αv-Integrin isoform expression in primary human tumors and brain metastases.

UNLABELLED: To determine whether metastasis to brain is associated with altered expression patterns of integrins, we investigated the expression of αvß3, αvß5, αvß6 and αvß8 integrins in primary malignancies and metastases to brain of breast, lung and renal carcinomas and in malignant melanoma. Inhibitors of αv integrins are currently in clinical trials for glioblastoma. The role of integrins in the process of brain metastasis from other human tumors is unknown. Immunohistochemistry with novel integrin subtype specific rabbit monoclonal antibodies was performed on tissue microarrays of archival material of surgical biopsies taken from primary tumors and brain metastases. Integrin αvß3 expression was increased in brain metastases compared to primary tumors of breast adenocarcinoma, non-small cell lung cancer, renal clear cell cancer and malignant cutaneous melanoma (all p < 0.01). Similarly, integrin αvß8 expression was increased in brain metastases compared to primary tumors of breast cancer (p < 0.0001), lung cancer (p < 0.01) and renal cancer (p < 0.0001), with a similar trend in metastatic melanoma. Integrin αvß5 was expressed in most primary tumors (98% breast cancer; 67% lung cancer; 90% renal cancer; 89% melanoma) and showed a stronger expression in brain metastases compared to primary tumors from lung cancer and melanoma (p < 0.05). Also integrin αvß6 expression was increased in brain metastases compared to primary breast cancer (p < 0.001). CONCLUSIONS: The stronger αv-integrin expression in brain metastases, especially of αvß3 and αvß8 integrins, suggests that certain αv integrin are involved in the process of brain metastasis. αv Integrins may be therapeutic targets for patients with metastatic cancer in brain.

Rorγt+ innate lymphocytes and γδ T cells initiate psoriasiform plaque formation in mice.

Psoriasis is a common, relapsing inflammatory skin disease characterized by erythematous scaly plaques. Histological manifestations of psoriasis include keratinocyte dysregulation and hyperproliferation, elongated rete ridges, and inflammatory infiltrates consisting of T cells, macrophages, dendritic cells, and neutrophils. Despite the availability of new effective drugs to treat psoriasis, the underlying mechanisms of pathogenesis are still poorly understood. Recent studies have shown that Aldara cream, used to treat benign skin abnormalities, triggers psoriasis-like disease in humans and mice and have implicated Th17 cells in disease initiation. Using this as a model, we found a predominant role for the Th17 signature cytokines IL-17A, IL-17F, and IL-22 in psoriasiform plaque formation in mice. Using gene-targeted mice, we observed that loss of Il17a, Il17f, or Il22 strongly reduced disease the severity of psoriasis. However, we found that Th17 cells were not the primary source of these pathogenic cytokines. Rather, IL-17A, IL-17F, and IL-22 were produced by a skin-invading population of γδ T cells and RORγt(+) innate lymphocytes. Furthermore, our findings establish that RORγt(+) innate lymphocytes and γδ T cells are necessary and sufficient for psoriatic plaque formation in an experimental disease model that closely resembles human psoriatic plaque formation.

Primary oligodendrocyte death does not elicit anti-CNS immunity.

Anti-myelin immunity is commonly thought to drive multiple sclerosis, yet the initial trigger of this autoreactivity remains elusive. One of the proposed factors for initiating this disease is the primary death of oligodendrocytes. To specifically test such oligodendrocyte death as a trigger for anti-CNS immunity, we inducibly killed oligodendrocytes in an in vivo mouse model. Strong microglia-macrophage activation followed oligodendrocyte death, and myelin components in draining lymph nodes made CNS antigens available to lymphocytes. However, even conditions favoring autoimmunity-bystander activation, removal of regulatory T cells, presence of myelin-reactive T cells and application of demyelinating antibodies-did not result in the development of CNS inflammation after oligodendrocyte death. In addition, this lack of reactivity was not mediated by enhanced myelin-specific tolerance. Thus, in contrast with previously reported impairments of oligodendrocyte physiology, diffuse oligodendrocyte death alone or in conjunction with immune activation does not trigger anti-CNS immunity.

Vascular CXCR4 expression - a novel antiangiogenic target in gastric cancer?

BACKGROUND: G-protein-coupled receptors (GPCRs) are prime candidates for novel cancer prevention and treatment strategies. We searched for differentially expressed GPCRs in node positive gastric carcinomas. METHODOLOGY/PRINCIPAL FINDINGS: Differential expression of GPCRs in three node positive vs. three node negative intestinal type gastric carcinomas was analyzed by gene array technology. The candidate genes CXCL12 and its receptor CXCR4 were validated by real-time reverse-transcription polymerase chain reaction in an independent set of 37 gastric carcinomas. Translation was studied by immunohistochemistry in 347 gastric carcinomas using tissue microarrays as well as in 61 matching lymph node metastases. Protein expression was correlated with clinicopathological patient characteristics and survival. 52 GPCRs and GPCR-related genes were up- or down-regulated in node positive gastric cancer, including CXCL12. Differential expression of CXCL12 was confirmed by RT-PCR and correlated with local tumour growth. CXCL12 immunopositivity was negatively associated with distant metastases and tumour grade. Only 17% of gastric carcinomas showed CXCR4 immunopositive tumour cells, which was associated with higher local tumour extent. 29% of gastric carcinomas showed CXCR4 positive tumour microvessels. Vascular CXCR4 expression was significantly associated with higher local tumour extent as well as higher UICC-stages. When expressing both, CXCL12 in tumour cells and CXCR4 in tumour microvessels, these tumours also were highly significantly associated with higher T- and UICC-stages. Three lymph node metastases revealed vascular CXCR4 expression while tumour cells completely lacked CXCR4 in all cases. The expression of CXCL12 and CXCR4 had no impact on patient survival. CONCLUSIONS/SIGNIFICANCE: Our results substantiate the significance of GPCRs on the biology of gastric carcinomas and provide evidence that the CXCL12-CXCR4 pathway might be a novel promising antiangiogenic target for the treatment of gastric carcinomas.

Mining tissue microarray data to uncover combinations of biomarker expression patterns that improve intermediate staging and grading of clear cell renal cell cancer.

PURPOSE: Tumor stage and nuclear grade are the most important prognostic parameters of clear cell renal cell carcinoma (ccRCC). The progression risk of ccRCC remains difficult to predict particularly for tumors with organ-confined stage and intermediate differentiation grade. Elucidating molecular pathways deregulated in ccRCC may point to novel prognostic parameters that facilitate planning of therapeutic approaches. EXPERIMENTAL DESIGN: Using tissue microarrays, expression patterns of 15 different proteins were evaluated in over 800 ccRCC patients to analyze pathways reported to be physiologically controlled by the tumor suppressors von Hippel-Lindau protein and phosphatase and tensin homologue (PTEN). Tumor staging and grading were improved by performing variable selection using Cox regression and a recursive bootstrap elimination scheme. RESULTS: Patients with pT2 and pT3 tumors that were p27 and CAIX positive had a better outcome than those with all remaining marker combinations. A prolonged survival among patients with intermediate grade (grade 2) correlated with both nuclear p27 and cytoplasmic PTEN expression, as well as with inactive, nonphosphorylated ribosomal protein S6. By applying graphical log-linear modeling for over 700 ccRCC for which the molecular parameters were available, only a weak conditional dependence existed between the expression of p27, PTEN, CAIX, and p-S6, suggesting that the dysregulation of several independent pathways are crucial for tumor progression. CONCLUSIONS: The use of recursive bootstrap elimination, as well as graphical log-linear modeling for comprehensive tissue microarray (TMA) data analysis allows the unraveling of complex molecular contexts and may improve predictive evaluations for patients with advanced renal cancer.

The role of vascular CXCR4 expression in colorectal carcinoma.

AIMS: The chemokine receptor CXCR4 plays a prominent role in the biology of many different tumours, promoting angiogenesis and metastasis. The impact of CXCR4 expression on tumour biology has been described in various gastrointestinal malignancies, but data on its in situ expression and clinicopathological correlations are sparse. Using a novel specific rabbit anti-CXCR4 antibody, the aim was to assess CXCR4 expression immunohistochemically on tissue microarrays generated from 402 colorectal cancers (CRCs) and compare it with CXCL12 expression and various clinicopathological parameters. METHODS AND RESULTS: CXCR4-expressing tumour cells were observed in 31% of the cases, and expression correlated only with blood vessel invasion (P = 0.049). Furthermore, CXCR4 was found in tumour microvessels in 25% of CRCs. This pattern of CXCR4 expression correlated significantly with T- (P = 0.008), N- (P = 0.009), M- (P = 0.043), L- (P = 0.014) and V-category (P = 0.043) as well as with International Union Against Cancer (UICC) stage (P = 0.001). Furthermore, in node negative CRCs, vascular CXCR4 expression was an independent adverse prognostic factor [hazard ratio 2.87 (1.31-6.29), P = 0.009]. No correlation with CXCL12 expression was found. CONCLUSIONS: Our data provide evidence that CXCR4 plays an important role in tumour angiogenesis of CRC. Therefore, the CXCR4 pathway is a promising therapeutic target for anti-angiogenic therapies in CRC.

Homogeneous MGMT immunoreactivity correlates with an unmethylated MGMT promoter status in brain metastases of various solid tumors.

The O(6)-methylguanine-methyltransferase (MGMT) promoter methylation status is a predictive parameter for the response of malignant gliomas to alkylating agents such as temozolomide. First clinical reports on treating brain metastases with temozolomide describe varying effects. This may be due to the fact that MGMT promoter methylation of brain metastases has not yet been explored in depth. Therefore, we assessed MGMT promoter methylation of various brain metastases including those derived from lung (n = 91), breast (n = 72) kidney (n = 49) and from malignant melanomas (n = 113) by methylation-specific polymerase chain reaction (MS-PCR) and MGMT immunoreactivity. Fifty-nine of 199 brain metastases (29.6%) revealed a methylated MGMT promoter. The methylation rate was the highest in brain metastases derived from lung carcinomas (46.5%) followed by those from breast carcinoma (28.8%), malignant melanoma (24.7%) and from renal carcinoma (20%). A significant correlation of homogeneous MGMT-immunoreactivity (>95% MGMT positive tumor cells) and an unmethylated MGMT promoter was found. Promoter methylation was detected in 26 of 61 (43%) tumors lacking MGMT immunoreactivity, in 17 of 63 (27%) metastases with heterogeneous MGMT expression, but only in 5 of 54 brain metastases (9%) showing a homogeneous MGMT immunoreactivity. Our results demonstrate that a significant number of brain metastases reveal a methylated MGMT-promoter. Based on an obvious correlation between homogeneous MGMT immunoreactivity and unmethylated MGMT promoter, we hypothesize that immunohistochemistry for MGMT may be a helpful diagnostic tool to identify those tumors that probably will not benefit from the use of alkylating agents. The discrepancy between promoter methylation and a lack of MGMT immunoreactivity argues for assessing MGMT promoter methylation both by immunohistochemical as well as by molecular approaches for diagnostic purposes.

Wnt signalling inhibits neural differentiation of embryonic stem cells by controlling bone morphogenetic protein expression.

Wnt signalling plays an important role in both embryonic development and in tumourigenesis. Activation of the signalling cascade by wnt, but also mutations of the adenomatous polyposis coli (APC) protein and of the phosphorylation domain of beta-catenin, result in accumulation of active beta-catenin in the nucleus, where it binds to TCF/LEF transcription factors. We studied the effect of wnt signalling in embryonic stem cells by either inactivating APC or by introducing a dominant active form of beta-catenin. Both resulted in inhibition of neural differentiation in vitro and after brain grafting and in activation of downstream targets of wnt signalling, such as cyclins, c-myc, and bone morphogenetic proteins (BMP). Neural differentiation could be partially restored by the addition of the BMP antagonist noggin. This suggests a mechanism regulating the fate of differentiating embryonic stem cells.