c-Met is a prognostic marker and potential therapeutic target in clear cell renal cell carcinoma.

BACKGROUND: Activation of the c-Met pathway occurs in a range of malignancies, including papillary renal cell carcinoma (RCC). Its activity in clear cell RCC is less clear. We investigated c-Met expression and inhibition in a large cohort of RCC tumors and cell lines. METHODS: c-Met protein expression was determined by automated quantitative analysis (AQUA) on a tissue microarray (TMA) constructed from 330 RCC tumors paired with adjacent normal renal tissue. c-Met expression and selective inhibition with SU11274 and ARQ 197 were studied in clear cell RCC cell lines. RESULTS: Higher c-Met expression was detected in all RCC subtypes than in the adjacent normal renal tissue (P < 0.0001). Expression was highest in papillary and sarcomatoid subtypes, and high-grade and stage tumors. Higher c-Met expression correlated with worse disease-specific survival [risk ratio = 1.36; 95% confidence interval (CI) 1.08-1.74; P = 0.0091] and was an independent predictor of survival, maintained in clear cell subset analyses. c-Met protein was activated in all cell lines, and proliferation (and colony formation) was blocked by SU11274 and ARQ 197. CONCLUSIONS: c-Met is associated with poor pathologic features and prognosis in RCC. c-Met inhibition demonstrates in vitro activity against clear cell RCC. Further study of ARQ 197 with appropriate biomarker studies in RCC is warranted.

Time-dependent mediators of HPA axis activation following live Escherichia coli.

The hypothalamus-pituitary-adrenal (HPA) axis is activated during an immune challenge to liberate energy and modulate immune responses via feedback and regulatory mechanisms. Inflammatory cytokines and prostaglandins are known contributors to HPA activation; however, most previous studies only looked at specific time points following LPS administration. Since whole bacteria have different immune stimulatory properties compared with LPS, the aim of the present studies was to determine whether different immune products contribute to HPA activation at different times following live Escherichia coli challenge. Sprague-Dawley rats were injected intraperitoneally with E. coli (2.5 × 10(7) CFU) and a time course of circulating corticosterone, ACTH, inflammatory cytokines, and PGE(2) was developed. Plasma corticosterone peaked 0.5 h after E. coli and steadily returned to baseline by 4 h. Plasma PGE(2) correlated with the early rise in plasma corticosterone, whereas inflammatory cytokines were not detected until 2 h. Pretreatment with indomethacin, a nonselective cyclooxygenase inhibitor, completely blocked the early rise in plasma corticosterone, but not at 2 h, whereas pretreatment with IL-6 antibodies had no effect on the early rise in corticosterone but attenuated corticosterone at 2 h. Interestingly, indomethacin pretreatment did not completely block the early rise in corticosterone following a higher concentration of E. coli (2.5 × 10(8) CFU). Further studies revealed that only animals receiving indomethacin prior to E. coli displayed elevated plasma and liver cytokines at early time points (0.5 and 1 h), suggesting prostaglandins suppress early inflammatory cytokine production. Overall, these data indicate prostaglandins largely mediate the early rise in plasma corticosterone, while inflammatory cytokines contribute to maintaining levels of corticosterone at later time points.

Rat strain differences in restraint stress-induced brain cytokines.

Induction of brain cytokines during times of stress has potent effects on altering behavior, mood, and cognitive functioning. Currently, it is unknown why exposure to some stressors such as tailshock and footshock elevate brain cytokines, while exposure to swim, predator odor, and restraint stress do not. Recent data indicate that brain noradrenergic signaling mediates brain cytokine production suggests magnitude of norepinephrine release during stress may be critical in initiating brain cytokine production. The aim of the current study was to investigate stress-induced brain cytokines between rat strains that differ in their magnitude of stress responsiveness as measured by brain norepinephrine and HPA responses. Sprague-Dawley and Fischer rats were placed in a restraint bag for 1 h or 2 h and sacrificed immediately following stressor termination. Exposure to restraint significantly elevated hypothalamic interleukin (IL)-1ß and IL-1 receptor type (R) 2 mRNA after 1 h and IL-1ß protein after 2 h in the high stress responsive Fischer rats, but not in Sprague-Dawley rats. IL-6, IL-1R1, Il-1 receptor antagonist (RA), and cyclooxygenase (Cox)-2 mRNA were not altered and neither there was expression of any cytokines in the hippocampus or circulating cytokines in either strain. Administration of desipramine (a norepinephrine reuptake inhibitor) to Sprague-Dawley rats was sufficient either alone or in combination with stress to increase IL-1ß mRNA in the hypothalamus and desipramine combined with stress was sufficient to increase IL-1R2 mRNA in the hypothalamus. These data support our hypothesis that there is a critical threshold of brain norepinephrine necessary to stimulate brain cytokines, which may help to explain why severe stressors are more commonly reported to induce brain cytokines. These data also suggest an organisms' susceptibility to stress-induced brain cytokine production, depends on responsiveness and regulation of noradrenergic neurons.

The CCK(2) receptor antagonist, YF476, inhibits Mastomys ECL cell hyperplasia and gastric carcinoid tumor development.

YF476 is a potent and highly selective cholecystokin 2 (CCK(2)) receptor antagonist of the benzodiazepine class. It inhibits gastric neuroendocrine enterochromaffin-like (ECL) cell secretion, proliferation and spontaneous formation of gastric neuroendocrine tumors (carcinoids) in cotton rats. The Mastomys rodent species exhibits a genetic predisposition to gastric ECL neuroendocrine tumor formation which can be accelerated by acid suppression and induction of hypergastrinemia. In this respect, it mimics the human condition of atrophic gastritis, hypergastrinemia and gastric carcinoid development. We investigated whether YF476 could inhibit acid suppression-induced ECL cell hyperplasia and neoplasia in this model. In addition, we examined whether YF476 could reverse established ECL cell hyperplasia and neoplasia. Targeting the CCK(2) receptor during Loxtidine-induced hypergastrinemia resulted in a reduction in ECL cell secretion (plasma and mucosal histamine, and histidine decarboxylase (HDC) transcripts, p<0.05) and proliferation (numbers of HDC-positive cells, connective tissue growth factor (CTGF) and cyclin D1 transcription). This was associated with a decrease in ECL cell hyperplasia and a 60% reduction in gastric ECL cell microcarcinoid (tumors <0.3mm in size) formation. YF476 inhibited ECL cell neoplasia (gastric carcinoid) in animals with hyperplasia, inhibited the formation of ECL cell tumors when co-administered with Loxtidine and reversed the growth and developement of gastric ECL cell carcinoids in long-term acid suppressed Mastomys. Variable importance analysis using a logistic multinomial regression model indicated the effects of YF476 were specific to the ECL cell and alterations in ECL cell function reflected inhibition of transcripts for HDC, Chromogranin A (CgA), CCK(2) and the autocrine growth factor, CTGF. We conclude that specifically targeting the CCK(2) receptor inhibits gastrin-mediated ECL cell secretion and ECL cell proliferation and tumor development in vivo.

Growth factor receptor-bound protein-7 (Grb7) as a prognostic marker and therapeutic target in breast cancer.

BACKGROUND: Growth factor receptor-bound protein-7 (Grb7) is an adapter-type signaling protein recruited to various tyrosine kinases, including HER2/neu. Grb7-specific inhibitors are in early development. As with other targeted therapies, response to therapy might be associated with target expression. MATERIALS AND METHODS: Tissue microarrays containing 638 primary breast cancer specimens with 15-year patient follow-up were employed to assess Grb7 expression using our Automated QUantitative Analysis method; cytokeratin defines pixels as breast cancer (tumor mask) within the histospot, and Grb7 expression within the mask is measured with Cy5-conjugated antibodies. RESULTS: High Grb7 expression was strongly associated with decreased survival in the entire cohort and in the node-positive subset (P = 0.0034 and P = 0.0019, respectively). On multivariable analysis, it remained an independent prognostic marker (P = 0.01). High Grb7 was strongly associated with high HER2/neu, and coexpression of these molecules was associated with worse prognosis than HER2/neu overexpression alone. CONCLUSIONS: High Grb7 defines a subset of breast cancer patients with decreased survival, indicating that Grb7 might be a valuable prognostic marker and drug target. Coexpression with HER2/neu indicates that cotargeting these molecules might be an effective approach for treating HER2/neu-positive breast cancers. Future studies using Grb7-targeting agents should include assessment of Grb7 levels.

More than skin deep: atherosclerosis as a systemic manifestation of psoriasis.

There is now growing evidence that psoriasis, like other inflammatory diseases such as rheumatoid arthritis and systemic lupus erythematosus, is a systemic disorder that is associated with enhanced atherosclerosis and risk of coronary artery disease. Here we summarize the available epidemiological evidence for this association and analyse pathogenic features that are common to psoriasis and atherosclerosis. Further prospective studies are urgently needed to extend knowledge of the risk of cardiovascular morbidity and mortality in patients with psoriasis and to confirm the degree to which treatment of psoriasis reduces this risk. Nevertheless, existing data are sufficient to indicate that severe psoriasis should be more widely recognized as a potential risk factor for cardiovascular disease and should be considered with the established factors when formulating strategies for the management of cardiovascular risk.

Microvessel area using automated image analysis is reproducible and is associated with prognosis in breast cancer.

Microvessel density may be one measure of tumor associated angiogenesis but is methodologically difficult to standardize and reproduce. We used our automated quantitative image analysis system, AQUA, to more objectively assess microvessel area. Cytokeratin and CD31 were used to create tumor and vessel compartments respectively with AQUA. Microvessel area was defined as CD31 compartment area normalized to the tissue spot area (CD31 area/area of entire tissue spot). Consecutive breast cancer whole sections were stained with CD31 to compare pathologist-based microvessel density with AQUA microvessel area. Microvessel areas of 3-fold redundant tissue microarrays of 652 primary breast cancers were also assessed. CD34 and factor VIII-related antigen were also tested. There was nearly linear correlation between pathologist's microvessel density and AQUA microvessel area with regression coefficient R = 0.846. On the redundant arrays, of the 67% evaluable cases, 52% were microvessel area high and 48% low with good reproducibility of scores (Spearman rho 0.551). AQUA microvessel area was associated with larger tumors, node positivity, and estrogen receptor negativity, with 20 year survival at the univariate and multivariate levels (P < .0001 and P = .0121, respectively). CD34 or factor VIII-related antigen were more heterogenous, had poor association with CD31, and did not correlate with outcome. AQUA-based microvessel area was significantly correlated with both standard breast cancer prognostic parameters as well as with clinical outcome. In the future, it may also allow the use of the AQUA-based algorithms to quantify the expression of angiogenic biomarkers to either tumor or microvessel area-specific compartments.

Human tissue kallikrein 7, a novel biomarker for advanced ovarian carcinoma using a novel in situ quantitative method of protein expression.

BACKGROUND: Kallikreins, a subgroup of the serine protease enzyme family, are considered important prognostic biomarkers in cancer. Here, we sought to determine the prognostic value of kallikrein 7 (hk7) in ovarian cancer using a novel method of compartmentalized in situ protein analysis. PATIENTS AND METHODS: A tissue array composed of 150 advanced-stage ovarian cancers, uniformly treated with surgical debulking followed by platinum-paclitaxel (Taxol) combination chemotherapy, was constructed. For evaluation of kallikrein 7 protein expression, we used an immunofluorescence-based method of automated in situ quantitative measurement of protein analysis (AQUA). RESULTS: Mean follow-up time of the cohort was 34.35 months. One hundred and twenty eight of 150 cases had sufficient tissue for AQUA. In univariate survival analysis, low tumor hk7 expression was associated with better outcome for overall survival (OS) and disease-free survival in 3 years (P values 0.032 and 0.037, respectively). In multivariate survival analysis, adjusting for well-characterized prognostic variables, low tumor hk7 expression level was the most significant predictor variable for OS (95% confidence interval 0.125-0.729, P = 0.007). CONCLUSIONS: High tumor hk7 protein expression is associated with inferior patient outcome in ovarian cancer. hk7 may represent a promising prognostic factor in ovarian cancer.

HSP90 as a marker of progression in melanoma.

BACKGROUND: HSP90 chaperones molecules critical for cell survival and malignant progression, including mutated B-raf. HSP90-targeting agents are in clinical trials. No large studies have been conducted on expression of HSP90 in melanomas. MATERIALS AND METHODS: Tissue microarrays containing 414 nevi, 198 primary and 270 metastatic melanomas were assessed using our automated quantitative analysis (AQUA) method of in situ protein measurement; we use S-100 to define pixels as melanocytes (tumor mask) within the array spot, and measure HSP90 expression within the mask using Cy5-conjugated antibodies. RESULTS: HSP90 expression was higher in melanomas than nevi (P < 0.0001) and higher in metastatic than primary specimens (P < 0.0001). No association was seen between high HSP90 expression and survival in the primary or metastatic patient subsets. In primary melanomas, high HSP90 expression was associated with higher Clark level (P = 0.0167) and increased Breslow depth (P < 0.0001). CONCLUSIONS: HSP90 expression was significantly higher in tumors than nevi and was associated with disease progression, indicating that it might be a valuable drug target in melanoma, as well as a useful diagnostic marker. Prospective studies are needed to confirm the diagnostic role of HSP90, as well as the predictive role of HSP90 expression in patients treated with HSP90 inhibitors.

CTGF, intestinal stellate cells and carcinoid fibrogenesis.

AIM: To investigate the role of small intestinal carcinoid tumor-derived fibrotic mediators, TGFbeta1 and CTGF, in the mediation of fibrosis via activation of an "intestinal" stellate cell. METHODS: GI carcinoid tumors were collected for Q RT-PCR analysis of CTGF and TGFbeta1. Markers of stellate cell desmoplasia were identified in peritoneal fibrosis by immunohistochemistry and stellate cells cultured from fresh resected fibrotic tissue. CTGF and TGFbeta1 were evaluated using quantitative tissue array profiling (AQUA analysis) in a GI carcinoid tissue microarray (TMA) with immunostaining and correlated with clinical and histologically documented fibrosis. Serum CTGF was analyzed using a sandwich ELISA assay. RESULTS: Message levels of both CTGF and TGFbeta1 in SI carcinoid tumors were significantly increased (> 2-fold, P < 0.05) versus normal mucosa and gastric (non-fibrotic) carcinoids. Activated stellate cells and markers of stellate cell-mediated fibrosis (vimentin, desmin) were identified in histological fibrosis. An intestinal stellate cell was immunocytochemically and biochemically characterized and its TGFbeta1 (10-7M) initiated CTGF transcription response (> 3-fold, P < 0.05) demonstrated. In SI carcinoid tumor patients with documented fibrosis, TMA analysis demonstrated higher CTGF immunostaining (AQUA Score: 92 +/- 8; P < 0.05), as well as elevated TGFbeta1 (90.6 +/- 4.4, P < 0.05). Plasma CTGF (normal 12.5 +/- 2.6 ng/mL) was increased in SI carcinoid tumor patients (31 +/- 10 ng/mL, P < 0.05) compared to non-fibrotic GI carcinoids (< 15 ng/mL). CONCLUSION: SI carcinoid tumor fibrosis is a CTGF/TGFbeta1-mediated stellate cell-driven fibrotic response. The delineation of the biology of fibrosis will facilitate diagnosis and enable development of agents to obviate its local and systemic complications.

Analysis of p53 protein expression levels on ovarian cancer tissue microarray using automated quantitative analysis elucidates prognostic patient subsets.

BACKGROUND: p53 protein is regarded as a valuable prognostic marker in cancer with a potential use as a molecular target. Here, we sought to determine the prognostic value of p53 in ovarian cancer using a novel method of compartmentalized in situ protein analysis. PATIENTS AND METHODS: A tissue array composed of 141 advanced stage ovarian cancers uniformly treated was constructed. For evaluation of p53 protein expression, we used an immunofluorescence-based method of automated in situ quantitative measurement of protein analysis (AQUA). RESULTS: High nuclear p53 expression levels were associated with better outcome for overall survival (OS) (P = 0.0023) and disease-free survival (P = 0.0338) at 5-years. High cytoplasmic p53 expression levels were associated with better outcome for OS (P = 0.0002). In multivariable analysis, high nuclear and high cytoplasmic p53 level with International Federation of Gynecology and Obstetrics (FIGO) stage were the most significant predictor variables for OS and high nuclear p53 level with FIGO stage were the significant predictor variables for disease-free survival. CONCLUSIONS: Assessment of the prognostic value of p53 protein levels using conventional immunohistochemistry is limited by the nonquantitative nature of the method. AQUA provides precise estimation of p53 protein levels and was able to elucidate the association of p53 protein levels and ovarian cancer prognosis.

Role of CCN2/CTGF in the proliferation of Mastomys enterochromaffin-like cells and gastric carcinoid development.

Mastomys enterochromaffin-like (ECL) cell proliferation is initially gastrin driven, but once neoplasia develops, cells become gastrin autonomous. We hypothesized that CCN2 (CTGF), a mitogenic growth factor, may regulate ECL cell proliferation. A Mastomys GeneChip database was examined (dCHIP) to identify CCN2 expression levels. CCN2 in normal and tumor ECL cell preparations obtained using FACS (100 nM acridine orange) was examined by real-time PCR. CCN2 protein was identified in mucosal and ECL cell preparations by immunohistochemistry. Short-term cultured cells were stimulated with either CCN2 or CCN2 + EGF, and proliferation was measured (MTT assay). The ERK1/2 inhibitor PD-98059 (0.1-100 microM) was assessed in terms of CCN2 (1 ng/ml)-mediated proliferation and ERK1/2 phosphorylation. CCN2 transcript and protein was then examined in clinical gastric carcinoids. The ccn2 transcript was upregulated in tumor samples compared with the normal mucosa (+2.36-fold, P < 0.01). PCR demonstrated that ccn2 was not expressed in FACS-prepared (>98% pure) normal ECL cells but was elevated in tumor ECL cell fractions (41.3 +/- 10.7-fold). Immunostaining of the Mastomys gastric mucosa and FACS preparations confirmed that CCN2 protein was present in ECL tumors but not in normal ECL cells. Neither CCN2 nor CCN2 + EGF stimulated normal ECL cell proliferation. CCN2 stimulated tumor proliferation (EC50 approximately 0.01 ng/ml); EGF significantly augmented (P < 0.01) CCN2-induced tumor cell proliferation (EC50 = 20 pg/ml). PD-98059 inhibited CCN2-induced proliferation (-12 +/- 3%, P < 0.05) and ERK1/2 phosphorylation (-34 +/- 5%, P < 0.05) in tumor cells. In clinical samples, both CCN2 transcript and protein were elevated in gastrin-autonomous carcinoids (P < 0.02) compared with the normal mucosa. In conclusion, CCN2 may be a proliferative regulator of Mastomys ECL neoplastic proliferation once these cells become autonomous of gastrin regulation. Identification of CCN2 in gastric carcinoid tissue may be useful both as an indicator of ECL cell transformation and may define gastrin autonomy, a criteria of gastric carcinoid malignancy.

Updates from the Fourth International Congress 'psoriasis: from gene to clinic', the Royal College of Physicians, London, UK, 1-3 December 2005.

The fourth 'Psoriasis: From Gene to Clinic' meeting was held in December 2005, its international status confirmed by the presence of almost 300 delegates from Europe, North America, Asia and Australasia. The meeting was co-organized by Jonathan Barker (St John's Institute of Dermatology, London) and Chris Griffiths (University of Manchester), who once again deserve congratulations for the success of their initiative. As its title suggests, the meeting was targeted at both clinical and basic scientists with a special interest in psoriasis, but in preparing this report the writer has selected presentations that caught his attention and that he felt able to review in a manner that might be of interest to the general readership of this Journal.

Automated quantitative analysis of DCC tumor suppressor protein in ovarian cancer tissue microarray shows association with beta-catenin levels and outcome in patients with epithelial ovarian cancer.

BACKGROUND: The deleted in colorectal cancer (DCC) protein, the product of DCC tumor suppressor gene, is frequently altered in cancer. Preclinical data demonstrate that DCC regulates beta-catenin levels. Here, we sought to determine the association of DCC with beta-catenin protein levels, clinicopathological parameters and patient outcome in ovarian cancer using a method of in situ compartmentalized protein analysis. METHODS: A tissue array composed of 150 advanced-stage ovarian cancers, treated with surgical debulking and platinum-paclitaxel (Taxol) combination chemotherapy, was constructed. For evaluation of protein expression, we used an immunofluorescence-based method of automated in situ quantitative measurement of protein analysis (AQUA). RESULTS: One hundred and twelve patients (74%) had sufficient tissue for AQUA. The median follow-up time for the entire cohort was 33 months. Patients with low nuclear DCC expression had a 3-year progression-free survival (PFS) rate of 0% compared with 33% of those with high DCC expression (P = 0.0067). In multivariate analysis, low nuclear DCC expression level retained its prognostic significance for PFS. Between DCC and beta-catenin, a significant relationship was found, where tumors with low DCC had low beta-catenin and vice versa (P = 0.003). CONCLUSIONS: Low nuclear DCC levels predict for poor patient outcome in epithelial ovarian cancer. DCC may exert its antitumor function, in part, through regulation of beta-catenin levels.

Role of the neurogranin concentrated in spines in the induction of long-term potentiation.

Synaptic plasticity in CA1 hippocampal neurons depends on Ca2+ elevation and the resulting activation of calmodulin-dependent enzymes. Induction of long-term depression (LTD) depends on calcineurin, whereas long-term potentiation (LTP) depends on Ca2+/calmodulin-dependent protein kinase II (CaMKII). The concentration of calmodulin in neurons is considerably less than the total concentration of the apocalmodulin-binding proteins neurogranin and GAP-43, resulting in a low level of free calmodulin in the resting state. Neurogranin is highly concentrated in dendritic spines. To elucidate the role of neurogranin in synaptic plasticity, we constructed a computational model with emphasis on the interaction of calmodulin with neurogranin, calcineurin, and CaMKII. The model shows how the Ca2+ transients that occur during LTD or LTP induction affect calmodulin and how the resulting activation of calcineurin and CaMKII affects AMPA receptor-mediated transmission. In the model, knockout of neurogranin strongly diminishes the LTP induced by a single 100 Hz, 1 s tetanus and slightly enhances LTD, in accord with experimental data. Our simulations show that exchange of calmodulin between a spine and its parent dendrite is limited. Therefore, inducing LTP with a short tetanus requires calmodulin stored in spines in the form of rapidly dissociating calmodulin-neurogranin complexes.

beta-Catenin functions mainly as an adhesion molecule in patients with squamous cell cancer of the head and neck.

BACKGROUND: beta-catenin, depending on subcellular localization, plays a dual role in carcinogenesis: as a signaling factor (in the nucleus) and as an adhesion molecule (in cell membrane). In this study, we sought to determine the role of beta-catenin in head and neck carcinogenesis. METHODS: First, we studied the incidence of mutations of beta-catenin in a cohort of 60 head and neck squamous cell cancers (HNSCC). We subsequently evaluated the protein expression levels of beta-catenin in a cohort of oropharyngeal squamous cell cancer tissue microarray using a novel in situ method of quantitative protein analysis and correlated those with cyclin D1 levels and clinical and pathologic data. RESULTS: The mean follow-up time for survivors was 45 months and for all patients was 35 months. We found no mutations in the cohort of 60 HNSCC. beta-catenin displayed primarily membranous expression pattern. Patients with high tumor-node-metastasis stage were more likely to have high expression of beta-catenin (P = 0.040). Patients with low beta-catenin expression had a local recurrence rate of 79% compared with 29% for patients with high beta-catenin tumors (P = 0.0021). Univariate Cox regression revealed a hazard ratio for low beta-catenin tumors of 3.6 (P = 0.004). Kaplan-Meier analysis showed that patients with low beta-catenin expressing tumors trended toward worse 5-year disease-free survival (P = 0.06). In multivariate analysis, only beta-catenin expression status was an independent prognostic factor (P = 0.044) for local recurrence. Tumors with high beta-catenin had low cyclin D1 and vice versa (P = 0.007). CONCLUSIONS: The absence of activating beta-catenin mutations combined with the inverse correlation between beta-catenin levels with cyclin D1 levels and outcome suggest that beta-catenin mainly functions as an adhesion and not signaling molecule in HNSCC.

Henoch-Schönlein purpura following hepatitis B vaccination.

The use of recombinant hepatitis B vaccines has led to the effective prevention of hepatitis B infection and its chronic sequelae in immunocompetent individuals. Whilst rare, a variety of serious adverse effects have been reported following vaccination including cutaneous vasculitis in eight previous cases. We describe a case of Henoch-Schönlein purpura developing after hepatitis B vaccination.