TWEAK and TNFα, Both TNF Ligand Family Members and Multiple Sclerosis-Related Cytokines, Induce Distinct Gene Response in Human Brain Microvascular Endothelial Cells.

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a member of the TNF ligand family involved in various diseases including brain inflammatory pathologies such as multiple sclerosis. It has been demonstrated that TWEAK can induce cerebrovascular permeability in an in vitro model of the blood-brain barrier. The molecular mechanisms playing a role in TWEAK versus TNFα signaling on cerebral microvascular endothelial cells are not well defined. Therefore, we aimed to identify gene expression changes in cultures of human brain microvascular endothelial cells (hCMEC/D3) to address changes initiated by TWEAK exposure. Taken together, our studies highlighted that gene involved in leukocyte extravasation, notably claudin-5, were differentially modulated by TWEAK and TNFα. We identified differential gene expression of hCMEC/D3 cells at three timepoints following TWEAK versus TNFα stimulation and also found distinct modulations of several canonical pathways including the actin cytoskeleton, vascular endothelial growth factor (VEGF), Rho family GTPases, and phosphatase and tensin homolog (PTEN) pathways. To our knowledge, this is the first study to interrogate and compare the effects of TWEAK versus TNFα on gene expression in brain microvascular endothelial cells.

Metal Imbalance in Neurodegenerative Diseases with a Specific Concern to the Brain of Multiple Sclerosis Patients.

There is increasing evidence that deregulation of metals contributes to a vast range of neurodegenerative diseases including multiple sclerosis (MS). MS is a chronic inflammatory disease of the central nervous system (CNS) manifesting disability and neurological symptoms. The precise origin of MS is unknown, but the disease is characterized by focal inflammatory lesions in the CNS associated with an autoimmune reaction against myelin. The treatment of this disease has mainly been based on the prescription of immunosuppressive and immune-modulating agents. However, the rate of progressive disability and early mortality is still worrisome. Metals may represent new diagnostic and predictive markers of severity and disability as well as innovative candidate drug targets for future therapies. In this review, we describe the recent advances in our understanding on the role of metals in brain disorders of neurodegenerative diseases and MS patients.

Positive FDG-PET/CT of the pleura twenty years after talc pleurodesis: three cases of benign talcoma.

The FDG-PET (fluorine-18 fluorodeoxyglucose positron emission tomography) scan is used with increasing frequency to investigate pleural abnormalities and to determine the possibility of neoplastic invasion. However, false-positive findings are not uncommon and talc pleurodesis has been reported to cause hypermetabolic pleural thickenings up to 5 years after the procedure. We report the cases of 3 patients (2 of whom had a history of asbestos exposure) requiring talc pleurodesis for recurrent pneumothoraces between 1988 and 1990, who were investigated in 2011 for pleural abnormalities. Avid pleural thickening on FDG-PET scan mimicking pleural cancer was found, but this was deemed secondary to the pleurodesis. Talc pleurodesis generates inflammation which promotes pleural adhesions. This inflammatory reaction could decrease with time, as in other inflammatory processes. Since talc is not metabolized by the body, the FDG-PET scan can remain positive, most likely because of a foreign-body granulomatous reaction, even 20 years later. It is important to be aware of this possibility and to question patients with pleural abnormalities about past procedures and mention such procedures to the colleagues who are responsible for interpreting metabolic imaging. Follow-up of hypermetabolic pleural lesions attributed to talc pleurodesis is important for the detection of new pleural lesions or neoplastic evolution.

Hypoxia-inducible factor-1α as prognostic marker.

INTRODUCTION: Hypoxia-inducible factor-1α (HIF-1α) is a key player in the signaling pathway that mediates a complex and pleiotropic range of adaptive responses to hypoxia. It serves as cellular hypoxia sensor and plays a critical role in physiologic processes including glucose metabolism, iron metabolism, erythropoiesis, angiogenesis, cell survival and apoptosis, but also, pathologic processes such as carcinogenesis, progression and metastasis of many cancers. With the recent advent of new molecular targeted therapies, there is a growing need of molecular understanding of physiology and physiopathology and increased demand of diagnosis, prognosis and follow-up markers. AREAS COVERED: This paper reviews the biology of regulation of HIF-1α, its physiological and physiopathological effects. EXPERT OPINION: The authors discuss the potential diagnosis and the prognosis significance of HIF-1α that was evaluated in recent studies.

Analysis of T-cell receptor-γ gene rearrangements using heteroduplex analysis by high-resolution microcapillary electrophoresis.

Determination of T-cell clonality has an important additional value for diagnosis of T-cell lymphomas. Various molecular methods have been developed, including polymerase chain reaction (PCR) of T-cell receptor γ (TCRγ). The detection of PCR products usually relies commonly on either GeneScan (GS) analysis or heteroduplex (HD) analysis by polyacrylamide gel electrophoresis (PAGE). These techniques have their disadvantages, being relatively time-consuming and laborious or requiring expensive equipment. Here, we propose an alternative method that combines multiplex PCR and HD analysis by microcapillary electrophoresis (ME) on the Agilent 2100 Bioanalyzer. The sensitivity of the method was determined with clonal PEER T-cell line DNA dilution in polyclonal DNA and was evaluated as 1-5%. Fifty-three samples from patients with T-cell lymphoproliferative disorders were analyzed by HD analysis using ME and GS analyses. Comparison of the two techniques showed them to be highly concordant (93% similarity). The rate of clonality detection by GS analysis was higher than HD analysis by ME, but none of the discordant patients (n=5) has yet developed lymphoma. HD analysis by ME to reveal TCRγ gene rearrangements in clinical specimens was consistent with clinical data and the outcome of patients. Detection of T-cell clonality by HD analysis with ME is sensitive, practical, safe and represents a potential alternative to PAGE and GS analysis.

Hypoxia inducible factor 1alpha gene (HIF-1alpha) splice variants: potential prognostic biomarkers in breast cancer.

BACKGROUND: Hypoxia-inducible factor 1 (HIF-1) is a master transcriptional regulator of genes regulating oxygen homeostasis. The HIF-1 protein is composed of two HIF-1alpha and HIF-1beta/aryl hydrocarbon receptor nuclear translocator (ARNT) subunits. The prognostic relevance of HIF-1alpha protein overexpression has been shown in breast cancer. The impact of HIF-1alpha alternative splice variant expression on breast cancer prognosis in terms of metastasis risk is not well known. METHODS: Using real-time quantitative reverse transcription PCR assays, we measured mRNA concentrations of total HIF-1alpha and 4 variants in breast tissue specimens in a series of 29 normal tissues or benign lesions (normal/benign) and 53 primary carcinomas. In breast cancers HIF-1alpha splice variant levels were compared to clinicopathological parameters including tumour microvessel density and metastasis-free survival. RESULTS: HIF-1alpha isoforms containing a three base pairs TAG insertion between exon 1 and exon 2 (designated HIF-1alphaTAG) and HIF-1alpha736 mRNAs were found expressed at higher levels in oestrogen receptor (OR)-negative carcinomas compared to normal/benign tissues (P = 0.009 and P = 0.004 respectively). In breast carcinoma specimens, lymph node status was significantly associated with HIF-1alphaTAG mRNA levels (P = 0.037). Significant statistical association was found between tumour grade and HIF-1alphaTAG (P = 0.048), and total HIF-1alpha (P = 0.048) mRNA levels. HIF-1alphaTAG mRNA levels were also inversely correlated with both oestrogen and progesterone receptor status (P = 0.005 and P = 0.033 respectively). Univariate analysis showed that high HIF-1alphaTAG mRNA levels correlated with shortened metastasis free survival (P = 0.01). CONCLUSIONS: Our results show for the first time that mRNA expression of a HIF-1alphaTAG splice variant reflects a stage of breast cancer progression and is associated with a worse prognosis.See commentary: http://www.biomedcentral.com/1741-7015/8/45.

[High-throughput quantification of tissue microarrays: identification of candidate target proteins in inflammatory breast cancer]. / La lecture automatisée à haut débit des micro-biopsies en pathologie: mise en évidence de la surexpression de c-Met et PI3K dans les cancers du sein inflammatoire.

Inflammatory breast carcinoma (IBC) is a rare but very aggressive tumour phenotype. Increased c-Met protein expression correlates with reduced survival and a higher metastatic risk in many human malignancies, including breast cancer Several studies have shown that c-Met protein is targetable by specific drugs. Here we compared c-Met expression in IBC (n = 41) and non IBC (n = 480). Two microarrays of IBC and non IBC tissues were constructed and standardized. C-Met, P13K and E-cadherin were immunodetected (Ven-tana Benchmark Autostainer) on serial sections. The results were quantified with an automated image analysis device (SAMBA Technologies) by immunoprecipitate densitometry of each core section (0.6 microns thick). We found that (i) c-Met is significantly overexpressed in IBC compared to non IBC (p < 0. 001), (ii) P13K is also overexpressed (p < 0.001) in IBC, suggesting that overexpressed c-Met is functionally active, at least through the PI3K signal transduction pathway ; and (iii) E-cadherin is paradoxically overexpressed in IBC. We conclude that c-Met may constitute a target for specific therapy in patients with poor-prognosis malignancies like IBC Automated image analysis of TMA is a valuable tool for high-throughput quantification of the immunohistochemical expression of the tumor proteome.

Overexpression of c-Met and of the transducers PI3K, FAK and JAK in breast carcinomas correlates with shorter survival and neoangiogenesis.

c-Met is responsible for cell motility and tumour spreading. c-Met expression and signal transducers reflecting c-Met functionality were investigated in breast carcinomas, in correlation with patient outcome and tumour vasculature. Tissue microarrays of 930 breast carcinomas were constructed, categorised according to patients' follow-up (4- to 10-year follow-up; median, 6.5 years). Standardised immunocytochemical procedures were performed using anti-c-Met, -PI3K, -FAK, -JAK, and -CD146, -FYN and an automated autostainer (Ventana). High-throughput densitometry measuring the extent of immunoprecipitates was assessed by image analysis (SAMBA). c-Met overexpression correlated with poor survival along with PI3K and FAK reflecting c-Met functionality and CD146 and FYN expression in endothelial cells. Automated quantification of immunocytochemical precipitates using image analysis was shown to provide an objective means of measuring cellular proteins that are potentially relevant for current practice in pathological diagnosis and for specific therapy combining inhibitors of both c-Met and downstream transducer pathways, and of tumour angiogenesis.

Poor prognosis in breast carcinomas correlates with increased expression of targetable CD146 and c-Met and with proteomic basal-like phenotype.

Genomic studies have led to new taxonomic classifications of breast carcinomas. Proteomic investigations using tissue microarrays have yielded complementary results and are useful in identifying potential molecular targets for specific therapies. Searching for new drug targets is particularly important for tumors of poor prognosis, such as breast tumors that lack estrogen receptors and HER2 amplification; in these tumors, certain molecules probably play a significant role in tumor spreading through the stromal microvasculature. We investigated 930 breast carcinomas categorized according to patients' survival (range of follow-up = 4-10 years; median follow-up = 6.5 years) using (1) automated immunohistochemical procedures (Ventana, Cedex, France) with tissue microarrays (Alphelys, Plaisir, France) and (2) quantification of immunoprecipitates assessed by automated image analysis densitometry (SAMBA, Meylan, France). Expression of c-Met and CD146 and that of signaling transducers PI3K, FAK, and FYN were compared in living and deceased patients. Expression of some proteins recently reported to be characteristic of basal cell carcinomas was also assessed, namely, CK5-6, caveolin-1, carbonic anhydrase IX, p63, and CD117; these also constitute potential targets for therapies for aggressive tumors. Overexpression of these proteins was observed in deceased or metastatic patients (P < .01 to P < .00001), particularly node-negative patients (except for FYN, p63, and CD146). c-Met and CD146 are involved in tumor spreading, and our results suggest that they probably play an important role in patients' death, along with other proteins involved in hypoxia (carbonic anhydrase IX) and other cell functions or structures (caveolin-1, CD117, CK5-6, and p63) that are expressed in an aggressive subtype of basal cell carcinoma for which no specific therapy is available.

FGFR1 and WT1 are markers of human prostate cancer progression.

BACKGROUND: Androgen-independent prostate adenocarcinomas are responsible for about 6% of overall cancer deaths in men. METHODS: We used DNA microarrays to identify genes related to the transition between androgen-dependent and androgen-independent stages in the LuCaP 23.1 xenograft model of prostate adenocarcinoma. The expression of the proteins encoded by these genes was then assessed by immunohistochemistry on tissue microarrays (TMA) including human prostate carcinoma samples issued from 85 patients who had undergone radical prostatectomy. RESULTS: FGFR1, TACC1 and WT1 gene expression levels were associated with the androgen-independent stage in xenografts and human prostate carcinoma samples. MART1 protein expression was correlated with pT2 tumor stages. CONCLUSION: Our results suggest that each of these four genes may play a role, or at least reflect a stage of prostate carcinoma growth/development/progression.

Toxic death-case after capecitabine + oxaliplatin (XELOX) administration: probable implication of dihydropyrimidine deshydrogenase deficiency.

This report here is the case of a 52-year-old male patient who suffered from extremely severe haematological toxicities (G4 neutropenia, G4 thrombocytopenia) while undergoing Xelox (Xeloda + Oxaliplatin) treatment for his multifocal hepatocarcinoma. Despite appropriate supportive treatment, his condition quickly deteriorated and led to death. It was hypothesized that dihydropyrimidine deshydrogenase (DPD) gene polymorphism could be, at least in part, responsible for this fatal outcome. To test this hypothesis, both phenotypic and genotypic studies were undertaken, and fully confirmed the DPD-deficient status of this patient. Uracil to dihydrouracil ratio in plasma was evaluated as a surrogate marker for DPD deficiency, and showed values out of the range previously recorded from a reference, non-toxic population. Interestingly, the canonical IVS14+1G>A single nucleotide polymorphism, usually associated with the most severe toxicities reported with 5-fluorouracil (5-FU), was not found in this patient, but further investigations showed instead a heterozygosity for the 1896C>T mutation located in the exon 14 of the DPYD gene. Taken together, the data strongly suggest for the first time that a toxic-death case after capecitabine-containing protocol could be, at least in part, linked with a DPD-deficiency syndrome. The case reported here warrants therefore systematic detection of patients at risk, including when oral capecitabine is scheduled.

[Plasmablastic lymphoma in a patient with HIV infection: an unusual case located in the skin]. / Lymphome plasmoblastique du sujet infecté par le VIH: a propos d'un cas très inhabituel de localisation cutanée.

We report the case of a plasmablastic lymphoma involving the skin in a 45 year-old HIV-positive patient. Plasmablastic lymphoma was first described in 1997 and is considered to be a diffuse large B-cell lymphoma with a unique immunophenotype and a predilection for the oral cavity. In this case, the tumor was revealed by multiple purple cutaneous nodules predominantly localized on the trunk and proximal parts of the limbs. A skin biopsy led to the diagnosis of plasmablastic lymphoma in view of the presence of a dense nodular infiltrate invading the dermis and subcutaneous fat composed of large cells that expressed neither the leucocyte common antigen nor the B- and T-cell antigens CD20 and CD3, but which showed a strong immunostaining with plasma cell marker VS38c. Most of the cells expressed Kappa light chain of immunoglobulins, they did not express Lambda light chain. In situ hybridization with EBER probe revealed detection of Epstein Barr virus in about 15 % of tumor cells. The clinical course was aggressive and rapidly fatal. Despite one cycle chemotherapy the patient died four months after presentation. HIV-associated plasmablastic lymphoma is a poor prognosis malignancy that may resist typing due to the lack of expression of commonly used lymphoid markers.

Conservative treatment of cervical intraepithelial neoplasia using a cold-knife section technique.

OBJECTIVE: The purpose of this study was to evaluate a conservative cold-knife section technique for treatment of cervical intraepithelial neoplasia (CIN). This procedure can be adapted to patient age, preservation of childbearing potential and extent of dysplasia. DESIGN: Prospective study. SETTING: Gynecological Oncology Department in French Public Hospital. POPULATION: A total of 460 women treated for CIN between 1985 and 1999 were included. METHODS: A conservative cold-knife cervical section followed by blanket suture reconstruction was used in all cases. MAIN OUTCOME MEASURES: Immediate operative results, recurrence and reproductive function were assessed. RESULTS: The mean length of the cervical specimen was 11.4 mm (range, 4-22 mm). Mean specimen thickness was strongly correlated with age: 10.6 +/- 4.1 mm in women <40 years versus 12.1 in women >40 years; p < 0.001. Complete excision was achieved in 395 cases (85.8%). Post-operative bleeding was observed in 5 cases (1.1%). The mean duration of follow-up was 62 months (range, 12.3-156.5 months). Recurrences developed in 26 patients (6.6%) including CIN 1 in 9 cases, CIN 2 in 9 and CIN 3 in 8. No patient developed carcinoma. The actuarial risk of recurrence was 2.4% (+/- S.D., 0.9) at 24 months and 7.8% (+/-S.D., 1.9) at 60 months. A total of 52 pregnancies were observed in 39 patients. No case of de novo infertility was reported post-operatively. Amenorrhea was noted in 1 patient (0.1%) and dysmenorrhea in 1 patient (0.1%). CONCLUSIONS: This conservative cold-knife section technique is effective for treatment of CIN with low morbidity and little adverse effect on childbearing potential. Exposure of the squamocolumnar junction (SCJ) greatly facilitates follow-up.

Overexpression of hypoxia-inducible factor HIF-1alpha predicts early relapse in breast cancer: retrospective study in a series of 745 patients.

Hypoxia-inducible factor-1alpha (HIF-1alpha) is a transcription factor that is involved in tumour growth and metastasis by regulating genes involved in response to hypoxia. HIF-1alpha protein overexpression has been shown in a variety of human cancers, but only 2 studies have documented the prognostic relevance of HIF-1alpha expression in breast cancer. The aim of our study was to determine accurately the impact of HIF-1alpha expression on prognosis in a large series (n = 745) of unselected patients with invasive breast cancer in terms of overall survival, local recurrence and distant metastasis risk. HIF-1alpha expression was investigated using immunohistochemical assays on frozen sections, and correlated with patients' outcome (median follow-up = 13.5 years). Univariate (Kaplan-Meier) analysis showed that high levels of HIF-1alpha expression (cutoff = 10%) significantly correlated with poor overall survival (p = 0.019). HIF-1alpha expression correlated with high metastasis risk among the whole group of patients (p = 0.008). Multivariate analysis (Cox model) showed that the HIF-1alpha predictive value was independent of other current prognostic indicators. Moreover among node negative ones, HIF-1alpha expression was also significantly predictive of metastasis risk (p = 0.03) and of relapse (p = 0.035). All the data suggest that HIF-1alpha is associated with a worse prognosis in patients with invasive breast carcinoma. Furthermore HIF-1alpha immunodetection may be considered as a potential indicator for selecting patients who could benefit from specific therapies interfering with HIF-1alpha pathway.

Comparison of the prognosis indication of VEGFR-1 and VEGFR-2 and Tie2 receptor expression in breast carcinoma.

The degree of angiogenesis in breast cancer has previously been shown to be an indicator of prognosis, and tumor microvasculature is a candidate target for new antiangiogenic therapies. The aim of this study was to investigate the prognostic value of vascular endothelial growth factor (VEGF) receptors, VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1), and Tie2/tek receptor tyrosine kinase in breast carcinoma. VEGF receptors and Tie2 expression was investigated using immunohistochemical assays with monoclonal antibodies on frozen sections in a series of 918 and 909 patients respectively. VEGFR-1 and VEGFR-2 and Tie2 were correlated with long-term (median, 11.3 years) patients' outcome. Univariate (Kaplan-Meier) analysis showed that VEGFR-1 positive tumor surface (cutoff = 5%) was significantly correlated with high metastasis risk (p=0.03) and relapse (p<0.01) in all patients, and in those with node negative tumors (p<0.001 and p<0.01 respectively), but not with overall survival. In contrast Tie2 positive tumor surface (cutoff = 7%) was significantly correlated with poor overall survival (p=0.025) and also with high metastasis risk particularly among node negative patients (p<0.01). Moreover, Tie2 immunoexpression was significantly predictive of relapse (p=0.003) in the node negative subgroup (p=0.02). In multivariate analysis (Cox model), VEGFR-1 and Tie2 immunoexpressions were identified as independent prognostic indicators. In contrast, univariate analysis showed that VEGFR-2 positive tumor surface (cutoff = 10%) was not correlated with survival or with metastasis and relapse risk. Our results suggest that VEGFR-1 and Tie2 immunohistochemical expression permits the identification of patients with poor outcome, and particularly node negative ones with a high risk for metastasis and relapse. VEGFR-1 and Tie2 immunodetection may also be considered as potential tools for selecting patients who could benefit in the future from specific antiangiogenic therapy interfering with VEGFR-1 and Tie2 activation pathways.

[Immunohistochemical expression of PTEN antigen: a new tool for diagnosis of early endometrial neoplasia]. / L'expression immunohistochimique de l'antigène PTEN: un nouvel outil diagnostique dans les néoplasies débutantes de l'endomètre.

We examined immunohistochemical PTEN protein expression in endometrial samples obtained from perimenopausal women with uterine bleeding in order to diagnose early endometrial neoplasia. Paraffin sections of endometrial samples (biopsies, endometrium resection, hysterectomy) were reviewed and tested for immunocytochemical PTEN expression by epithelial cells. Immunohistochemical studies were performed with the Ventana Benchmark device and the immunoperoxidase technique with monoclonal anti-PTEN (6H2.1/Cascade Biosciences). We studied 45 samples of proliferative endometria, 42 samples of atypical hyperplasia (neoplasia), and 55 samples of endometrial carcinomas (30 endometrioid, 5 serous papillary, 10 clear cell, 5 adenosquamous, and 5 MMMT). A strong positive PTEN immunoreaction was observed in all 45 normal proliferative endometria, 2/30 endometrioid carcinomas, and 23/25 non endometrioid carcinomas. In contrast, PTEN immunoexpression was negative or weak in 40/42 atypical hyperplasia and 28/30 endometrioid carcinomas. Negative or weak PTEN expression correlated with endometrial neoplasia (atypical proliferation) (p < 0.001). Thus (i) PTEN immunoexpression in endometrial paraffin sections is a new diagnostic tool, distinguishing PTEN-positive cyclic abnormal monoclonal proliferative endometrium (anovulatory, non atypical, simple and complex hyperplasia) from PTEN-negative (or decreased) endometrial neoplasia, and especially early endometrial neoplasia (atypical simple and complex hyperplasia, in situ carcinoma, superficial carcinoma with focal invasion). (ii) Given the lack of reproducibility of histological identification of atypical (precancerous invasive) and non atypical (precancerous non invasive) endometrial hyperplasia, the new criteria (PTEN-negative crowded glands, gland/stroma ratio > 55%, nuclear pleomorphism, in areas > 1 mm) recently proposed to diagnose early endometrial neoplasia appear to be clinically relevant.