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The goal of this analysis was to characterize the survival impact of angiogenesis in the patients with high-risk breast cancer, particularly the predictive impact on benefit from dose intensification of adjuvant chemotherapy. Formalin-fixed tissue sample of 152 patients treated as part of the WSG AM-01 trial by either high-dose or conventional dose-dense chemotherapy were analyzed. Angiogenic activity was measured using microvessel count and vascular surface area (VSA) determined by the expression of vascular markers CD31 (n = 128) and CD105/endoglin (n = 130). Protein molecular breast cancer subclasses were analyzed by k-means clustering (k = 5). The univariate impact of factors on event-free (EFS) and overall survival (OS) was tested by log-rank statistics and quantified by univariate Cox analysis. Multivariate survival analysis included factors significant in univariate analysis, as well as interactions was performed for EFS. Both VSA/CD31 (P = 0.004) and VSA/CD105 (P = 0.003) were significantly higher among cases with increased Ki-67. A significant association with molecular subtypes was also found for VSA/CD105: in patients with basal-like/Her-2 subtypes, mean was 1.72 versus 1.24 in patients with other subtypes (P < 0.001). Elevated VSA/CD105 was associated with both significantly decreased EFS (P = 0.01) and OS (P = 0.02). Increased tumor size and positive Her-2 status were also prognostic for poorer EFS. The benefit of dose intensification for EFS was seen in those low-VSA/CD105 patients. The result was evident both in univariate and in multivariate survival analysis including all factors that were significant at the univariate level. Expression of angiogenesis markers may mirror or confer resistance to chemotherapy in the patients with breast cancer, particularly within the context of dose intensified chemotherapy. Highly angiogenic tumors may not derive sufficient benefit from dose intensification of chemotherapy alone. Our findings may serve as a rationale for further exploring anti-angiogenic treatment options in the patients with such highly angiogenic tumor subtypes.

Hypoxia is an important condition in the tumor cell microenvironment and approximately 1% to 1.5% of the genome is transcriptionally responsive to hypoxia with hypoxia-inducible factor-1 (HIF-1) as a major mediator of transcriptional activation. Tumor hypoxia is associated with a more aggressive phenotype of many cancers in adults, but data on pediatric tumors are scarce. Because, by immunohistochemistry, HIF-1alpha expression was readily detectable in 18 of 28 primary Ewing's sarcoma family tumors (ESFT), a group of highly malignant bone-associated tumors in children and young adults, we studied the effect of hypoxia on ESFT cell lines in vitro. Intriguingly, we found that EWS-FLI1 protein expression, which characterizes ESFT, is upregulated by hypoxia in a HIF-1alpha-dependent manner. Hypoxia modulated the EWS-FLI1 transcriptional signature relative to normoxic conditions. Both synergistic as well as antagonistic transcriptional effects of EWS-FLI1 and of hypoxia were observed. Consistent with alterations in the expression of metastasis-related genes, hypoxia stimulated the invasiveness and soft agar colony formation of ESFT cells in vitro. Our data represent the first transcriptome analysis of hypoxic ESFT cells and identify hypoxia as an important microenvironmental factor modulating EWS-FLI1 expression and target gene activity with far-reaching consequences for the malignant properties of ESFT., ((c)2010 AACR.)

PURPOSE EWS-ETS fusion genes are the driving force in Ewing's sarcoma pathogenesis. Because of the variable breakpoint locations in the involved genes, there is heterogeneity in fusion RNA and protein architecture. Since previous retrospective studies suggested prognostic differences among patients expressing different EWS-FLI1 fusion types, the impact of fusion RNA architecture on disease progression and relapse was studied prospectively within the Euro-E.W.I.N.G. 99 clinical trial. PATIENTS AND METHODS Among 1,957 patients who registered before January 1, 2007, 703 primary tumors were accessible for the molecular biology study. Fusion type was assessed by polymerase chain reaction on frozen (n = 578) or paraffin-embedded materials (n = 125). The primary end point was the time to disease progression or relapse. Results After exclusion of noninformative patients, 565 patients were entered into the prognostic factor analysis comparing type 1 (n = 296), type 2 (n = 133), nontype 1/nontype 2 EWS-FLI1 (n = 91) and EWS-ERG fusions (n = 45). Median follow-up time was 4.5 years. The distribution of sex, age, tumor volume, tumor site, disease extension, or histologic response did not differ between the four fusion type groups. We did not observe any significant prognostic value of the fusion type on the risk of progression or relapse. The only slight difference was that the risk of progression or relapse associated with nontype 1/nontype 2 EWS-FLI1 fusions was 1.38 (95% CI, 0.96 to 2.0) times higher than risk associated with other fusion types, but it was not significant (P = .10). CONCLUSION In contrast to retrospective studies, the prospective evaluation did not confirm a prognostic benefit for type 1 EWS-FLI1 fusions.

Despite tremendous effort and progress in the diagnostics of pancreatic cancer with respect to imaging techniques and molecular genetics, only very few patients can be cured by surgery leading to a 5-year survival rate of only 3%. Especially the lack of chemotherapeutical options in this entity requires a better understanding of the molecular mechanisms leading to pancreatic carcinoma growth and progression in order to develop novel treatment regimens. To identify signaling pathways that are critical for this tumor entity, we compared six well-established pancreatic cancer cell lines (Capan-1, Capan-2, HUP-T3, HUP-T4, KCL-MOH, PaTu-8903) with colon cancer cell lines and tumor cell lines of non-epithelial origin by expression profiling. For this purpose we employed Human Genome Focus Arrays representing about 8500 well annotated human genes. We identified 353 genes with significantly high expression in the group of pancreatic carcinomas. Based on Gene Ontology annotations these genes are especially involved in Rho protein signal transduction, proteasome activator activity, cell motility, apoptotic program, and cell-cell adhesion processes indicating these pathways to be interesting candidates for the design of targeted therapies. Most pancreatic carcinomas are characterized by mutations in the TP53 and the KRAS genes and the absence of microsatellite instability, which could also be confirmed for our panel of pancreatic carcinoma cell lines. Looking for individual differences within this group that may be responsible for more or less aggressive behavior, we identified genomic amplifications at the 8q22.1 and the 8q24.22 loci to be associated with enhanced gene transcription. Because we have previously shown that gains of genomic material from the long arm of chromosome 8 have an adverse effect on the outcome of pancreatic carcinoma patients, we conclude that functional analysis of amplified genes at 8q22 and/or 8q24 may lead to an improved understanding of pancreatic carcinoma progression.

Purpose: To characterize the prognostic and predictive impact of protein expression profiles in high-risk breast cancer patients who had previously been shown to benefit from high-dose chemotherapy (HDCT) in comparison to dose-dense chemotherapy (DDCT)., Experimental Design: The expression of 34 protein markers was evaluated using tissue microarrays containing paraffin-embedded breast cancer samples from 236 patients who were randomized to the West German Study Group AM01 trial., Results: (a) 24 protein markers of the initial panel of 34 markers were sufficient to identify five profile clusters (subtypes) by K-means clustering: luminal-A (27%), luminal-B (12%), HER-2 (21%), basal-like (13%) cluster, and a so-called "multiple marker negative" (MMN) cluster (27%) characterized by the absence of specifying markers. (b) After DDCT, HER-2 and basal-like groups had significantly worse event-free survival [EFS; hazard ratio (HR), 3.6 [95% confidence interval (95% CI), 1.65-8.18; P = 0.001] and HR, 3.7 (95% CI, 1.68-8.48; P < 0.0001), respectively] when compared with both luminal groups. (c) After HDCT, the HR was 1.5 (95% CI, 0.76-3.05) for EFS in the HER-2 subgroup and 1.1 (95% CI, 0.37-3.32) in the basal-like subgroup, which indicates a better outcome for patients in the HER-2 and basal-like subgroups who received HDCT. The MMN cluster showed a trend to a better EFS after HDCT compared with DDCT., Conclusions: Protein expression profiling in high-risk breast cancers identified five subtypes, which differed with respect to survival and response to chemotherapy: In contrast to luminal-A and luminal-B subtypes, HER-2 and basal-like subgroups had a significant predictive benefit, and the MMN cluster had a trend to a predictive benefit, both from HDCT when compared with DDCT.

Clear cell sarcoma of soft tissue (CCSST) represents a highly malignant tumor of the musculoskeletal system that is characterized by the chromosomal translocation t(12;22)(q13;q12) of the Ewing sarcoma gene (EWSR1) and activating transcription factor 1 (ATF1). In a former microarray expression study, we identified ERBB3, a member of the epidermal growth factor receptor (EGFR) family, as a promising new diagnostic marker in the differential diagnosis of CCSST. Here we show that, besides ErbB3, all CCSST cell lines (n = 8) also express the ErbB2 receptor or the ErbB4 receptor, representing an adequate coreceptor of ErbB3. The phosphorylation status of ErbB3 revealed these receptor pairs to be either constitutively activated in CCSST cells with high neuregulin-1 (NRG1) expression (n = 4) or activatable by exogenic NRG1 in cells showing low amounts of NRG1 mRNA (n = 4). Exogenous NRG1 stimulated the growth of a subset of CCSST cells but did not affect the kinetics of another subset. This difference was not strictly dependent on endogenous NRG1 expression; however, the growth-inhibiting effect of the pan-ErbB tyrosine kinase inhibitor CI-1033 or PD158780 clearly correlated with NRG1 expression indicating an autocrine growth stimulation loop which may constitute an interesting target of new therapeutic strategies in this tumor entity.

Using Affymetrix oligonucleotide microarrays, we analyzed mRNA gene expression patterns of 12 primary pediatric rhabdomyosarcomas (RMS) and 11 Ewing's sarcomas (EWS), which belong to the small round blue cell tumors (SRBCTs). Diagnostic classification of these cancers is frequently complicated by the highly similar appearance in routine histology, and additional molecular markers could significantly improve tumor classification. A combination of three independent statistical approaches (t-test, SAM, k-nearest neighborhood analysis) resulted in 101 highly significant probe sets that clearly discriminate between EWS and RMS. We identified novel marker transcripts that have not been previously associated with either RMS or EWS yet, including CITED2, glypican 3 (GPC3), and cyclin D1 (CCND1). Expression levels for selected candidate genes were validated by quantitative real-time reverse-transcription PCR. Furthermore, to identify biologically meaningful trends, functional annotations were assigned to 946 genes differentially expressed between EWS and RMS (t-test). Genes involved in protein biosynthesis (n = 28) and complex assembly (n = 9), lipid metabolism (n = 23), energy generation (n = 22), and mRNA processing (n = 11) were expressed significantly higher in EWS. Thus, functional annotation of tumor-specific genes reveals detailed insights into tumor biology and differentiation-specific expression patterns and gives important clues related to the possible cellular origin of these pediatric tumors. Supplementary material for this article is available at the International Journal of Cancer website at http://www.interscience.wiley.com/jpages/0020-7136/suppmat/index.html., (Copyright 2004 Wiley-Liss, Inc.)

We identified patterns of differentially-expressed genes in cell lines derived from several pediatric solid tumors. Affymetrix Human Cancer G110 Arrays, carrying 1,700 cancer-associated genes, were applied to a panel of 11 cell lines originating from Ewing tumors (ETs), neuroblastomas, and malignant melanoma of soft parts. Hierarchical clustering clearly differentiated these 3 entities and revealed groups of 75, 102, and 36 gene probe-sets exhibiting tumor-type specific up-regulation in these cell lines, respectively. Whereas ET lines demonstrated increased expression of microtubule-associated protein tau (MAPT), protein phosphatase 1 regulatory subunit 1A (PPP1R1A), NIMA (never in mitosis gene a)-related kinase 2 (NEK2), and cyclin D1 (CCND1), neuroblastoma samples exhibited high expression of wingless-type mouse mammary tumor virus integration site family member 11 (WNT11), Drosophila frizzled homolog 2 (FZD2), and adenomatous polyposis coli (APC) which are involved in regulating free beta-catenin levels. These genes likely maintain tumor-specific characteristics and participate in key downstream regulatory mechanisms. We also correlated the expression levels of up-regulated genes in ETs with their chromosomal localization and compared these data to the comparative genomic hybridization profiles of the cell lines. We demonstrate that gains of genetic material contribute essentially to differential gene expression.