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Supplementary Table 1 from Protein Expression Profiling in High-Risk Breast Cancer Patients Treated with High-Dose or Conventional Dose–Dense Chemotherapy

Supplementary Figure 1 from Protein Expression Profiling in High-Risk Breast Cancer Patients Treated with High-Dose or Conventional Dose–Dense Chemotherapy

Supplementary Table 1 from Expression Profiling of t(12;22) Positive Clear Cell Sarcoma of Soft Tissue Cell Lines Reveals Characteristic Up-Regulation of Potential New Marker Genes Including ERBB3

Supplementary Table 2 from Expression Profiling of t(12;22) Positive Clear Cell Sarcoma of Soft Tissue Cell Lines Reveals Characteristic Up-Regulation of Potential New Marker Genes Including ERBB3

Clear cell sarcoma of soft tissue (CCSST), also known as malignant melanoma of soft parts, represents a rare lesion of the musculoskeletal system usually affecting adolescents and young adults. CCSST is typified by a chromosomal t(12;22)(q13;q12) translocation resulting in a fusion between the Ewing sarcoma gene (EWSR1) and activating transcription factor 1 (ATF1), of which the activity in nontransformed cells is regulated by cyclic AMP. Our aim was to identify critical differentially expressed genes in CCSST tumor cells in comparison with other solid tumors affecting children and young adults to better understand signaling pathways regulating specific features of the development and progression of this tumor entity. We applied Affymetrix Human Genome U95Av2 oligonucleotide microarrays representing ∼12,000 genes to generate the expression profiles of the CCSST cell lines GG-62, DTC-1, KAO, MST2, MST3, and Su-CC-S1 in comparison with 8 neuroblastoma, 7 Ewing tumor, and 6 osteosarcoma cell lines. Subsequent hierarchical clustering of microarray data clearly separated all four of the tumor types from each other and identified differentially expressed transcripts, which are characteristically up-regulated in CCSST. Statistical analysis revealed a group of 331 probe sets, representing ∼300 significant (P < 0.001) differentially regulated genes, which clearly discriminated between the CCSST and other tumor samples. Besides genes that were already known to be highly expressed in CCSST, like S100A11 (S100 protein) or MITF (microphthalmia-associated transcription factor), this group shows an obvious portion of genes that are involved in cyclic AMP response or regulation, in pigmentation processes, or in neuronal structure and signaling. Comparison with other expression profile analyses on neuroectodermal childhood tumors confirms the high robustness of this strategy to characterize tumor entities based on their gene expression. We found the avian erythroblastic leukemia viral oncogene homologue 3 (ERBB3) to be one of the most dramatically up-regulated genes in CCSST. Quantitative real-time PCR and Northern blot analysis verified the mRNA abundance and confirmed the absence of the inhibitory transcript variant of this gene. The protein product of the member of the epidermal growth factor receptor family ERBB3 could be shown to be highly present in all of the CCSST cell lines investigated, as well as in 18 of 20 primary tumor biopsies. In conclusion, our data demonstrate new aspects of the phenotype and the biological behavior of CCSST and reveal ERBB3 to be a useful diagnostic marker.

Background Ewing's sarcoma (ES) is the second most common bone or soft-tissue sarcoma in childhood and adolescence and features a high propensity to metastasize. The six-transmembrane epithelial antigen of the prostate 1 (STEAP1) is a membrane-bound mesenchymal stem cell marker highly expressed in ES. Here, we investigated the role of STEAP1 as an immunohistological marker for outcome prediction in patients with ES. Patients and methods Membranous STEAP1 immunoreactivity was analyzed using immunohistochemistry in 114 primary pre-chemotherapy ES of patients diagnosed from 1983 to 2010 and compared with clinical parameters and patient outcome. Median follow-up was 3.85 years (range 0.43–17.51). Results A total of 62.3% of the ES samples displayed detectable STEAP1 expression with predominant localization of the protein at the plasma membrane. High membranous STEAP1 immunoreactivity was found in 53.5%, which correlated with better overall survival (P = 0.021). Accordingly, no or low membranous STEAP1 expression was identified as an independent risk factor in multivariate analysis (hazard ratio 2.65, P = 0.036). Conclusion High membranous STEAP1 expression predicts improved outcome and may help to define a specific subgroup of ES patients, who might benefit from adapted therapy regimens.

Identification of factors to detect chemotherapy-resistant tumours at diagnosis is a first priority for risk-adapted therapy in the oncology of children and young adults, where more individualized, effective, and less toxic treatments are highly desirable. In this study, we analysed the miRNAs discriminating Ewing's sarcoma (EWS) patients with different clinical outcomes in order to identify new indicators of prognosis. miRNA expression was investigated in 49 primary EWSs by using the Agilent human miRNA microarray v.2 and/or qRT-PCR. Statistical power of the samples studied for miRNA expression was verified, indicating adequate sample size. Microarray analysis defined a signature of five miRNAs (miR-34a, miR-23a, miR-92a, miR-490-3p, and miR-130b) as an independent predictor of risk for disease progression and survival. Validation analysis in the extended sample set indicated that both miR-34a and miR-490-3p achieved sufficient statistical power to predict prognosis. Results were particularly robust for miR-34a, which appeared associated with either event-free or overall survival and emerged as a significant predictor also after multivariate analysis. Patients with the highest expression of miR-34a did not experience adverse events in 5 years; in contrast, patients with the lowest expression recurred within 2 years. High expression of miR34a can be detected also in paraffin-embedded tissues by in situ hybridization, thus contributing to an easy routine evaluation of this miRNA. Functional analysis of miR-34a in EWS cell lines indicated that when miR-34a expression was enforced, cells were less proliferative, less malignant, and sensitized to doxorubicin and vincristine. Expression of miR-34a could be increased in p53wt cells by treatment with nutlin-3a. Accordingly, nutlin-3a synergizes with doxorubicin. Overall, our data indicate that miR-34a expression is a strong predictor of outcome in EWS. Restoration of miR-34a activity may be useful to decrease malignancy and increase tumour sensitivity to current drugs, so sparing excessive long-term toxicity to EWS patients.

Background. Alveolar Rhabdomyosarcomas (RMA) are characterized by chromosomal translocations, fusing the PAX3 or PAX7 gene with FKHR in about 85%. Previous studies have suggested that the fusion type is associated with prognosis. In order to investigate the predictive value of the PAX-FKHR fusion status on disease outcome of patients with RMA treated in the CWS trials we performed a retrospective analysis. Procedure. Between 1986 and 2004, out of 446 patients with RMA treated in four consecutive CWS trials, tumor samples from 126 patients were available for RT-PCR analysis. Survival depending on fusion status in context with known clinical risk-factors was analyzed. Results. Out of 126 samples, 121 had adequate quality for PAX-FKHR fusion status analysis. PAX-FKHR fusions were detected in 101 samples: 60% PAX3-FKHR and 24% PAX7-FKHR fusions, 17% were fusion-negative. There was no significant difference in survival between patients with PAX3-FKHR versus PAX7-FKHR positive tumors. The fusion transcript negative cohort showed a more favorable outcome than the fusion transcript positive cohort among patients with metastatic disease. From the established clinical risk-factors none was associated with a significantly higher risk of failure or death in a multivariate analysis. Conclusions. PAX-FKHR fusion type was not a significant predictor for survival in our analysis. Moreextensive molecular analyses are needed to identify features with prognostic relevance and useful therapeutic impact. Pediatr Blood Cancer 2011; 57: 406-414. (C) 2011 Wiley-Liss, Inc. (Less)

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.

Usage of cancer cell lines has repeatedly generated conflicting results provoked by differences among subclones or contamination with mycoplasm or other immortal mammalian cells. To overcome these limitations, we decided within the EuroBoNeT consortium to characterize a common set of cell lines including osteosarcomas (OS), Ewing sarcomas (ES), and chondrosarcomas (CS). DNA fingerprinting was used to guarantee the identity of all of the cell lines and to distinguish subclones of osteosarcoma cell line HOS. Screening for homozygous loss of 38 tumor suppressor genes by MLPA revealed deletion of CDKN2A as the most common event (15/36), strictly associated with absence of the CDKN2A (p16) protein. Ten cell lines showed missense mutations of the TP53 gene while another set of nine cell lines showed mutations resulting in truncation of the TP53 protein. Cells harboring missense mutations expressed high levels of nuclear TP53, while cell lines with nonsense mutations showed weak/absent staining for TP53. TP53wt cell lines usually expressed the protein in 2-10% of the cells. However, seven TP53wt osteosarcomas were negative for both mRNA and protein expression. Our analyses shed light on the correlation between immunohistochemical and genetic data for CDKN2A and TP53, and confirm the importance of these signaling pathways. The characterization of a substantial number of cell lines represents an important step to supply research groups with proven models for further advanced studies on tumor biology and may help to make results from different laboratories more comparable.

Peripheral primitive neuroectodermal tumor/Ewing's tumors are rare bone and soft tissue malignancies with a highly aggressive clinical course and early metastases occurring at multiple peripheral sites. Here, we present for the first time a case of a 46-year-old man with a primary peripheral primitive neuroectodermal tumor/Ewing's tumor of the testis. The diagnosis of peripheral primitive neuroectodermal tumor/Ewing's tumor was established by histology, immunohistochemistry, and molecular pathology. The tumor revealed a rapid progress in 2 months' time. Therefore, the patient was included in the EURO-E.W.I.N.G.99 study and was placed on chemotherapy. However, the tumor progressed during ongoing therapy, and the patient died in March 2008. In conclusion, though being reported here for the first time, peripheral primitive neuroectodermal tumor/Ewing's tumors should be considered in the differential diagnosis of blue round cell tumors of the testis. A rapid and correct diagnosis of this entity is crucial for fast and accurate therapy, which is stressed by the fatal case presented here.

Only few clinical factors predict the prognosis of patients with Ewing tumors. Unfavorable outcome is associated with primary metastatic disease, age > 15 years, tumor volume above 200 ml, and the histological response to chemotherapy. The aim of this study was to elucidate the prevalence and clinical impact of microsatellite instability (MSI) together with the relation between MSI and mismatch repair protein expression in Ewing tumors. DNA from 61 primary Ewing tumors and 11 Ewing tumor cell lines was extracted and microsatellite analysis for the detection of instability or loss of heterozygosity was performed for the five markers of the Bethesda panel BAT25, BAT26, D5S346, D2S123, and D17S250, which represents the established marker panel for the analysis of hereditary non-polyposis colorectal carcinoma (HNPCC) patients. In addition, single nucleotide repeat regions of the two tumor genes BAX and transforming growth factor receptor II (TGFBR2) were also included. All of the 61 samples were suitable for LOH analysis and 55 for the determination of MSI-status. LOH of these microsatellite markers was detected in 9 of the 61 patients (14.8%). Over all, genetic instability, i.e. MSI and/or LOH, was detected in 17 tumors (27.9%). One out of the 11 tumor cell lines (STA ET1) was characterized by instability of all the five Bethesda markers, while from primary tumor samples, only one showed MSI in more than one microsatellite marker (D5S346 and D17S250, MSI-high). Eight of the fifty-five patients (14.5%) showed instability of one microsatellite locus (MSI-low). No instability was detected in BAT26, D2S123, BAX and TGFBR2. There was no significant correlation between MSI and loss of expression of mismatch repair proteins MLH1, MSH2, or MSH6. The impairment of the p53 signaling pathway (expression of TP53 and/or MDM2 by immunohistochemistry) was significantly associated with reduced overall survival (15 of 49 patients (30.6%), P = 0.0410, log-rank test). We conclude that MSI is not prevalent in Ewing tumor and that the nature of instability differs from the form observed in colorectal carcinoma, the model tumor of MSI. This is documented by the different pattern of MSI (no BAT26 instability) in Ewing tumors and the lack of a strict correlation between MSI-high and loss of expression of MSH2, MSH6 and MLH1.

The t(11;22) translocation is a diagnostic hallmark of various small round-cell tumors. This study correlates the performance of fluorescence in situ hybridization (FISH) and reverse transcription polymerase chain reaction (RT-PCR) in the detection of this translocation analyzing paraffin-embedded tissue specimens. As negative control samples, 10 cases of normal colon mucosa and 10 cases of colon carcinoma tissue were analyzed by FISH to determine a valid cutoff value for the diagnosis of a t(11;22) translocation. The mean number of false-positive nuclei differed significantly between disomic and polysomic control group cases (P=0.002). Therefore, the cutoff value was determined considering the pitfall polysomy. The analysis group consisted of 20 cases from the University of Düsseldorf and 10 cases from the University of Bonn. These cases were analyzed using PCR (Düsseldorf) and FISH (Bonn) using a single-blinded approach. Twenty-two cases (73.3%) were concordant in both methods. Five cases (16.7%) were discrepant, showing a positive result in FISH whereas PCR was negative. Three cases (10.0%) were analyzed by FISH, and PCR failed for nonoptimized tissue preparation. In conclusion, the detection of t(11;22) translocation is critically dependent on a thoroughly defined cutoff value for FISH and on appropriate tissue preparation for both methods. We recommend FISH as a sensitive screening tool in the detection of t(11;22) followed by subsequent PCR amplification of the specific chimeric transcript.

In all, 85% of Ewing's sarcoma family tumors (ESFT), a neoplasm of unknown histogenesis, express EWS-FLI1 transcription factor gene fusions. To characterize direct target genes avoiding artificial model systems, we cloned genomic DNA from ESFT chromatin precipitating with EWS-FLI1. We now present a comprehensive list of 99 putative transcription factor targets identified, for the first time, by a hypothesis-free approach based on physical interaction. Gene-derived chromatin fragments co-precipitating with EWS-FLI1 were nonrandomly distributed over the human genome and localized predominantly to the upstream region and the first two introns of the genes. At least 20% of putative direct EWS-FLI1 targets were neural genes. One-third of genes recovered showed a significant ESFT-specific expression pattern and were found to be altered upon RNAi-mediated knockdown of EWS-FLI1. Among them, MK-STYX, encoding a MAP kinase phosphatase-like protein, was consistently expressed in ESFT. EWS-FLI1 was found to drive MK-STYX expression by binding to a single ETS binding motif within the first gene intron. MK-STYX serves as precedence for successful recovery of direct EWS-FLI1 targets from the authentic ESFT cellular context, the most relevant system to study oncogenic mechanisms for the discovery of new therapeutic targets in this disease.

Secretory carcinomas (SCA) are distinguished from infiltrating ductal carcinomas (IDC) of the breast by their characteristic histomorphology and more favorable prognosis and by the expression of a chimeric tyrosine kinase that is encoded by the ETV6-NTRK3 fusion gene. On this basis, we evaluated 13 SCAs (12 of them with ETV6-NTRK3 gene fusion) by molecular and immunohistochemical (IHC) methods. DNA was obtained from 8 of 13 microdissected SCAs and was analyzed for genetic alterations (GA) by comparative genomic hybridization (CGH). IHC staining was performed for estrogen receptor (ER), progesterone receptor (PR), HER2/neu, and Ki-67 (MIB1) in all 13 cases. Molecular and immunohistochemical results in SCAs were compared with previous data regarding immunohistochemical and molecular characteristics of IDCs. An average of 2.0 GAs (range: 0 to 6) were detected, including recurrent gains of chromosome 8q (37.5%) and 1q (25%) and losses of 22q (25%). Four of 13 (31%) SCAs were positive for ER, and 2 were positive for PR. The mean MIB1-labeling index was 11.4% (range

Clear cell sarcoma (CCS) is associated with the EWS/ATF1 oncogene that is created by chromosomal fusion of the Ewings Sarcoma oncogene (EWS) and the cellular transcription factor ATF1. The melanocytic character of CCS suggests that the microphthalmia-associated transcription factor (Mitf), a major inducer of melanocytic differentiation, may be miss-expressed in CCS. Accordingly, we show that the mRNA and protein of the melanocyte-specific isoform of Mitf (Mitf-M) are present in several cultured CCS cell lines (Su-ccs-1, DTC1, Kao, MST-1, MST-2 and MST-3). The above cell lines thus provide a valuable experimental resource for examining the role of Mitf-M in both CCS and melanocyte differentiation. Melanocyte-specific expression of Mitf-M is achieved via an ATF-dependent melanocyte-specific cAMP-response element in the Mitf-M promoter, and expression of Mitf-M in CCS cells suggests that EWS/ATF1 (a potent and promiscuous activator of cAMP-inducible promoters) may activate the Mitf-M promoter. Surprisingly, however, the Mitf-M promoter is not activated by EWS/ATF1 in transient assays employing CCS cells, melanocytes or nonmelanocytic cells. Thus, our results indicate that Mitf-M promoter activation may require an appropriate chromosomal context in CCS cells or alternatively that the Mitf-M promoter is not directly activated by EWS/ATF1.

Osteosarcomas are the most frequent bone sarcomas. The molecular chromosomal aberrations in osteosarcomas were analyzed by comparative genomic hybridization (CGH). We studied 47 frozen tumors (41 primary samples, 6 relapses) in osteosarcoma patients registered in the Cooperative Osteosarcoma Study (COSS) protocol. Genomic imbalances were detected in 40 of 41 primary tumors and 6 of 6 relapsed tumors. Gains were more frequent than losses (ratio of 1.3:1). The median number of changes was 16 and 12 in primary and relapsed osteosarcomas, respectively. The median number of aberrations in primary high-grade osteosarcomas (17.0) was significantly higher than in low- or intermediate-grade osteosarcoma subtypes (3.0) (p = 0.038). The most frequent gains included 8q, 1p21-p31 and 1q21-q24, and the most frequent losses were 10q, 5q and 13q. High-level gains were observed on 8q23-q24, 17p13 and 1q21-q24. A gain of 19p (p < 0.001) or loss of 9p (p = 0.027) was more frequent in poor responders than in good responders. Univariate analysis revealed that patients with primary metastases (p = 0.002), poor histologic responses (p = 0.005), high-level gains of 19p (p = 0.012) or losses of 13q14 (p = 0.042) had significantly lower event-free survival (EFS), whereas patients with a loss of 5q (p = 0.007) or a loss of 10q21-22 (p = 0.017) had significantly higher EFS than patients without these aberrations. Multivariate analysis demonstrated that primary metastasis, loss of 13q14 and loss of 5q were independent prognostic factors. The findings of our study seem to be useful for evaluating the prognosis of patients and may finally lead to treatment strategies based on genetic background of osteosarcoma. © 2002 Wiley-Liss, Inc.

Telomerase activity (TA) has been shown to correlate with poor clinical outcome in various tumour entities, indicating that tumours expressing this enzyme may be more aggressive and that TA may be a useful prognostic marker. For breast cancer, however, TA is a controversial prognostic marker; whereas some studies suggest an association between TA and disease outcome, others do not find this association. This study used tissue microarrays (breast carcinoma prognosis arrays) containing 611 samples (each 0.6 mm in diameter) from the tumour centre of paraffin-embedded breast carcinomas to analyse the catalytic subunit of telomerase, human telomerase reverse-transcriptase (hTERT), and the internal RNA component (hTR), which are the core components of the telomerase holoenzyme complex. hTERT protein expression was obtained by immunohistochemistry (human anti-telomerase antibody Ab-2, Calbiochem), and hTR RNA was measured by radioactive in situ hybridization. hTERT and hTR expression were determined semi-quantitatively and graded (scores 1-4). Clinical data, such as histological subtype, pT stage, tumour diameter, pN stage, BRE grade, tumour-specific survival (in months), patient's age and others, were available for statistical analysis. A statistically significant correlation was found between tumour-specific survival (overall survival) and hTERT expression (p < 0.0001) or hTR expression (p = 0.00110). Tumours with higher scores (scores 3, 4) for hTR and/or hTERT were associated with a worse prognosis. In multivariate analysis, hTERT expression was an independent prognostic factor. Previous studies, focusing on analysis of TA in smaller numbers of fresh-frozen breast carcinomas by the TRAP assay, gave controversial results with respect to TA as a prognostic marker. Using tissue microarrays from 611 breast carcinomas, this study has demonstrated that increased expression levels of the telomerase core components, hTERT and hTR, are associated with lower overall survival. These findings suggest that TA should be included in future validation studies as a prognostic marker in breast cancer.

Telomere maintenance is regarded as a key mechanism in overcoming cellular senescence in tumor cells and in most cases is achieved by the activation of telomerase. However there is at least one alternative mechanism of telomere lengthening (ALT) which is characterized by heterogeneous and elongated telomeres in the absence of telomerase activity (TA). We evaluated the prevalence of TA, gene expression of telomerase subunits and ALT in relation to telomere morphology and function in matrix producing bone tumors and in osteosarcoma cell lines and present evidence of a direct association of ALT with telomere dysfunction and chromosomal instability. Telomere fluorescence in situ hybridization (T-FISH) in ALT cells revealed elongated and shortened telomeres, partly in unusual configurations and loci, dicentric marker chromosomes and signal-free chromosome ends. Free ends give rise to end-to-end associations and may induce breakage-fusion-bridge cycles resulting in an increased number of complex chromosomal rearrangements, as detected by multiplex-FISH (M-FISH). We propose that ALT cannot be seen as an equivalent to telomerase activity in telomere maintenance. Its association with telomere dysfunction and chromosomal instability may have major implications for tumor progression.

PURPOSE: We have recently demonstrated that telomerase activity (TA) is an independent prognostic factor in neuroblastomas. In the present study, the prognostic impact of TA and gene expression of the three major telomerase subunits is evaluated by molecular and immunohistochemical techniques in fresh-frozen and paraffin-embedded tissues. PATIENTS AND METHODS: One hundred thirty-three neuroblastomas of all stages were analyzed for TA. The TA levels of 75 neuroblastoma cases were correlated with gene expression of telomerase subunits hTRT, human telomerase RNA (hTR), and telomerase protein 1 (TP1) by quantitative reverse transcriptase polymerase chain reaction (RT-PCR), using an innovative approach on the LightCycler instrument (Roche Diagnostics, Mannheim, Germany). For selected cases, the applicability of RT-PCR and immunohistochemistry for hTRT expression analysis was investigated in paraffin-embedded tissues. TA and subunit expression patterns were correlated with traditional prognostic indicators and disease outcome. RESULTS: TA was present in a total of 39 (29.3%) of 133 neuroblastomas and in 31 (29.8%) of 104 initial neuroblastomas without cytotoxic pretreatment. TA was significantly correlated with both event-free and overall survival (P < .0001). Furthermore, we found a significant correlation between expression levels of TA and hTRT (P < .0001) as well as hTR (P < .001). Multivariate analysis revealed only TA and tumor stage but not serum lactate dehydrogenase, MYCN amplification, or age at diagnosis as independent prognostic factors. CONCLUSION: The significant correlation with clinical outcome strongly recommends that analysis of TA be incorporated into the clinical investigation of each individual neuroblastoma at the time of diagnosis. Because the mere presence or absence of TA without further quantification is sufficient basis for predicting disease outcome, the telomeric repeat amplification protocol assay could be complemented with but not replaced by analysis of hTRT or hTR expression.

Background. As traditional parameters do not ensure completely accurate prognostic grouping in neuroblastoma (NB), new molecular markers are needed for assessing the individual patient’s prognosis more precisely. Procedure, Results, and Conclusions. Based on 133 NB, we show that telomerase activity (TA) is a powerful, independent prognostic marker for all stages and is capable of differentiating between good and poor outcome in putative ‘favorable’ clinical or biological subgroups of NB patients. Analysis of gene and protein expression of telomerase subunits suggests that the presence or absence of TA in NB is strongly correlated with expression levels of both the catalytic subunit hTERT and the internal RNA component (hTR). Med. Pediatr. Oncol. 35: 651‐655, 2000. © 2000 Wiley-Liss, Inc.

Background Chemokine receptor CXCR4, together with its ligand CXCL12, plays critical roles in cancer progression, including growth, metastasis and angiogenesis. Ewing sarcoma is a sarcoma with poor prognosis despite current therapies, particularly for patients with advanced-stage disease. Lungs and bone (marrow), organs of predilection for (primary/metastatic) Ewing sarcoma, represent predominant CXCL12 sources. Methods To gain insight into the role of the CXCR4-CXCL12 axis in Ewing sarcoma, CXCR4, CXCL12 and hypoxia-inducible factor-1α protein expression was studied in therapy-naïve and metastatic tumors by immunohistochemistry. CXCR4 function was assessed in vitro, by flow cytometry and proliferation/ cell viability assays, in the presence of recombinant CXCL12 and/or CXCR4-antagonist AMD3100 or under hypoxic conditions. Results Whereas CXCR4 was predominantly expressed by tumor cells, CXCL12 was observed in both tumor and stromal areas. Survival analysis revealed an (expression level-dependent) negative impact of CXCR4 expression (p in vivo hypoxia-inducible factor-1α expression and culture of cells under hypoxic conditions revealed no role for hypoxia in CXCR4 expression. Conclusions Together, our results imply a crucial role for the CXCR4-CXCL12 axis in auto- and/or paracrine growth stimulation. Integration of CXCR4-targeting strategies into first- and/or second-line treatment regimens may represent a promising treatment option for Ewing sarcoma.

Purpose: Targeted therapies using the anti-EGFR antibodies panitumumab (Pmab) or cetuximab (Cmab) are currently restricted to patients with metastatic colorectal adenocarcinoma whose tumours do not show a mutation in KRAS. However, recent retrospective studies indicated that patients with tumours mutated in codon 13 of KRAS may benefit from treatment with Cmab in contrast to patients with tumours mutated in KRAS codon 12. Methods: To study the functional impact of the subtype of KRAS mutations on the efficiency of EGFR-targeted therapies, we correlated the KRAS mutation status of 15 colorectal carcinoma cell lines with the in vitro sensitivity of these cells to Cmab/Pmab. Mutations in the potential predictive biomarkers BRAF and PIK3CA as well as protein expression of EGFR and PTEN were also determined. Results: Four out of seven KRAS-mutated cell lines were characterised by the p.G13D mutation. Treatment of these cells using Cmab/Pmab induced a significant growth inhibition in contrast to cell lines showing a KRAS mutation at codon 12 or 61. Out of the eight KRAS wild-type cell lines, five were insensitive to Cmab/Pmab. These cell lines were characterised either by BRAF mutation or by absence of EGFR or PTEN protein expression. Conclusions: Since KRAS p.G13D-mutated tumour cells may respond to EGFR-targeted therapy, we suggest including subtype analysis of KRAS mutations in prospective clinical trials. In KRAS wild-type tumour cells, BRAF mutations and loss of EGFR or PTEN expression may lead to resistance to EGFR-targeted therapy and should be considered as additional negative predictive biomarkers. © 2012 Springer-Verlag Berlin Heidelberg.

Identification of factors to detect chemotherapy-resistant tumours at diagnosis is a first priority for risk-adapted therapy in the oncology of children and young adults, where more individualized, effective, and less toxic treatments are highly desirable. In this study, we analysed the miRNAs discriminating Ewing's sarcoma (EWS) patients with different clinical outcomes in order to identify new indicators of prognosis. miRNA expression was investigated in 49 primary EWSs by using the Agilent human miRNA microarray v.2 and/or qRT-PCR. Statistical power of the samples studied for miRNA expression was verified, indicating adequate sample size. Microarray analysis defined a signature of five miRNAs (miR-34a, miR-23a, miR-92a, miR-490-3p, and miR-130b) as an independent predictor of risk for disease progression and survival. Validation analysis in the extended sample set indicated that both miR-34a and miR-490-3p achieved sufficient statistical power to predict prognosis. Results were particularly robust for miR-34a, which appeared associated with either event-free or overall survival and emerged as a significant predictor also after multivariate analysis. Patients with the highest expression of miR-34a did not experience adverse events in 5 years; in contrast, patients with the lowest expression recurred within 2 years. High expression of miR34a can be detected also in paraffin-embedded tissues by in situ hybridization, thus contributing to an easy routine evaluation of this miRNA. Functional analysis of miR-34a in EWS cell lines indicated that when miR-34a expression was enforced, cells were less proliferative, less malignant, and sensitized to doxorubicin and vincristine. Expression of miR-34a could be increased in p53wt cells by treatment with nutlin-3a. Accordingly, nutlin-3a synergizes with doxorubicin. Overall, our data indicate that miR-34a expression is a strong predictor of outcome in EWS. Restoration of miR-34a activity may be useful to decrease malignancy and increase tumour sensitivity to current drugs, so sparing excessive long-term toxicity to EWS patients.

Cancer cell lines represent in vitro models for studying malignancies, general cell biology, drug discovery and more. Whether they can be considered as exact representative models of the parental tumors remains uncertain given the acquisition of additional ex vivo changes of the cells and the lack of tissue architecture and stroma. Previously, within the EuroBoNeT consortium, we characterized a collection of bone sarcoma cell lines on genomic and proteomic level. Here, we address the phenotypical and functional characterization of the unique set of osteosarcoma cell lines (n=19) in vitro and in vivo. For functional analysis of differentiation capacity, cells were stimulated towards osteoblasts, adipocytes and chondrocytes. Furthermore, all cell lines were injected subcutaneously and intramuscularly into nude mice to assay their in vivo tumor formation capacity as well as for phenotypical analysis of the tumors. All formed tumors were further characterized histologically and immunohistochemically. Out of 19 cell lines, 17 (89%) showed adipogenic differentiation, 13/19 (68%) could differentiate towards osteoblasts and in 6/19 (32%) cell lines chondrogenic differentiation was evident. About half of the cell lines (8/19, 42%) produced tumors in vivo after subcutaneous and intramuscular injections. Several cell lines showed invasion into adjacent tissues and one tumor developed several lung metastases. The use of cell lines, especially in cancer research, is of paramount importance. Here, we identify comprehensively characterized osteosarcoma cell lines, which robustly represent clinical osteosarcoma providing researchers useful in vitro and in vivo models to study the genetics and functional characteristics of this highly malignant neoplasm.

EWS-FLI1 is a chromosome translocation-derived chimeric transcription factor that has a central and rate-limiting role in the pathogenesis of Ewing's sarcoma. Although the EWS-FLI1 transcriptomic signature has been extensively characterized on the mRNA level, information on its impact on non-coding RNA expression is lacking. We have performed a genome-wide analysis of microRNAs affected by RNAi-mediated silencing of EWS-FLI1 in Ewing's sarcoma cell lines, and differentially expressed between primary Ewing's sarcoma and mesenchymal progenitor cells. Here, we report on the identification of hsa-mir-145 as the top EWS-FLI1-repressed microRNA. Upon knockdown of EWS-FLI1, hsa-mir-145 expression dramatically increases in all Ewing's sarcoma cell lines tested. Vice versa, ectopic expression of the microRNA in Ewing's sarcoma cell lines strongly reduced EWS-FLI1 protein, whereas transfection of an anti-mir to hsa-mir-145 increased the EWS-FLI1 levels. Reporter gene assays revealed that this modulation of EWS-FLI1 protein was mediated by the microRNA targeting the FLI1 3'-untranslated region. Mutual regulations of EWS-FLI1 and hsa-mir-145 were mirrored by an inverse correlation between their expression levels in four of the Ewing's sarcoma cell lines tested. Consistent with the role of EWS-FLI1 in Ewing's sarcoma growth regulation, forced hsa-mir-145 expression halted Ewing's sarcoma cell line growth. These results identify feedback regulation between EWS-FLI1 and hsa-mir-145 as an important component of the EWS-FLI1-mediated Ewing's sarcomagenesis that may open a new avenue to future microRNA-mediated therapy of this devastating malignant disease.

Identification of patient selection criteria and understanding of the potential mechanisms involved in the development of resistance are crucial for an appropriate and successful design of clinical trials with anti-insulin-like growth factor (IGF)-1R therapies. Few Ewing's sarcomas are highly sensitive to IGF-1R targeting and understanding the reason why, may hold the secret to improve successful treatments. In this paper, we show that a major mechanism of resistance to highly specific inhibitors of IGF-1R, either antibodies or tyrosine kinase inhibitors may involve enhanced insulin receptor (IR)-A homodimer formation and IGF-2 production. Resistant cells are able to switch from IGF-1/IGF-1R to IGF-2/IR-A dependency to maintain sustained activation of AKT and ERK1/2, proliferation, migration and metastasis. These cells also showed higher proliferative response to insulin, in keeping with a switch towards insulin pathways sustaining proliferation and malignancy, rather than metabolism. Our findings demonstrate a role for IR-A in eliciting intrinsic and adaptive resistance to anti-IGF-1R therapies. Thus, we indicate that tumors with low IGF-1R:IR ratio are unlikely to greatly benefit from anti-IGF-1R therapies and that the efficacy of anti-IGF-1R therapies should be evaluated in relationship to the IR-A:IGF-1R ratio in cancer cells. Moreover, we provide evidences supporting IR-A as an important target in sarcoma therapy.

Background Hypoxia regulates gene expression via the transcription factor HIF (Hypoxia-Inducible Factor). Little is known regarding HIF expression and function in primary bone sarcomas. We describe HIF expression and phenotypic effects of hypoxia, hypoglycaemia and HIF in Ewing's sarcoma and osteosarcoma. Methods HIF-1α and HIF-2α immunohistochemistry was performed on a Ewing's tumour tissue array. Ewing's sarcoma and osteosarcoma cell lines were assessed for HIF pathway induction by Western blot, luciferase assay and ELISA. Effects of hypoxia, hypoglycaemia and isoform-specific HIF siRNA were assessed on proliferation, apoptosis and migration. Results 17/56 Ewing's tumours were HIF-1α-positive, 15 HIF-2α-positive and 10 positive for HIF-1α and HIF-2α. Expression of HIF-1α and cleaved caspase 3 localised to necrotic areas. Hypoxia induced HIF-1α and HIF-2α in Ewing's and osteosarcoma cell lines while hypoglycaemia specifically induced HIF-2α in Ewing's. Downstream transcription was HIF-1α-dependent in Ewing's sarcoma, but regulated by both isoforms in osteosarcoma. In both cell types hypoglycaemia reduced cellular proliferation by ≥ 45%, hypoxia increased apoptosis and HIF siRNA modulated hypoxic proliferation and migration. Conclusions Co-localisation of HIF-1α and necrosis in Ewing's sarcoma suggests a role for hypoxia and/or hypoglycaemia in in vivo induction of HIF. In vitro data implicates hypoxia as the primary HIF stimulus in both Ewing's and osteosarcoma, driving effects on proliferation and apoptosis. These results provide a foundation from which to advance understanding of HIF function in the pathobiology of primary bone sarcomas.

Alveolar Rhabdomyosarcomas (RMA) are characterized by chromosomal translocations, fusing the PAX3 or PAX7 gene with FKHR in about 85%. Previous studies have suggested that the fusion type is associated with prognosis. In order to investigate the predictive value of the PAX-FKHR fusion status on disease outcome of patients with RMA treated in the CWS trials we performed a retrospective analysis.Between 1986 and 2004, out of 446 patients with RMA treated in four consecutive CWS trials, tumor samples from 126 patients were available for RT-PCR analysis. Survival depending on fusion status in context with known clinical risk-factors was analyzed.Out of 126 samples, 121 had adequate quality for PAX-FKHR fusion status analysis. PAX-FKHR fusions were detected in 101 samples: 60% PAX3-FKHR and 24% PAX7-FKHR fusions, 17% were fusion-negative. There was no significant difference in survival between patients with PAX3-FKHR versus PAX7-FKHR positive tumors. The fusion transcript negative cohort showed a more favorable outcome than the fusion transcript positive cohort among patients with metastatic disease. From the established clinical risk-factors none was associated with a significantly higher risk of failure or death in a multivariate analysis.PAX-FKHR fusion type was not a significant predictor for survival in our analysis. More extensive molecular analyses are needed to identify features with prognostic relevance and useful therapeutic impact.

Osteosarcomas are the most common primary malignant tumor of bone, and almost all conventional osteosarcomas are high-grade tumors with complex karyotypes. We have examined DNA copy number changes in 36 osteosarcoma tumors and 20 cell lines using microarray-based comparative genomic hybridization. The most frequent minimal recurrent regions of gain identified in the tumor samples were in 1q21.2-q21.3 (78% of the samples), 1q21.3-q22 (78%), and 8q22.1 (72%). Minimal recurrent regions in 10q22.1-q22.2 (81%), 6q16.1 (67%), 13q14.2 (67%), and 13q21.1 (67%) were most frequently lost. A small region in 3q13.31 (2.1 Mb) containing the gene limbic system-associated membrane protein (LSAMP) was frequently deleted (56%). LSAMP has previously been reported to be a candidate tumor suppressor gene in other cancer types. The deletion was validated using fluorescence in situ hybridization, and the expression level and promoter methylation status of LSAMP were investigated using quantitative real-time reverse transcription PCR and methylation-specific PCR, respectively. LSAMP showed low expression compared to two normal bone samples in 6/15 tumors and 5/9 cell lines with deletion of 3q13.31, and also in 5/14 tumors and 3/11 cell lines with normal copy number or gain. Partial or full methylation of the investigated CpG island was identified in 3/30 tumors and 7/20 cell lines. Statistical analyses revealed that loss of 11p15.4-p15.3 and low expression of LSAMP (both P = 0.011) were significantly associated with poor survival. Our results show that LSAMP is a novel candidate tumor suppressor gene in osteosarcomas.

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License.-- et al., [BACKGROUND]: Ewing sarcoma is a paradigm of solid tumour -bearing chromosomal translocations resulting in fusion proteins that act as deregulated transcription factors. Ewing sarcoma translocations fuse the EWS gene with an ETS transcription factor, mainly FLI1. Most of the EWS-FLI1 target genes still remain unknown and many have been identified in heterologous model systems. [METHODS]: We have developed a stable RNA interference model knocking down EWS-FLI1 in the Ewing sarcoma cell line TC71. Gene expression analyses were performed to study the effect of RNA interference on the genetic signature of EWS-FLI1 and to identify genes that could contribute to tumourigenesis. [RESULTS]: EWS-FLI1 inhibition induced apoptosis, reduced cell migratory and tumourigenic capacities, and caused reduction in tumour growth. IGF-1 was downregulated and the IGF-1/IGF-1R signalling pathway was impaired. PBK/TOPK (T-LAK cell-originated protein kinase) expression was decreased because of EWS-FLI1 inhibition. We showed that TOPK is a new target gene of EWS-FLI1. TOPK inhibition prompted a decrease in the proliferation rate and a dramatic change in the cell's ability to grow in coalescence. [CONCLUSION]: This is the first report of TOPK activity in Ewing sarcoma and suggests a significant role of this MAPKK-like protein kinase in the Ewing sarcoma biology. © 2009 Cancer Research., CIC-IBMCC and HHU belong to NoE Eurobonet, FP6-2004-Lifescihealth-5, proposal number 018814, European Commission. Work at CIC is also funded by Instituto de Salud Carlos III, Spanish Ministry of Science and Innovation-FEDER (PI052524; RD06/0020/0059, CD6/ 00001). Herrero-Martín was a recipient of a pre-doctoral fellowship from the Departamento de Educación, Junta de Castilla y León, Spain. This work has been done within the Acción Transversal en Cáncer program and the cooperative agreement between ISCIII and FICUS.

Ewing sarcoma (EWS) is a tumour most commonly arising in bone, although on occasion in soft tissue, with a poor prognosis in patients with refractory or relapsed disease, despite multimodal therapy. Immunotherapeutic strategies based on tumour-reactive T and/or natural killer cells may improve the treatment of advanced-stage EWS. Since cellular immune recognition critically depends on human leukocyte antigen (HLA) expression, knowledge about HLA expression in EWS is crucial in the design of cellular immunotherapeutic strategies. Constitutive and IFNgamma-induced HLA class I expression was analysed in EWS cell lines (n = 6) by flow cytometry, using antibodies against both monomorphic and allele-specific antigens. Expression of antigen processing pathway components and beta-2 microglobulin (beta2m) was assessed by western blot. Expression of class II transactivator (CIITA), and its contribution to HLA class II expression, was evaluated by qRT-PCR, transduction assays, and flow cytometry. beta2m/HLA class I and class II expression was validated in EWS tumours (n = 67) by immunofluorescence. Complete or partial absence of HLA class I expression was observed in 79% of EWS tumours. Lung metastases consistently lacked HLA class I and sequential tumours demonstrated a tendency towards decreased expression upon disease progression. Together with absent or low constitutive expression levels of specific HLA class I loci and alleles, and differential induction of identical alleles by IFNgamma in different cell lines, these results may reflect the existence of an immune escape mechanism. Inducible expression of TAP-1/-2, tapasin, LMP-2/-7, and the beta2m/HLA class I complex by IFNgamma suggests that regulatory mechanisms are mainly responsible for heterogeneity in constitutive class I expression. EWSs lack IFNgamma-inducible HLA class II, due to lack of functional CIITA. The majority of EWS tumours, particularly if advanced-stage, exhibit complete or partial absence of both classes of HLA. This knowledge will be instrumental in the design of cellular immunotherapeutic strategies for advanced-stage EWS.

Although p53 is the most frequently mutated gene in cancer, half of human tumors retain wild-type p53, whereby it is unknown whether normal p53 function is compromised by other cancer-associated alterations. One example is Ewing's sarcoma family tumors (ESFT), where 90% express wild-type p53. ESFT are characterized by EWS-FLI1 oncogene fusions. Studying 6 ESFT cell lines, silencing of EWS-FLI1 in a wild-type p53 context resulted in increased p53 and p21WAF1/CIP1 levels, causing cell cycle arrest. Using a candidate gene approach, HEY1 was linked to p53 induction. HEY1 was rarely expressed in 59 primary tumors, but consistently induced upon EWS-FLI1 knockdown in ESFT cell lines. The NOTCH signaling pathway targets HEY1, and we show NOTCH2 and NOTCH3 to be expressed in ESFT primary tumors and cell lines. Upon EWS-FLI1 silencing, NOTCH3 processing accompanied by nuclear translocation of the activated intracellular domain was observed in all but one p53-mutant cell line. In cell lines with the highest HEY1 induction, NOTCH3 activation was the consequence of JAG1 transcriptional induction. JAG1 modulation by specific siRNA, NOTCH-processing inhibition by either GSI or ectopic NUMB1, and siRNA-mediated HEY1 knockdown all inhibited p53 and p21WAF1/CIP1 induction. Conversely, forced expression of JAG1, activated NOTCH3, or HEY1 induced p53 and p21WAF1/CIP1. These results indicate that suppression of EWS-FLI1 reactivates NOTCH signaling in ESFT cells, resulting in p53-dependent cell cycle arrest. Our data link EWS-FLI1 to the NOTCH and p53 pathways and provide a plausible basis both for NOTCH tumor suppressor effects and oncogenesis of cancers that retain wild-type p53. [Cancer Res 2008;68(17):7100–9]

In Ewing's sarcoma family of tumours (ESFT), the clinically most adverse prognostic parameters are the presence of tumour metastasis at time of diagnosis and poor response to neoadjuvant chemotherapy. To identify genes differentially regulated between metastatic and localised tumours, we analysed 27 ESFT specimens using Affymetrix microarrays. Functional annotation of differentially regulated genes revealed 29 over-represented pathways including PDGF, TP53, NOTCH, and WNT1-signalling. Regression of primary tumours (n = 20) induced by polychemotherapy was found to be correlated with the expression of genes involved in angiogenesis, apoptosis, ubiquitin proteasome pathway, and PI3 kinase and p53 pathways. These findings could be confirmed by in vitro cytotoxicity assays. A set of 46 marker genes correctly classifies these 20 tumours as responding versus non-responding. We conclude that expression signatures of initial tumour biopsies can help to identify ESFT patients at high risk to develop tumour metastasis or to suffer from a therapy refractory cancer., This work was supported by grants from the ‘Forschungskommission der Medizinischen Fakultaet Duesseldorf’, and the ‘Madeleine Schickedanz-KinderKrebs-Stiftung’. The Departments of Pathology (Heinrich-Heine-University, Duesseldorf, Germany) and the Laboratory of Molecular Pathology (Universidad de Salamanca-CSIC, Spain) are partners of the EuroBoNeT consortium, a European Commission granted Network of Excellence for studying the pathology and genetics of bone tumours.

Renal cell carcinoma (RCC) occurring in renal allografts after cadaveric kidney transplantation has rarely been observed. RCC accounts for 2.3% of all malignancies in the general population, but up to 4.8% of malignancies in renal transplant recipients. Most have been reported in the patient's own diseased kidneys, whereas RCC in the renal allograft occur in only 10%. Here, we describe an organ-preserving surgical technique of a malignant renal tumor in a kidney allograft using a harmonic scalpel (Ultracision) for tumor enucleation. Furthermore we demonstrate by DNA microsatellite analysis the tumor's genetic origin as donor related. Collectively, we suggest that patients with a well defined low grade RCC in the kidney allograft and altogether low malignancy and good allograft function should only undergo an organ-preserving procedure and short-term postoperative screening.

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.

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.

High CD99 expression levels and rearrangements of the EWS gene with ETS transcription factor genes characterize the Ewing's sarcoma family of tumors (ESFT). CD99 is a cell surface glycoprotein whose engagement has been implicated in cell proliferation as well as upregulation and transport of several transmembrane proteins in hematopoietic cells. In ESFT, antibody ligation of CD99 induces fast homotypic cell aggregation and cell death although its functional role in these processes remains largely unknown. Here, using an RNAi approach, we studied for the first time the consequences of modulated CD99 expression in six different ESFT cell lines, representing the most frequent variant forms of EWS gene rearrangement. CD99 suppression resulted in growth inhibition and reduced migration of ESFT cells. Among genes whose expression changes in response to CD99 modulation, the potassium-channel modulatory factor KCMF1 was consistently upregulated. In a series of 22 primary ESFT, KCMF1 expression levels inversely correlated with CD99 abundancy. Cells forced to express ectopic KCMF1 showed a similar reduction in migratory ability as CD99 silenced ESFT cells. Our results suggest that in ESFT, high CD99 expression levels contribute to the malignant properties of ESFT by promoting growth and migration of tumor cells and identify KCMF1 as a potential metastasis suppressor gene downregulated by high constitutive CD99 expression in ESFT.

Since most tumours escape replicative senescence by re-activation of the enzyme telomerase, telomerase is a promising target in the treatment of cancer and a promising marker for diagnosis and therapeutic response. We evaluated the effects of doxorubicin, one of the most active drugs in the treatment of Ewing's sarcoma, on telomerase in the human Ewing's sarcoma cell line STA-ET-1 in vitro and in STA-ET-1 xenografts in vivo. Telomerase activity (TA) was examined by TRAP-assay and real-time PCR. Real-time PCR was also used to quantify the mRNA expression of the catalytic subunit of telomerase (hTERT). In vitro growth inhibition was determined by the MTT-assay. Tumour xenografts were analyzed for tumour volume, apoptosis, necrosis, and proliferation. Doxorubicin concentrations that inhibited in vitro growth of STA-ET-1 by 50% compared to untreated controls ranged between 0.14 microM after 24 h and 0.01 microM after 72 h. Compared to untreated controls doxorubicin reduced TA in STA-ET-1 at toxic concentrations, but increased TA at non-toxic concentrations. In comparison with untreated xenografts, TA was reduced to 65% and hTERT expression dropped to 25% within 72 h in xenografts treated with 17.5 mg/kg doxorubicin i.p.; both recovered to initial values after 264 h. The rate of proliferating cells dropped to 70% within 96 h and increased thereafter. The highest rates of necrosis and apoptosis were seen after 96 h. hTERT expression co-varied significantly with proliferation but not with TA, apoptosis, and necrosis. No correlation was observed between TA, proliferation, apoptosis and necrosis. The results suggest doxorubicin induces down-regulation of hTERT gene expression that at least in part modulates TA in these tumours.

The proto-oncogene c-KIT (CD117) is highly expressed in normal breast epithelium and is decreased in invasive breast cancer. In this study, we analyzed the protein expression and the mutational status of c-KIT in ductal carcinoma in situ (DCIS) of the breast and correlated these findings with nuclear grade, architectural pattern, and expression of HER-2, estrogen receptor (ER)-alpha, and progesterone receptor (PR). C-KIT, HER-2, ER, and PR expression were analyzed immunohistochemically in 106 cases of paraffin-embedded DCIS (85 pure DCIS and 21 DCIS with concurrent carcinoma). Direct sequencing of exons 9 and 11 of the c-KIT gene was performed to analyze the hot spot mutational regions in representative cases. C-KIT expression was found in 55 (52.8%) of all DCIS, correlating with high nuclear grade (P < .0001), comedonecrosis (P < .0001), and solid growth pattern (P = .001). Furthermore, c-KIT expression was strongly associated with HER-2 positivity (P < .0001) and was significantly lower in ER- or PR-positive cases (P = .001 and P = .006, respectively). C-KIT expression alone or co-expression with HER-2 in pure DCIS did not differ significantly from DCIS with invasive component (P = .09). Mutational analysis in 6 c-KIT-positive DCIS revealed no activating mutations in exons 9 or 11. Our findings suggest that the expression of c-KIT protein might define a subset of poorly differentiated, HER-2-positive DCIS with decreased expression of steroid hormone receptors, comedonecrosis, and a solid growth pattern. The implications of c-KIT and HER-2 co-expression for breast carcinogenesis should be further evaluated.

Purpose Peripheral primitive neuroectodermal tumours (PNETs) are rare and highly aggressive soft-tissue malignancies originating from the neural crest. As yet, a gastric PNET has not been reported. Here, we present the case of a 14-year-old boy in whom we detected a large polypoid submucosal gastric tumour as a cause of severe anaemia and weight loss. Methods Histology, immunocytochemistry and molecular pathology were the basis to establish the diagnosis of PNET. Gastroendosonography, computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET) and somatostatin receptor scintigraphy were performed for staging. Results In biopsy samples, small round blue tumour cells were detected clearly expressing CD99, CD117 (c-kit), S100, neurofilament, and neuron-specific enolase. The diagnosis of a PNET was confirmed by detection of the characteristic EWS/FLI-1 fusion gene, resulting in a reciprocal translocation t(11;22)(q24;q12). Three distinct liver metastases were detected by CT, MRI, and PET. The tumour failed to respond to neoadjuvant polychemotherapy with vincristine, etoposide, doxorubicin, and ifosfamide. Subtotal gastrectomy was performed and, surprisingly, we found diffuse metastatic infiltration of the liver that had not been detected by preoperative staging. Due to the diffuse metastatic disease the young patient's prognosis has to be considered very poor. Because of the tumour's intense expression of CD117 (c-kit), the patient is now treated with the tyrosine kinase inhibitor imatinib (STI571). Conclusion Although PNETs are rare malignancies, they should be considered in the differential diagnosis of submucosal gastric tumours in adolescents with clinical alarm symptoms.

Clear cell sarcoma of soft tissue (CCSST), also known as malignant melanoma of soft parts, represents a rare lesion of the musculoskeletal system usually affecting adolescents and young adults. CCSST is typified by a chromosomal t(12;22)(q13;q12) translocation resulting in a fusion between the Ewing sarcoma gene (EWSR1) and activating transcription factor 1 (ATF1), of which the activity in nontransformed cells is regulated by cyclic AMP. Our aim was to identify critical differentially expressed genes in CCSST tumor cells in comparison with other solid tumors affecting children and young adults to better understand signaling pathways regulating specific features of the development and progression of this tumor entity. We applied Affymetrix Human Genome U95Av2 oligonucleotide microarrays representing ∼12,000 genes to generate the expression profiles of the CCSST cell lines GG-62, DTC-1, KAO, MST2, MST3, and Su-CC-S1 in comparison with 8 neuroblastoma, 7 Ewing tumor, and 6 osteosarcoma cell lines. Subsequent hierarchical clustering of microarray data clearly separated all four of the tumor types from each other and identified differentially expressed transcripts, which are characteristically up-regulated in CCSST. Statistical analysis revealed a group of 331 probe sets, representing ∼300 significant (P < 0.001) differentially regulated genes, which clearly discriminated between the CCSST and other tumor samples. Besides genes that were already known to be highly expressed in CCSST, like S100A11 (S100 protein) or MITF (microphthalmia-associated transcription factor), this group shows an obvious portion of genes that are involved in cyclic AMP response or regulation, in pigmentation processes, or in neuronal structure and signaling. Comparison with other expression profile analyses on neuroectodermal childhood tumors confirms the high robustness of this strategy to characterize tumor entities based on their gene expression. We found the avian erythroblastic leukemia viral oncogene homologue 3 (ERBB3) to be one of the most dramatically up-regulated genes in CCSST. Quantitative real-time PCR and Northern blot analysis verified the mRNA abundance and confirmed the absence of the inhibitory transcript variant of this gene. The protein product of the member of the epidermal growth factor receptor family ERBB3 could be shown to be highly present in all of the CCSST cell lines investigated, as well as in 18 of 20 primary tumor biopsies. In conclusion, our data demonstrate new aspects of the phenotype and the biological behavior of CCSST and reveal ERBB3 to be a useful diagnostic marker.

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.

Telomerase activity (TA) has been shown to correlate with poor clinical outcome in various tumour entities, indicating that tumours expressing this enzyme may be more aggressive and that TA may be a useful prognostic marker. For breast cancer, however, TA is a controversial prognostic marker; whereas some studies suggest an association between TA and disease outcome, others do not find this association. This study used tissue microarrays (breast carcinoma prognosis arrays) containing 611 samples (each 0.6 mm in diameter) from the tumour centre of paraffin-embedded breast carcinomas to analyse the catalytic subunit of telomerase, human telomerase reverse-transcriptase (hTERT), and the internal RNA component (hTR), which are the core components of the telomerase holoenzyme complex. hTERT protein expression was obtained by immunohistochemistry (human anti-telomerase antibody Ab-2, Calbiochem), and hTR RNA was measured by radioactive in situ hybridization. hTERT and hTR expression were determined semi-quantitatively and graded (scores 1-4). Clinical data, such as histological subtype, pT stage, tumour diameter, pN stage, BRE grade, tumour-specific survival (in months), patient's age and others, were available for statistical analysis. A statistically significant correlation was found between tumour-specific survival (overall survival) and hTERT expression (p0.0001) or hTR expression (p = 0.00110). Tumours with higher scores (scores 3, 4) for hTR and/or hTERT were associated with a worse prognosis. In multivariate analysis, hTERT expression was an independent prognostic factor. Previous studies, focusing on analysis of TA in smaller numbers of fresh-frozen breast carcinomas by the TRAP assay, gave controversial results with respect to TA as a prognostic marker. Using tissue microarrays from 611 breast carcinomas, this study has demonstrated that increased expression levels of the telomerase core components, hTERT and hTR, are associated with lower overall survival. These findings suggest that TA should be included in future validation studies as a prognostic marker in breast cancer.

Background Whereas in sporadic human malignancies mutations of the TP53 tumor-suppressor gene occur in cancers of almost every organ and histologic subtype, patients with an inborn TP53 defect are at high risk to develop, in particular, soft tissue and bone sarcomas, brain tumors, leukaemias, adrenocortical tumors, and breast cancer. To demonstrate the usefulness of microarray technology applied to TP53 sequencing in pediatric tumors, we investigated young patients suffering from tumors typical of the Li-Fraumeni context who were, therefore, candidates for harboring inborn defects in tumor-suppressor genes. Procedure Six individuals were studied, including typical Li-Fraumeni patients as well as patients without any family history of cancer. DNA samples were independently analyzed for TP53 mutations by GeneChip® and standard automated laser fluorescence (ALF®) sequencing technology. Results The tumor and corresponding constitutional DNA samples clearly showed identical mutations which were confirmed by ALF sequencing. All coding exons (exons 2–11) of TP53 were analyzed simultaneously. The entire sequencing procedure and data analysis was carried out within 24 hr. Conclusions The GeneChip®TP53-sequencing assay may be feasible for routine molecular genetic diagnostics in determining the TP53 status of childhood-tumor patients and in enabling a disease management based on the genetic background of the individual. Med. Pediatr. Oncol. 2002;38:247–253. © 2002 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.

Fibroadenoma and phyllodes tumour of the breast are both fibroepithelial tumours. Although progression to epithelial malignancy has been described, the behaviour of most fibroadenomas is benign. Phyllodes tumours, on the other hand, can display locally destructive growth and can even metastasize. A relationship between the two tumours has been suggested in the literature. This study investigated the clonality of both the stroma and the epithelium of these fibroepithelial tumours and attempted to construct a model in which fibroadenoma can progress in both an epithelial and a stromal direction. Fibroadenomas (n=25) and phyllodes tumours (n=12) were selected for analysis. Tissue was microdissected and analysed for clonality using a polymerase chain reaction (PCR)-based assay targeted at an X-linked polymorphic marker, the human androgen receptor gene (HUMARA). Nineteen fibroadenomas and nine phyllodes tumours could be analysed. Normal-appearing epithelium, hyperplastic epithelium, and stroma removed from fibroadenomas were polyclonal. As expected, carcinoma in situ (CIS) removed from four fibroadenomas was monoclonal. Three areas of apparent stromal expansion within fibroadenoma were monoclonal, suggesting stromal progression. Mostly, the stroma of phyllodes tumours was monoclonal and the epithelium polyclonal. In two cases, however, the epithelium seemed to be monoclonal, whereas in three other cases the stromal component was polyclonal. These findings indicate that fibroadenoma can progress in an epithelial direction to CIS and in a stromal direction to phyllodes tumour.

Ewing tumors are characterized by reciprocal translocations involving the EWS gene on 22q12 fused to ETS transcription-factor family members. Little is known about further aberrations contributing to tumor development and progression. Sixty-two frozen tumors with known EWS rearrangements (52 primary tumors, 10 relapses) of ET patients registered in the EICESS protocol were analyzed by comparative genomic hybridization (CGH). The median number of changes in 52 primary and 10 relapsed cases was 2.5 and 5.0 per tumor (P = 0.153). Frequent abnormalities included gains of chromosomes 8, 12, 20, and 1q and losses of 16q and 19q. Neither number nor type of aberration was associated with histology, tumor size, disease stage, tumor localization, or histologic tumor response to chemotherapy. Among the 52 primary tumors, 26 with Type I fusion (EWS exon 7 to FLI1 exon 6) and 26 with other fusion types had a median of 2.0 and 3.0 aberrations per tumor, respectively (P = 0.031). Combinations of gains of chromosomes 8 and 12, gains of chromosome 20, and either gains of 8q or 18q and losses of 16q and 17p frequently occurred. The cumulative overall survival (OAS) was different between 35 patients with

GG-62 is a cell line previously thought to be derived from an atypical Ewing tumor (ET). Reverse-transcriptase polymerase chain reaction revealed an in-frame fusion between the Ewing sarcoma gene ( EWS) codon 325 and the activating transcription factor 1 gene ( ATF1) codon 65 which permits the production of chimeric EWS-ATF1 oncoproteins. We also identified the genomic breakpoint resulting from a reciprocal t(12;22)(q13;q12), which is the hallmark of malignant melanoma of soft parts (MMSP). We applied Affymetrix human cancer G110 arrays to compare the gene expression patterns of GG-62 and other cell lines derived from small blue round cell tumors of childhood. Hierarchical clustering of 463 differentially expressed genes distinguished GG-62 from the ETs, as well as the neuroblastomas, and revealed a cluster of 36 upregulated genes. Several of these genes are involved in signal transduction pathways that may be critical for maintaining cell transformation; some examples are avian erythroblastic leukemia viral oncogene homolog 3 ( ERBB3), neuregulin 1 ( NRG1), fibroblast growth factor 9 ( FGF9), and fibroblast growth factor receptor-1 ( FGFR1). Furthermore, genes near the chromosome-12q13 breakpoint exhibited increased expression of GG-62 including ERBB3, NR4A1 (nuclear receptor subfamily 4, group A, member 1), cyclin-dependent kinase 2 ( CDK2), and alpha 5 integrin ( ITGA5). Altogether our findings demonstrate the MMSP derivation of GG-62 and may shed light on the mechanisms of tumorigenesis in this rare disease.