demonstrated that bone marrow-derived cells (BMDC) can contribute to tumor angiogenesis. However, the role of BMDC in lymphangiogenesis has largely remained elusive. Here, we demonstrate by bone marrow transplantation/reconstitution and genetic lineage-tracing experiments that BMDC integrate into tumor-associated lymphatic vessels in the Rip1Tag2 mouse model of insulinoma and in the TRAMP-C1 prostate cancer transplantation model, and that the integrated BMDC originate from the myelomonocytic lineage. Conversely, pharmacological depletion of tumor-associated macrophages reduces lymphangiogenesis. No cell fusion events are detected by genetic tracing experiments. Rather, the phenotypical conversion of myeloid cells into lymphatic endothelial cells and their integration into lymphatic structures is recapitulated in two in vitro tube formation assays and is dependent on fibroblast growth factor-mediated signaling. Together, the results reveal that myeloid cells can contribute to tumor-associated lymphatic vessels, thus extending the findings on the previously reported role of hematopoietic cells in lymphatic vessel formation.

MGMT promoter methylation is prognostic but not predictive for outcome to adjuvant PCV chemotherapy in anaplastic oligodendroglial tumors: a report from EORTC Brain Tumor Group Study 26951.

PURPOSE: O6-methylguanine-methyltransferase (MGMT) promoter methylation has been shown to predict survival of patients with glioblastomas if temozolomide is added to radiotherapy (RT). It is unknown if MGMT promoter methylation is also predictive to outcome to RT followed by adjuvant procarbazine, lomustine, and vincristine (PCV) chemotherapy in patients with anaplastic oligodendroglial tumors (AOT). PATIENTS AND METHODS: In the European Organisation for the Research and Treatment of Cancer study 26951, 368 patients with AOT were randomly assigned to either RT alone or to RT followed by adjuvant PCV. From 165 patients of this study, formalin-fixed, paraffin-embedded tumor tissue was available for MGMT promoter methylation analysis. This was investigated with methylation specific multiplex ligation-dependent probe amplification. RESULTS: In 152 cases, an MGMT result was obtained, in 121 (80%) cases MGMT promoter methylation was observed. Methylation strongly correlated with combined loss of chromosome 1p and 19q loss (P = .00043). In multivariate analysis, MGMT promoter methylation, 1p/19q codeletion, tumor necrosis, and extent of resection were independent prognostic factors. The prognostic significance of MGMT promoter methylation was equally strong in the RT arm and the RT/PCV arm for both progression-free survival and overall survival. In tumors diagnosed at central pathology review as glioblastoma, no prognostic effect of MGMT promoter methylation was observed. CONCLUSION: In this study, on patients with AOT MGMT promoter methylation was of prognostic significance and did not have predictive significance for outcome to adjuvant PCV chemotherapy. The biologic effect of MGMT promoter methylation or pathogenetic features associated with MGMT promoter methylation may be different for AOT compared with glioblastoma.

Mutations leading to X-linked hypohidrotic ectodermal dysplasia affect three major functional domains in the tumor necrosis factor family member ectodysplasin-A.

Mutations in the epithelial morphogen ectodysplasin-A (EDA), a member of the tumor necrosis factor (TNF) family, are responsible for the human disorder X-linked hypohidrotic ectodermal dysplasia (XLHED) characterized by impaired development of hair, eccrine sweat glands, and teeth. EDA-A1 and EDA-A2 are two splice variants of EDA, which bind distinct EDA-A1 and X-linked EDA-A2 receptors. We identified a series of novel EDA mutations in families with XLHED, allowing the identification of the following three functionally important regions in EDA: a C-terminal TNF homology domain, a collagen domain, and a furin protease recognition sequence. Mutations in the TNF homology domain impair binding of both splice variants to their receptors. Mutations in the collagen domain can inhibit multimerization of the TNF homology region, whereas those in the consensus furin recognition sequence prevent proteolytic cleavage of EDA. Finally, a mutation affecting an intron splice donor site is predicted to eliminate specifically the EDA-A1 but not the EDA-A2 splice variant. Thus a proteolytically processed, oligomeric form of EDA-A1 is required in vivo for proper morphogenesis.