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PurposeThe transcription factor Fra-2 affects the invasive potential of breast cancer cells by dysregulating adhesion molecules in vitro. Previous results suggested that it upregulates the expression of E- and P-selectin ligands. Such selectin ligands are important members of the leukocyte adhesion cascade, which govern the adhesion and transmigration of cancer cells into the stroma of the host organ of metastasis. As so far, no in vivo data are available, this study was designed to elucidate the role of Fra-2 expression in a spontaneous breast cancer metastasis xenograft model.MethodsThe effect of Fra-2 overexpression in two stable Fra-2 overexpressing clones of the human breast cancer cell line MDA MB231 on survival and metastatic load was studied after subcutaneous injection into scid and E- and P-selectin-deficient scid mice.ResultsFra-2 overexpression leads to a significantly shorter overall survival and a higher amount of spontaneous lung metastases not only in scid mice, but also in E- and P-deficient mice, indicating that it regulates not only selectin ligands, but also selectin-independent adhesion processes.ConclusionThus, Fra-2 expression influences the metastatic potential of breast cancer cells by changing the expression of adhesion molecules, resulting in increased adherence to endothelial cells in a breast cancer xenograft model.

Objective At present, maintenance therapy with the antiangiogenic agent bevacizumab or with PARP-inhibitors represent two options for BRCA-wildtype ovarian cancer patients, after platinum-based first line chemotherapy. The identification of molecular markers to predict patient response to different maintenance therapies remains a major challenge. In the present study we analyzed the predictive potential of vascular endothelial growth factor C (VEGF-C) to identify ovarian cancer patients that might benefit from an antiangiogenic therapy. Methods 101 patients with primary epithelial ovarian cancer were analyzed for serum levels of VEGF-A,–C and CA-125 by ELISA. Serum levels were compared between patients with low pT-stage (pT1a-pT2c n = 11), healthy individuals (n = 27) and patients with higher pT-stage (> = pT3 n = 90). Adjusted ROC curves and an adjusted logistic regression model were carried out to evaluate the potential impact of VEGF-A and -C, as well as CA-125 serum level concentration on bevacizumab-therapy response, under consideration of covariates such as FIGO, pM, pN and residual tumor after surgery. Results A patient which has in comparison twice the VEGF-C concentration in serum, has a significant increased chance of response to bevacizumab by a factor of 2.79. Further, only VEGF-C serum levels were significantly higher in the group of patients with lower pT-stage compared to healthy individuals, whereas VEGF-A or CA-125 serum levels could not discriminate between healthy individuals and patients with ovarian cancer at low pT-stages. Conclusion VEGF-C serum level might serve as as a biomarker to evaluate treatment response under bevacizumab.

BackgroundMaturation of complex N-glycans involves the action of Golgi mannosidases and plays a major role in cancer progression. We recently showed a favourable prognostic role of α-mannosidase MAN1A1 in breast cancer mainly caused by alteration of certain adhesion molecules.MethodsWe analysed the protein expression of MAN1A1 in ovarian cancer (n = 204) using western blot and studied the impact of MAN1A1 itself and of MAN1A1-related glycosylation on the prognostic relevance of two adhesion molecules. Functional consequences of mannosidase inhibition using kifunensine and MAN1A1 knock out were investigated in ovarian cancer cells in vitro.ResultsPatients with high MAN1A1 expression in tumours showed significantly shorter RFS than those with low-MAN1A1 levels. Moreover, high MAN1A1 expression correlated significantly with advanced stage, lymph node involvement and distant metastasis. Further, the glycosylated adhesion molecule ALCAM reveals a significant adverse prognostic effect only in the presence of high MAN1A1 expression. In spheroid-formation assays, mannosidase inhibition and especially MAN1A1 knock out led to strong reduction of tumour cell aggregation.ConclusionsOur study demonstrates the unfavourable prognostic role of MAN1A1 in ovarian cancer, probably caused by an altered ability of spheroid formation, and the strong influence of this glycosylation enzyme on the prognostic impact of ALCAM.

The RAS family comprises three proto-oncogenes (H-RAS, K-RAS, and N-RAS) and is among the most widely studied of oncogenes. The present study aimed at investigating the clinical relevance of mRNA levels of the three isoforms in a large group of breast cancer patients with a long-term follow-up. 198 previously untreated patients were enrolled in the study. mRNA levels of K-RAS, H-RAS, and N-RAS were measured using microarray (Affymetrix HG-U133A). Elevated H-RAS levels were found significantly more frequently in patients with larger (p = 0.021) and ER-positive tumors (p = 0.048), while elevated K-RAS levels were associated with nodal positivity (p = 0.001) and HER2-positivity (p = 0.010). Patients with high N-RAS mRNA levels were more likely to be diagnosed with triple-negativity (p

BMC cancer 21(1), 920 (2021). doi:10.1186/s12885-021-08656-0, Published by Springer, Berlin ; Heidelberg

Aberrant sialylation of glycoproteins has been detected in many tumors, and upregulation of the beta-galactosamide alpha-2,6-sialyltransferase 1 (ST6GAL1) has been implicated with tumor aggressiveness and chemoresistance in experimental models. In our present study, we aimed to study the prognostic or predictive role of ST6GAL1 in ovarian carcinoma, using two independent ovarian cancer cohorts. ST6GAL1 mRNA levels were retrieved from a publicly available database (n = 517), and ST6GAL1 protein levels were analyzed by western blot analysis in a cohort of 204 ovarian tumor samples. The results were correlated with clinical and histological tumor parameters and follow-up information. High ST6GAL1 mRNA levels significantly correlated with lymphovascular invasion and shorter survival, whereas high ST6GAL1 protein expression was associated with advanced stage, distant metastasis and shorter recurrence-free intervals. In both cohorts the prognostic role was most pronounced in tumors without macroscopically visible residual tumor after surgery. In these cases, ST6GAL1 expression levels might help to identify cases with a higher risk of chemoresistance and metastatic relapse that might require an adapted therapeutic regime.

Background: Alterations in protein glycosylation have been related to malignant transformation and tumour progression. We recently showed that low mRNA levels of Golgi alpha-mannosidase MAN1A1 correlate with poor prognosis in breast cancer patients. Methods: We analysed the role of MAN1A1 on a protein level using western blot analysis (n=105) and studied the impact of MAN1A1-related glycosylation on the prognostic relevance of adhesion molecules involved in breast cancer using microarray data (n=194). Functional consequences of mannosidase inhibition using the inhibitor kifunensine or MAN1A1 silencing were investigated in breast cancer cells in vitro. Results: Patients with low/moderate MAN1A1 expression in tumours showed significantly shorter disease-free intervals than those with high MAN1A1 levels (P=0.005). Moreover, low MAN1A1 expression correlated significantly with nodal status, grading and brain metastasis. At an mRNA level, membrane proteins ALCAM and CD24 were only significantly prognostic in tumours with high MAN1A1 expression. In vitro, reduced MAN1A1 expression or mannosidase inhibition led to a significantly increased adhesion of breast cancer cells to endothelial cells. Conclusions: Our study demonstrates the prognostic role of MAN1A1 in breast cancer by affecting the adhesive properties of tumour cells and the strong influence of this glycosylation enzyme on the prognostic impact of some adhesion proteins.

Purpose Ovarian cancer (OvCa) progression mainly takes place by intraperitoneal spread. Adhesion of tumor cells to the mesothelial cells which form the inner surface of the peritoneum is a crucial step in this process. Cancer cells use in principle different molecules of the leukocyte adhesion cascade to facilitate adhesion. This cascade is initiated by selectin-ligand interactions followed by integrin - extracellular matrix protein interactions. Here we address the question whether all tumor cells predominantly employ selectin-dependent leukocyte-like adhesion cascade (SDAC) or whether they use integrin mediated adhesion for OvCa progression as well. Methods A comparative transcriptomic analysis of the human OvCa cell lines OVCAR8 and SKOV3 was performed. Intraperitoneal xenograft model of OVCAR8 cells was used to determine whether there is a correlation between SDAC gene expression and the metastatic potential of the control cells and the cells overexpressing c-Fos. Transcriptomic analysis of OVCAR8 and SKOV3 samples was performed using microarrays. Results One-third of the protein-coding genes involved in SDAC exhibited lower expression levels in OVCAR8 than in SKOV3 cells. In contrast to SKOV3 cells, c-Fos overexpression in OVCAR8 cells did not significantly influence the expression of SDAC genes. Intraperitoneal xenograft model of OVCAR8 cells unexpectedly demonstrated that the aggressiveness of OVCAR8 tumors was not depended on the c-Fos expression level and was comparable to that of SKOV3 control tumors. Gene expression analysis of tumors suggests that SKOV3-derived tumor progression was mainly depended on SDAC. Progression of OVCAR8 tumors relied on other cell adhesion molecules that do not interact with selectins. Conclusions High expression of c-Fos in ovarian cancer cells is not always associated with reduced metastatic potential. Low expression level of SDAC genes may not ensure low OvCa metastatic potential hence alternative adhesion mechanisms involving laminin-integrin interactions exist as well.

BMC cancer 21(1), 1024 (2021). doi:10.1186/s12885-021-08754-z, Published by BioMed Central, London

// Katharina Prieske 1, * , Stefan Prieske 1, * , Simon A. Joosse 2 , Fabian Trillsch 3 , Donata Grimm 1 , Eike Burandt 4 , Sven Mahner 3 , Barbara Schmalfeldt 1 , Karin Milde-Langosch 1 , Leticia Oliveira-Ferrer 1 and Linn Woelber 1 1 Department of Gynecology and Gynecologic Oncology, University Medical Centre Hamburg-Eppendorf, 20246 Hamburg, Germany 2 Department of Tumor Biology, University Medical Centre Hamburg-Eppendorf, 20246 Hamburg, Germany 3 Department of Gynecology and Obstetrics, University of Munich, 81377 Munich, Germany 4 Department of Pathology, University Medical Centre Hamburg-Eppendorf, 20246 Hamburg, Germany * These authors have contributed equally to this work Correspondence to: Katharina Prieske, email: k.prieske@uke.de Keywords: BRCA methylation, ovarian cancer, platinum, recurrence, high-grade Received: May 30, 2017 Accepted: July 25, 2017 Published: September 15, 2017 ABSTRACT Background: Approximately 20-25% of ovarian cancers are attributable to germline or somatic BRCA1/2 mutations, resulting in defects in the homologous recombination pathway. Inactivation of these genes can also be mediated by epigenetic changes, e.g., hypermethylation of CpG islands in the promoter regions. In such homologous recombination deficient tumors, platinum based chemotherapy is in general effective, however, loss of hypermethylation might lead to refractory disease. The aim of this study was to evaluate the stability of BRCA1 promoter hypermethylation in recurrent disease after platinum based chemotherapy. Methods: Tumor tissue from 76 patients with primary and 48 patients with platinum-sensitive recurrent high-grade ovarian cancer was collected. In a subgroup of 12 patients, ‘paired’ tumor tissue from primary and recurrent surgery was available. BRCA1 promoter methylation status was assessed using methylation specific polymerase chain reaction and was verified by Sanger Sequencing. Results: 73.7% (56/76) of primary and 20.8% (10/48) of recurrent tumors displayed BRCA1 promoter hypermethylation. BRCA1 promoter methylation status was not associated with progression-free- or overall survival. In the paired subgroup 83.3% (10/12) of the primary vs. 16.7% (2/12) of the recurrent tumors showed hypermethylation. In eight patients loss of BRCA1 hypermethylation was observed, whereas two patients had stable methylation status. Conclusions: Loss of BRCA1 promoter methylation may be a mechanism to restore BRCA1 function in recurrent disease. However, currently the clinical significance is still unclear and should be evaluated in prospective clinical trials.

Although altered glycosylation has been detected in human cancer cells decades ago, only investigations in the last years have enormously increased our knowledge about the details of protein glycosylation and its role in tumour progression. Many proteins, which are heavily glycosylated, i.e. adhesion proteins or proteases, play an important role in cancer metastasis that represents the crucial and frequently life-threatening step in progression of most tumour types. Compared to normal tissue, tumour cells often show altered glycosylation patters with appearance of new tumour-specific antigens. In this review, we give an overview about the role of glycosylation in tumour metastasis, describing recent results about O-glycans, N-glycans and glycosaminoglycans. We show that glycan structures, glycosylated proteins and glycosylation enzymes have influence on different steps of the metastatic process, including epithelial-mesenchymal transition (EMT), migration, invasion/intravasation and extravasation of tumour cells. Regarding the important role of cancer metastasis for patients survival, further knowledge about the consequences of altered glycosylation patterns in tumour cells is needed which might eventually lead to the development of novel therapeutic approaches.

// Sascha Kuerti 1, * , Leticia Oliveira-Ferrer 1, * , Karin Milde-Langosch 1 , Barbara Schmalfeldt 1 , Karen Legler 1 , Linn Woelber 1 , Katharina Prieske 1 , Sven Mahner 2 and Fabian Trillsch 2 1 Department of Gynaecology and Gynaecologic Oncology, University Medical Center Hamburg-Eppendorf, Hamburg 20246, Germany 2 Department of Obstetrics and Gynaecology, University of Munich, Munich 81377, Germany * These authors have contributed equally to this work Correspondence to: Fabian Trillsch, email: Fabian.Trillsch@med.uni-muenchen.de Keywords: ovarian cancer, (VEGF)-A, -C, -D, mesenchymal phenotype, tumour dissemination, lymph node metastases Received: February 20, 2017 Accepted: March 31, 2017 Published: May 18, 2017 ABSTRACT Background: Peritoneal dissemination and retroperitoneal lymph node involvement are main routes for progression of epithelial ovarian cancer (EOC). Vascular endothelial growth factor (VEGF) mediated angiogenesis has been identified as an important mechanism promoting tumour progression. Methods: Tumour tissue of 100 patients with EOC was analysed for protein expression of vascular endothelial growth factor (VEGF)-A, -C, -D by Western Blot analysis. Expression patterns in patients with ‘extensive intraperitoneal’ metastases (pT3c pN1 and pT3b-pT3c pN0, n=80) were compared to patients with ‘predominantly retroperitoneal’ metastases (pT1a-pT3b, pN1, n=20). Overall and progression-free survival was analysed by Kaplan-Meier method. Results: While no significant differences in expression levels among the different modes of metastases were noted for VEGF-A and -D, VEGF-C expression was significantly higher in the group of predominantly retroperitoneal metastases compared to the group with extensive intraperitoneal metastases. Patients with high VEGF-C expression had a significantly worse overall survival compared to patients with low expression levels. Conclusions: Retroperitoneal tumour progression in EOC patients is associated with high VEGF-C expression. VEGF-C may serve as a molecular marker to identify patients with assumed high risk for lymphatic metastases, who might benefit from specific treatment strategies.

Exosomes are membrane vesicles that mediate intercellular communication by transporting their molecular cargo from cell to cell. We investigated whether serum levels of exosomal miR-373, miR-200a, miR-200b and miR-200c and circulating exosomes have diagnostic and prognostic relevance in a cohort of 163 epithelial ovarian cancer (EOC) patients using TaqMan MicroRNA assays and ELISA. The serum concentrations of exosomal miR-373 (p = 0.0001), miR-200a (p = 0.0001), miR-200b (p = 0.0001) and miR-200c (p = 0.028) were significantly higher in EOC patients than healthy women. The levels of miR-200a (p = 0.0001), miR-200b (p = 0.0001) and miR-200c (p = 0.019) could distinguish between malignant and benign ovarian tumors. While the levels of miR-373 and miR-200a were increased in all FIGO/lymph node stages (p = 0.0001), the levels of miR-200b and miR-200c were higher in patients with FIGO stage III-IV (p = 0.0001, p = 0.008, respectively) including lymph node metastasis (p = 0.0001, p = 0.004, respectively) than FIGO stages I-II. The increased levels of miR-200b and miR-200c were also associated with CA125 values (p = 0.0001, p = 0.0001, respectively) and a shorter overall survival (p = 0.007, p = 0.017, respectively). The levels of exosomes were excessively elevated in EOC patients (p = 0.0001). In all three cohorts, they were positively associated with the serum levels of exosomal miR-373 (p = 0.004), miR-200a (p = 0.0001), miR-200b (p = 0.0001) and miR-200c (p = 0.008). In conclusion, the increased levels of exosomal miR-200b and miR-200c mainly observed in advanced EOC suggest that these microRNAs may be involved in tumor progression. The high concentrations of exosomes in EOC patients imply an excessive, active exosomal secretion in EOC.

OA gold Biomarkers for prognosis in radiotherapy-treated breast cancer patients are urgently needed and important to stratify patients for adjuvant therapies. Recently, a role of the receptor of hyaluronan-mediated motility (RHAMM) has been suggested for tumor progression. Our aim was (i) to investigate the prognostic value of RHAMM in breast cancer and (ii) to unravel its potential function in the radiosusceptibility of breast cancer cells. We demonstrate that RHAMM mRNA expression in breast cancer biopsies is inversely correlated with tumor grade and overall survival. Radiosusceptibility in vitro was evaluated by sub-G1 analysis (apoptosis) and determination of the proliferation rate. The potential role of RHAMM was addressed by short interfering RNAs against RHAMM and its splice variants. High expression of RHAMMv1/v2 in p53 wild type cells (MCF-7) induced cellular apoptosis in response to ionizing radiation. In comparison, in p53 mutated cells (MDA-MB-231) RHAMMv1/v2 was expressed sparsely resulting in resistance towards irradiation induced apoptosis. Proliferation capacity was not altered by ionizing radiation in both cell lines. Importantly, pharmacological inhibition of the major ligand of RHAMM, hyaluronan, sensitized both cell lines towards radiation induced cell death. Based on the present data, we conclude that the detection of RHAMM splice variants in correlation with the p53 mutation status could help to predict the susceptibility of breast cancer cells to radiotherapy. Additionally, our studies raise the possibility that the response to radiotherapy in selected cohorts may be improved by pharmaceutical strategies against RHAMM and its ligand hyaluronan.

Background: Peritoneal dissemination and retroperitoneal lymph node involvement are main routes for tumour spread of epithelial ovarian cancer (EOC), possibly determined by the intercellular connecting protein E-Cadherin (E-Cad) and its fragments. Methods: Tumour tissue of 105 advanced EOC patients was evaluated for protein expression of E-Cad, β-Catenin and Calpain by western blotting and immunohistochemistry. Expression patterns were compared between tumours with solely intraperitoneal (pT3c, pN0; n=41) and tumours with retroperitoneal metastases (pT1a-3c, pN1; n=64). Lysates of the EOC cell line SKOV3 and tumour tissue from the intraperitoneal group were tested for E-Cad expression following Calpain treatment. Results: E-Cad full-length (E-Cad-FL, 120 kDa) and two major fragments at 85 kDa (E-Cad-85) and 23 kDa (E-Cad-23) were detected by western blotting. E-Cad-85 expression was significantly higher in tumours with solely intraperitoneal metastases and correlated strongly with E-Cad-23 and the protease Calpain. Calpain-mediated cleavage was identified as a potential mechanism to generate E-Cad-85 from E-Cad-FL by treating lysates from SKOV3 cells and tumour tissue with this enzyme. Increased cytoplasmic localisation of β-Catenin in tumours with high E-Cad-85 expression corroborates that E-Cad-85 loses the binding site for β-Catenin after fragmentation, enabling tumour cluster formation and peritoneal dissemination. Conclusions: Calpain-mediated E-Cad fragmentation appears to promote intraperitoneal EOC progression. Understanding these mechanisms might eventually lead to new tailored subtype-specific diagnostic and therapeutic interventions.

Background An important component of the RAS signalling pathway, the RAS p21 oncogene, is frequently hyperactivated in breast cancer. Its expression in tumor tissue has been linked to poor clinical outcome. This study was designed to evaluate the clinical relevance of RAS p21 levels in peripheral blood in a large cohort of metastatic breast cancer patients. Methods Two hundred fifty-one patients with metastatic breast cancer were enrolled in this prospective, multicentre, open-label, non-randomized study. Blood samples were collected before start of first-line or later-line treatment. RAS p21 was determined using a sandwich-type ELISA immunoassay. For the determination of the cutoff, blood samples from age-matched healthy controls were analyzed. A value above 452 pg/ml was regarded as elevated (mean + 2 x SD). In the univariate survival analysis, two other cutoffs were considered as well (50th and 75th percentile of patients, i.e. 229 pg/ml and 320 pg/ml). Circulating tumor cells (CTCs) were detected using the CellSearch system. Results 29 of 251 (12%) patients had RAS p21 levels above the cut-off level of 452 pg/ml. Clinical-pathological parameters, such as hormone receptor and HER2 status, line of therapy and CTC status, did not correlate with RAS p21 levels. Elevated RAS p21 was significantly associated with shorter progression-free and overall survival in the univariate analysis (median PFS: 3.9 months [95%-CI: 1.8–6.0] for patients with elevated RAS p21 levels versus 8.5 months [95%-CI: 7.4–9.5] with non-elevated levels [p = 0.01]; median OS: 7.1 months [95%-CI: 0.3–14.2] versus not reached [p = 0.002], respectively). When RAS p21 cutoffs other than 452 pg/ml were considered, elevated RAS p21 was significantly associated with OS but not with PFS. Classical clinical-pathological factors were included into a multivariate Cox regression analysis. In addition, factors previously shown to influence survival in a univariate analysis, such as serum HER2, CAIX and TIMP1, were included as well. In the multivariate analysis, RAS p21, presence of ≥5 CTCs per 7.5 ml blood, higher grading and higher line of therapy remained independent predictors of shorter OS. Conclusions Metastatic breast cancer patients with elevated levels of circulating RAS p21 have significantly worse clinical outcome. Hypothetically, these patients might benefit from therapeutic strategies targeting RAS pathway. Trial registration Current Controlled Trials ISRCTN59722891 (DETECT); trial registration date: April, 17th 2010; the trial was registered retrospectively.

Metastasis-Associated in Colon Cancer-1(MACC1) was first identified as a transcriptional activator of the HGF/MET pathway. Deregulation of HGF/MET signaling is reported as a prognostic marker for tumorigenesis, early stage invasion, and metastasis which is associated with poor clinical outcome in breast cancer patients. The aim of the present study was to further investigate the prognostic or predictive value of MACC1 expression in breast cancer.We analyzed the MACC1 expression in 105 primary breast cancer samples by Western-Blot analysis and immunohistochemistry.A significant correlation of high MACC1 expression with shorter disease-free survival was found within the group of lymph-node-negative patients. Additionally, an association of high MACC1 expression and shorter disease-free survival was observed within estrogen receptor positive tumors and patients without adjuvant chemotherapy.Our results support a biologic role and potentially open the perspective for the use of MACC1 as a prognostic marker for treatment decision in breast cancer patients.

The underlying mechanisms of ovarian cancer (OvCa) dissemination are still poorly understood, and novel molecular markers for this cancer type are urgently needed. In search of adhesion molecules with prognostic relevance in OvCa, we compared tumors with good outcome (alive > 3 years) and those with poor outcome (dead n=517) as well as CEACAM1 protein expression by Western blot analysis in a cohort of 242 tumor samples. Further, CEACAM1 localization in tumour tissue was evaluated by immunohistochemistry and CEACAM1 splice variants by RT-PCR in representative tumours. In Kaplan–Meier analysis, high CEACAM1 mRNA levels significantly correlated with longer survival (p=0.008). By Western blot analysis in the second cohort, similar associations of high CEACAM1 protein levels with longer recurrence-free survival (RFS, p=0.035) and overall survival (OAS, p=0.004) were observed. In multivariate Cox regression analysis including clinical prognostic parameters, CEACAM1 mRNA or protein expression turned out as independent prognostic markers. Stratified survival analysis showed that high CEACAM1 protein expression was prognostic in node-negative tumors (p=0.045 and p=0.0002 for DFS and OAS) but lost prognostic significance in node-positive carcinomas. Similarly, high CEACAM1 mRNA expression did not show prognostic relevance in tumors with lymphatic invasion (L1) but was associated with longer survival in cases without lymphovascular involvement. Further analysis showed a predominance of 4S and 4L isoforms and mostly membraneous CEACAM1 localization in ovarian tumours. Our results suggest that CEACAM1 might be an independent favorable prognostic marker in OvCa, especially in the subgroup of patients with solely intraperitoneal metastasis.

Background: Owing to late diagnosis in advanced disease stages, prognosis of patients with epithelial ovarian cancer (EOC) is poor. The quantification of deregulated levels of microRNAs could facilitate earlier diagnosis and improve prognosis of EOC. Methods: Seven microRNAs (miR-7, miR-16, miR-25, miR-93, miR-182, miR-376a and miR-429) were quantified in the serum of 180 EOC patients and 66 healthy women by TaqMan PCR microRNA assays. Median follow-up time was 21 months. The effects of miR-7 and miR-429 on apoptosis, cell proliferation, migration and invasion were investigated in two (EOC) cell lines. Results: Serum levels of miR-25 (P=0.0001) and miR-93 (P=0.0001) were downregulated, whereas those of miR-7 (P=0.001) and miR-429 (P=0.0001) were upregulated in EOC patients compared with healthy women. The four microRNAs discriminated EOC patients from healthy women with a sensitivity of 93% and a specificity of 92%. The levels of miR-429 positively correlated with CA125 values (P=0.0001) and differed between FIGO I–II and III–IV stages (P=0.001). MiR-429 was an independent predictor of overall survival (P=0.011). Overexpressed miR-429 in SKOV3 cells led to suppression of cell migration (P=0.037) and invasion (P=0.011). Increased levels of miR-7 were associated with lymph node metastases (P=0.0001) and FIGO stages III–IV (P=0.0001). Overexpressed miR-7 in SKOV3 cells resulted in increased cell migration (P=0.001) and invasion (P=0.011). Additionally, the increased levels of miR-376a correlated with FIGO stages III–IV (P=0.02). Conclusions: Our data indicate the diagnostic potential of miR-7, miR-25, miR-93 and miR-429 in EOC and the prognostic potential of miR-429. This microRNA panel may be promising molecules to be targeted in the treatment of EOC.

Background: In a recent study based on cDNA microarray data of breast cancer patients, we could show that the mRNA expression of some glycosylation enzymes involved in synthesis and trimming of N-glycansis significantly associated with metastasis and recurrence in breast cancer patients suggesting an important role of N-glycosylation in tumour progression (Milde-Langosch et al., Breast Cancer Res Treat 2014). The aim of the present study was to further investigate this issue on a protein and glycoprotein level (1.) by western blot analysis of selected glycosylation enzymes and (2.) by lectin histochemistry using lectins which bind to O- and/or N-glycans. Methods: In order to confirm the role of two selected glycosylation enzymes, ribophorin (RPN1) and mannosidase (MAN1A1) on protein level, we analyzed their expression in a cohort of 196 breast cancer samples by western blot analysis. In addition, we studied tumour cell binding of various lectins, which recognize diverse O- or N-glycans (UEA1, HPA, PNA, GNA, PHA-L), using a tissue microarray with up to 293 evaluable breast cancer samples. These results were correlated with recurrence-free survival (RFS) and overall survival (OS) and data on vascular and lymphatic invasion. Results: By western blot analysis, the negative prognostic significance of ribophorin (RPN1), a key enzyme of N-glycosylation, was corroborated on a protein level. For MAN1A1 which is involved in trimming of N-glycan structures, two isoforms with different molecular weight could be detected on western blots, showing contradictory associations with OS. Protein levels of RPN1 and total MAN1A1 showed significant correlations to the respective mRNA expression data. By lectin histochemistry, we found significant correlations of lectin binding to lymph node metastasis, vascular invasion and lymphangiosis in breast cancer samples. Binding of PHA-L and GNA (binding to N-glycans) and PNA (binding to O- and N-glycans) was significantly associated with vascular invasion, whereas PNA and PHA-L binding correlated with lymph node involvement. In addition, PNA and HPA reactivity was associated with microscopically detected lymphangiosis. PNA binding also correlated with shorter RFS and OS. Conclusion: While the role of O-glycans in breast cancer metastasis has already been described, our data point to an additional strong impact of N-glycosylation on metastasis and progression of mammary carcinomas. Citation Format: Karin Milde-Langosch, Tosca Karius, Dina Schütze, Harriet Wikman, Isabell Witzel, Leticia Oliveira-Ferrer, Volkmar Müller, Udo Schumacher. N-glycosylation changes in tumour cells are associated with vascular invasion and metastasis in breast cancer patients [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-07-21.

Background Approximately 20-25% of ovarian cancers are attributable to germline or somatic BRCA1/2 mutations, resulting in defects in the homologous recombination pathway. Inactivation of these genes can also be mediated by epigenetic changes, e.g., hypermethylation of CpG islands in the promoter regions. In such homologous recombination deficient tumors, platinum based chemotherapy is in general effective, however, loss of hypermethylation might lead to refractory disease. The aim of this study was to evaluate the stability of BRCA1 promoter hypermethylation in recurrent disease after platinum based chemotherapy. Methods Tumor tissue from 76 patients with primary and 48 patients with platinum-sensitive recurrent high-grade ovarian cancer was collected. In a subgroup of 12 patients, ‘paired’ tumor tissue from primary and recurrent surgery was available. BRCA1 promoter methylation status was assessed using methylation specific polymerase chain reaction and was verified by Sanger Sequencing. Results 73.7% (56/76) of primary and 20.8% (10/48) of recurrent tumors displayed BRCA1 promoter hypermethylation. BRCA1 promoter methylation status was not associated with progression-free- or overall survival. In the paired subgroup 83.3% (10/12) of the primary vs. 16.7% (2/12) of the recurrent tumors showed hypermethylation. In eight patients loss of BRCA1 hypermethylation was observed, whereas two patients had stable methylation status. Conclusions Loss of BRCA1 promoter methylation may be a mechanism to restore BRCA1 function in recurrent disease. However, currently the clinical significance is still unclear and should be evaluated in prospective clinical trials.

In this study, we compared the blood serum levels of circulating cell-free and exosomal microRNAs, and their involvement in the molecular subtypes of breast cancer patients. Our analyses on cell-free miR-101, miR-372 and miR-373 were performed in preoperative blood serum of 168 patients with invasive breast cancer, 19 patients with benign breast diseases and 28 healthy women. MicroRNAs were additionally quantified in exosomes of 50 cancer patients and 12 healthy women from the same cohort. Relative concentrations were measured by quantitative TaqMan MicroRNA assays and correlated to clinicopathological risk factors. The concentrations of cell-free miR-101 (p=0.013) and miR-373 (p=0.024) were significantly different between patients with breast cancer and benign tumors. A prevalence of miR-101, miR-372 and miR-373 were found in exosomes. The levels of circulating exosomal (but not cell-free) miR-373 were higher in triple negative than luminal carcinomas (p=0.027). Also, estrogen-negative (p=0.021) and progesterone-negative (p=0.01) tumors displayed higher concentrations of exosomal miR-373 than patients with hormone-receptor positive tumors. Overexpression of miR-373 by transfection of MCF-7 cells showed downregulated protein expression of the estrogen receptor, and inhibition of apoptosis induced by camptothecin. Our data indicate that serum levels of exosomal miR-373 are linked to triple negative and more aggressive breast carcinomas.

Glycosylation of cellular proteins has important impact on their stability and functional properties, and glycan structures strongly influence cell adhesion. Many enzymes are involved in glycoconjugate synthesis and degradation, but there is only limited information about their role in breast cancer progression. Therefore, we retrieved RNA expression data of 202 glycosylation genes generated by microarray analysis (Affymetrix HG-U133A) in a cohort of 194 mammary carcinomas with long-term follow-up information. After univariate and multivariate Cox regression analysis, genes with independent prognostic value were identified. These were further analysed by Kaplan-Meier analysis and log-rank tests, and their prognostic value was validated in a second cohort of 200 tumour samples from patients without systemic therapy. In our first cohort, we identified 24 genes with independent prognostic value, coding for sixteen anabolic and eight catabolic enzymes. Functionally, these genes are involved in all important glycosylation pathways, namely O-glycosylation, N-glycosylation, O-fucosylation, synthesis of glycosaminoglycans and glycolipids. Eighteen genes also showed prognostic significance in chemotherapy-treated patients. In the second cohort, six of the 24 relevant genes were of prognostic significance (FUT1, FUCA1, POFUT1, MAN1A1, RPN1 and DPM1), whereas a trend was observed for three additional probesets (GCNT4, ST3GAL6 and UGCG). In a stratified analysis of molecular subtypes combining both cohorts, great differences appeared suggesting a predominant role of N-glycosylation in luminal cancers and O-glycosylation in triple-negative ones. Correlations of gene expression with metastases of various localizations point to a role of glycan structures in organ-specific metastatic spread. Our results indicate that various glycosylation reactions influence progression and metastasis of breast cancer and might thus represent potential therapeutic targets.

The RANK/RANKL/OPG pathway is well known for bone destruction in skeletal metastases but has also been implicated in osteoclast-independent roles in tumorigenesis and de novo metastasis. Experimental data suggest contribution of progesterone to tumorigenesis may be mediated by RANKL. Importantly, modulation of this pathway became possible through the availability of denosumab, an artificial counterpart of OPG, but significant gaps remain in the translation of preclinical findings on the pathway. We analyzed gene expression of RANK, RANKL and OPG from 40 Affymetrix datasets encompassing 4467 primary breast cancers and focused on ER positive disease. We did not observe a significant prognostic value of RANK and RANKL mRNA expression. In contrast, OPG was associated with a better prognosis among 1941 ER positive cancers (HR 0.64, 95% CI 0.53-0.77; P < 0.0001) using a cutoff from its highly bimodal expression. We detected considerable heterogeneity regarding the prognostic value of OPG between different datasets. This heterogeneity could neither be attributed to technical reasons nor to differences in standard clinical parameters or treatments of the cohorts. Interestingly, the prognostic value of the progesterone receptor and of OPG showed similar cohort specific effects. Still both factors were no surrogates for each other but contributed independent prognostic value in multivariate analyses. Thus, both OPG and PgR are independently associated with good prognosis in ER positive breast cancer. However both markers share common cohort specific differences in contrast to proliferation markers as Ki67 which may be based on the underlying biology.

The incidence of brain metastases in breast cancer patients has increased in the last years. However, the knowledge about tumor cell invasion in the brain is still very limited. Based on our recent study on cDNA microarray data of breast cancer patients, we hypothesized that two enzymes involved in the hyaluronan metabolism, namely, hyaluronan synthase 2 (HAS2) and hyaluronidase 1 (HYAL1) are associated with brain metastases formation. Protein expression levels of hyaluronan, HAS2, and HYAL1 were analyzed in primary breast cancer, and metastatic tissue samples from different localizations (brain, bone, skin, liver, and lung) were included in four different cohorts by immunohistochemistry. Correlations of expression levels with clinical and pathological parameters were performed within the individual cohorts. Higher HYAL1 expression was detected among primary tumors from patients with subsequent brain metastases compared with those without brain metastases (p = 0.011). Interestingly, brain metastatic tissue showed a significantly reduced HYAL1 expression compared with the corresponding primary tumor (p = 0.003). HYAL1 expression in brain metastases was also significantly lower than in skin, liver, and lung metastases. Further, hyaluronan staining in brain metastases was mainly located on the surface of the tumor cells, whereas in all other metastatic sites hyaluronan was only detected in the extracellular matrix. We could not show an association of HAS2 with the formation of brain metastases. In conclusion, our results suggest that the enzyme HYAL1 plays a role in tumor dissemination and brain-specific colonization, rather than in subsequent metastatic out-growth.

Background: C-Fos was initially described as oncogene, but was associated with favourable prognosis in ovarian cancer (OvCa) patients. The molecular and functional aspects underlying this effect are still unknown. Methods: Using stable transfectants of SKOV3 and OVCAR8 cells, proliferation, migration, invasion and apoptotic potential of c-FOS-overexpressing clones and controls were compared. Adherence to components of the extracellular matrix was analysed in static assays, and adhesion to E-selectin, endothelial and mesothelial cells in dynamic flow assays. The effect of c-FOS in vivo was studied after intraperitoneal injection of SKOV3 clones into SCID mice, and changes in gene expression were determined by microarray analysis. Results: Tumour growth after injection into SCID mice was strongly delayed by c-FOS overexpression, with reduction of lung metastases and circulating tumour cells. In vitro, c-FOS had only weak influence on proliferation and migration, but was strongly pro-apoptotic. Adhesion to components of the extracellular matrix (collagen I, IV) and to E-selectin, endothelial and mesothelial cells was significantly reduced in c-FOS-overexpressing OvCa cells. This corresponds to deregulation of adhesion proteins and glycosylation enzymes in microarray analysis. Conclusion: In addition to its known pro-apoptotic effect, c-FOS might influence OvCa progression by changing the adhesion of OvCa cells to peritoneal surfaces.

The ubiquitin C-terminal hydrolase L1 (UCH-L1) belongs to the family of deubiquitinating enzymes. It is overexpressed in various tumour entities and associated with metastases formation in some solid tumours. However, only limited information about its role in breast cancer is available. The aim of this study was to examine the UCH-L1 expression in primary breast cancer and to determine its relevance as a potential prognostic marker. We investigated both UCH-L1 mRNA expression in microarray data from 182 primary mammary carcinomas and UCH-L1 protein expression using a tissue microarray containing samples from 1,622 breast cancer patients. With both methods, high UCH-L1 expression correlated significantly with negative oestrogen receptor and progesterone receptor status and advanced tumour stage. Moreover by Kaplan–Meier analysis, high UCH-L1 mRNA and protein expression correlated with a significantly shorter overall survival. The data of our study suggest that high levels of UCH-L1 expression indicate a more aggressive tumour behaviour and might represent a potential target in breast cancer treatment.

The androgen receptor (AR) is intensively discussed as a prognostic and/or predictive marker in breast cancer patients.We evaluated the value of AR mRNA expression with the Affymetrix HG-U 133A array in 3 different cohorts: a cohort of breast cancer patients who received adjuvant treatment (cohort A; n = 165), a cohort of untreated breast cancer patients (cohort B; n = 200) and a cohort of chemotherapy-treated breast cancer patients with estrogen receptor (ER)-positive tumors (cohort C; n = 223).AR mRNA expression was associated with lower grading (Grades 1 and 2) as well as ER and progesterone receptor (PgR) positivity in all cohorts. In the treated cohort (cohort A), low androgen receptor expression was associated with shorter event-free survival (OR 2,34, 95 % CI 1.01-5.43, p = 0.047) which was not seen in the untreated cohort B. Subgroup analysis revealed that shorter survival of patients with low AR mRNA expression was observed mainly in the ER-positive subgroup of patients treated with adjuvant chemotherapy. In the validation cohort C we could confirm a benefit of chemotherapy for the group of tumors with high AR mRNA expression (5-year event-free survival (EFS) 74 % versus 57 %, p = 0.013). In this cohort, low AR mRNA expression was associated with shorter event-free survival also in multivariate analysis (OR 2.86, 95 % CI 1.29-6.35, p = 0.010) adjusted for HER2, ki-67, tumor size, age and tumor grade.We provide evidence that AR expression is associated with chemotherapy responsiveness in ER-positive patients.

Carbonic anhydrase IX (CAIX) is involved in pH homeostasis, growth and survival of tumor cells. Besides the membranous form of CAIX, a soluble form is detectable in serum (s-CAIX). Overexpression of CAIX in tumors offers the opportunity for therapeutic strategies such as CAIX targeting antibodies. The aim of this study was to examine the relationships of CAIX mRNA expression and s-CAIX levels with clinicopathological parameters and survival of patients with primary breast cancer.Tumor tissue of 169 primary breast cancer patients was analyzed for RNA expression by microarray analysis (Affymetrix HG-U133A). Concentration of s-CAIX was determined by ELISA in blood samples of 140 patients.In tumor tissue, CAIX mRNA signal intensities (MAS5 values) ranged from 34 to 2,513. Higher CAIX expression was associated with younger age (/≥50 years p = 0.040), negative hormone receptors (estrogen receptor p = 0.004; progesterone receptor p = 0.022) and positive nodal status (p = 0.001) as well as with shorter disease-free survival (p = 0.031). Concentrations of s-CAIX ranged from 56 to 1,500 pg/ml. There was no correlation between s-CAIX and CAIX mRNA levels as well as clinicopathological characteristics or outcome.In contrast to reported immunohistochemical studies, we performed RNA-based tissue analyses of CAIX expression and, for the first time, analyzed CAIX serum levels in primary breast cancer. The correlations between CAIX RNA expression and prognostic factors and patient outcome support a biologic role of CAIX in early breast cancer. A role of s-CAIX in primary breast cancer is not supported by our findings.

Objectives: Angiogenesis plays an important role in ovarian cancer. The interaction of platelet-derived growth factor receptor-beta (PDGFR-β) with vascular endothelial growth factor (VEGF) in the process of angiogenesis may represent an essential feature in the progression of the disease. Methods: Patients with epithelial ovarian cancer, who underwent primary surgery and platinum-based first-line chemotherapy, were included. A total of 133 serum samples from 39 patients were analyzed. Samples were prospectively collected at 4 time points: (1) before surgery, (2) after surgery and before chemotherapy, (3) during chemotherapy and (4) after chemotherapy. Serum PDGFR-β was quantified by ELISA. We analyzed the correlation of serum levels to chemotherapy response, progression-free and overall survival (PFS and OS) and the serum markers CA-125 and VEGF-165. Results: Serum concentration of PDGFR-β ranged between 4 and 72 ng/ml and increased significantly during first-line chemotherapy (p = 0.019). PDGFR-β serum concentrations showed an inverse correlation with CA-125 and VEGF-165 after chemotherapy (r = -0.495, p = 0.003 and r = -0.345, p = 0.04, respectively). Increased PDGFR-β serum levels after chemotherapy were significantly correlated with better PFS (p = 0.026) and OS (p = 0.013) in a univariate analysis. Conclusion: PDGFR-β might be a useful biomarker in terms of prognosis and could be important as antiangiogenic agents become a component of standard treatment in ovarian cancer.

In the present study, we investigated whether circulating cell-free microRNAs serve as potential biomarkers in epithelial ovarian cancer (EOC) patients. Circulating miR-373, miR-200a, miR-200b and miR-200c were quantified in a cohort of 60 EOC patients, 20 patients with benign ovarian diseases and 32 healthy women using quantitative TaqMan MicroRNA assays. The serum concentrations of cell-free miR-373, miR-200a, miR-200b and miR-200c were significantly higher in EOC patients than in healthy women (p = 0.0001). With a sensitivity of 83 % and a specificity of 100 %, the combination of miR-200a, miR-200b and miR-200c could differ between malignant and benign ovarian tumors (p = 0.0001). Elevated levels of these cell-free microRNAs could be detected in FIGO I-II and FIGO III-IV stages, grading G1-2 and G3 and lymph node-negative and -positive EOC. In conclusion, the increased serum levels of this microRNA panel have diagnostic value for distinguishing healthy controls and benign tumors from EOC.

In the present study, we investigated whether circulating cell-free microRNAs serve as potential biomarkers in epithelial ovarian cancer (EOC) patients. Circulating miR-373, miR-200a, miR-200b and miR-200c were quantified in a cohort of 60 EOC patients, 20 patients with benign ovarian diseases and 32 healthy women using quantitative TaqMan MicroRNA assays. The serum concentrations of cell-free miR-373, miR-200a, miR-200b and miR-200c were significantly higher in EOC patients than in healthy women (p = 0.0001). With a sensitivity of 83 % and a specificity of 100 %, the combination of miR-200a, miR-200b and miR-200c could differ between malignant and benign ovarian tumors (p = 0.0001). Elevated levels of these cell-free microRNAs could be detected in FIGO I–II and FIGO III–IV stages, grading G1-2 and G3 and lymph node-negative and -positive EOC. In conclusion, the increased serum levels of this microRNA panel have diagnostic value for distinguishing healthy controls and benign tumors from EOC.

Expression of the adhesion molecule L1-CAM (L1) has been shown to correlate with early recurrence in breast cancer. Here, we investigated whether L1-CAM expression of breast cancer cells might influence adherence to human pulmonary microvascular endothelial cells (HPMEC) and thus promote metastasis.MDA-MB231-Fra2 breast cancer cells that express high levels of L1-CAM (L1(high) cells) were stably transfected to generate clones with strong L1-CAM downregulation. Adhesion to activated HPMEC was studied in dynamic cell flow and static assays. Potential binding partners on endothelial cells were identified by blocking experiments and adhesion assays after coating of the flow channels with recombinant proteins.Adhesion of L1(high) cells to activated HPMEC was significantly higher compared to L1l(ow) clones under flow conditions. Blocking experiments and adhesion assays with recombinant proteins identified activated leucocyte cell adhesion molecule (ALCAM) or L1 itself, but not ICAM-1, as potential binding partners on endothelial cells. E-selectin blocking antibodies strongly diminished the adherence of breast cancer cells irrespective of their L1-CAM expression.Our experiments indicate that L1-CAM expression on breast cancer cells can promote adherence to activated endothelial cells by binding to endothelial L1-CAM or ALCAM. This mechanism might lead to increased metastasis and a poor prognosis in L1-CAM-positive carcinomas in vivo. Therefore, L1-CAM might be a suitable therapeutic target in breast cancers with a high L1-CAM expression.

Objective: Conflicting results have been reported about activated leukocyte cell adhesion molecule (ALCAM) expression in breast cancer and its prognostic value. Little is known about the role of ALCAM levels in the serum of breast cancer patients. Methods: We analyzed soluble ALCAM (sALCAM) levels in the serum of 157 primary breast cancer patients and 48 healthy women by ELISA. In addition, we determined ALCAM protein expression by Western blot analysis (n = 120) and mRNA expression by cDNA microarray analysis (n = 115) in the tumor tissue of corresponding patients. Results: sALCAM levels differed between patients and healthy controls (median 24.2 vs. 18.9 ng/ml, p < 0.001). We observed no correlation between serum levels and protein or mRNA expression in corresponding tumors (r < 0.1, p = n.s.). sALCAM levels were not correlated with histological type, grading, tumor stage, or patient age, but elevated sALCAM levels were associated with shorter disease-free survival (HR = 1.97, 95% CI 1.01–3.2, p = 0.043). Conclusions: Our results indicate that sALCAM can be detected in the serum of patients with primary breast cancer. Elevated serum levels might indicate more aggressive tumor behavior as they might be an independent factor for a worse prognosis in breast cancer patients.

Introduction:Therapeutic options in advanced or recurrent vulvar cancer are limited. The identification of new prognostic factors and markers for therapy stratification is therefore highly desirable. Carbonic anhydrase IX (CAIX) is up-regulated in various solid tumors and a promising new target. We therefore determined CAIX serum concentration and its prognostic relevance in correlation to intratumoral CAIX expression in patients with primary vulvar cancer.Methods:Thirty-one serum samples of patients with primary vulvar cancer were prospectively collected before surgery and analyzed for CAIX by enzyme-linked immunosorbent assay. In addition, intratumoral CAIX expression was determined by immunohistochemistry and correlation with serum CAIX and clinicopathological factors, and outcome was analyzed.Results:Preoperative serum concentration of CAIX ranged between 56 and 879 pg/mL (median, 147 pg/mL; mean, 237.29) and was significantly higher in patients with high intratumoral expression (median, 269 pg/mL vs 126 pg/mL,P= 0.03). High serum CAIX was not associated with any of the analyzed clinicopathological parameters. However, disease-free survival was shorter in patients with high preoperative serum CAIX (above median;P= 0.012). By immunohistochemistry, 26% of the tumors showed a moderate or strong expression of CAIX, whereas 74% showed weak or no expression. High intratumoral expression of CAIX was also associated with unfavorable disease-free survival (P= 0.043).Conclusions:Carbonic anhydrase IX serum concentration is higher in patients with high intratumoral expression, and elevated preoperative serum values are associated with unfavorable prognosis. Serum CAIX might therefore be an easily assessable marker to stratify patients for adjuvant therapy and potentially monitor response. Carbonic anhydrase IX is differentially expressed in vulvar cancer and potentially associated with negative outcome.

The activated leukocyte cell adhesion molecule (ALCAM) is overexpressed in many mammary tumors, but controversial results about its role and prognostic impact in breast cancer have been reported. Therefore, we evaluated the biologic effects of ALCAM expression in two breast cancer cell lines and a larger cohort of mammary carcinomas. By stable transfections, MCF7 cells with ALCAM overexpression and MDA-MB231 cells with reduced ALCAM levels were generated and analyzed in functional assays and cDNA microarrays. In addition, an immunohistochemical study on 347 patients with breast cancer with long-term follow-up and analysis of disseminated tumor cells (DTCs) was performed. In both cell lines, high ALCAM expression was associated with reduced cell motility. In addition, ALCAM silencing in MDA-MB231 cells resulted in lower invasive potential, whereas high ALCAM expression was associated with increased apoptosis in both cell lines. Among genes which were differentially expressed in clones with altered ALCAM expression, there was an overlap of 15 genes between both cell lines, among them cathepsin D, keratin 7, gelsolin, and ets2 whose deregulation was validated by western blot analysis. In MDA-MB231 cells, we observed a correlation with VEGF expression which was validated by enzyme-linked immuno sorbent assay (ELISA). Our IHC results on primary breast carcinomas showed that ALCAM expression was associated with an estrogen receptor-positive phenotype. In addition, strong ALCAM immunostaining correlated with nodal involvement and the presence of tumor cells in bone marrow. By Kaplan–Meier analysis, strong ALCAM expression in ductal carcinomas correlated with shorter recurrence-free intervals (P = 0.048) and overall survival (OAS, P = 0.003). Our results indicate that the biologic role of ALCAM in breast cancer is complex, but overexpression might be relevant for outcome in ductal carcinomas.

Members of the Fos protein family dimerise with Jun proteins to form the AP-1 transcription factor complex. They have a central function in proliferation and differentiation of normal tissue as well as in oncogenic transformation and tumour progression. We analysed the expression of c-Fos, FosB, Fra-1 and Fra-2 to investigate the function of Fos transcription factors in ovarian cancer. A total of 101 patients were included in the study. Expression of Fos proteins was determined by western blot analysis, quantified by densitometry and verified by immunohistochemistry. Reduced c-Fos expression was independently associated with unfavourable progression-free survival (20.6, 31.6 and 51.2 months for patients with low, moderate and high c-Fos expression; P=0.003) as well as overall survival (23.8, 46.0 and 55.5 months for low, moderate and high c-Fos levels; P=0.003). No correlations were observed for FosB, Fra-1 and Fra-2. We conclude that loss of c-Fos expression is associated with tumour progression in ovarian carcinoma and that c-Fos may be a prognostic factor. These results are in contrast to the classic concept of c-Fos as an oncogene, but are supported by the recently discovered tumour-suppressing and proapoptotic function of c-Fos in various cancer types.

Zusammenfassung: Ziele: "Activated leukocyte cell adhesion molecule" (ALCAM) ist ein Oberflächenimmunglobulin und wird in vielen Mammakarzinomen exprimiert. Es wird vermutet, dass ALCAM in der Tumorgenese und -progression eine Rolle spielt. Die Bedeutung des Adhäsionsmoleküls ALCAM für das Ansprechen auf eine taxanfreie adjuvante Chemotherapie wurde untersucht. Material und Methoden: Gewebeproben von 162Patientinnen mit Mammakarzinomen wurden im Hinblick auf die Expression des ALCAM-Proteins untersucht. Immunhistologische Untersuchungen (IHC) und Western-Blot-Analysen (WB) wurden mit einem monoklonalen Antikörper gegen ALCAM durchgeführt. Die Proteinmengen im WB wurden densitometrisch quantifiziert und mit klinischen sowie histologischen Parametern korreliert. Die Ergebnisse wurden auf mRNA-Ebene mithilfe der Microarray-Analysen (Affymetrix) validiert. Ergebnisse: In der normalen Brustdrüse wird ALCAM in den luminalen und den basalen Epithelzellen exprimiert. Die WB-Analysen von Mammakarzinomen zeigen eine positive Korrelation der ALCAM-Expression mit dem Östrogenrezeptorstatus (p=0,04). Bei Patientinnen, die eine taxanfreie Chemotherapie erhalten haben, ist eine hohe ALCAM-Expression (WB, Affymetrix) prädikitiv für das Ansprechen einer Chemotherapie. Der mediane mRNA-Level von ALCAM war bei Patientinnen, die während des Beobachtungszeitraums noch lebten, 4,5-fach höher als bei Patientinnen, die verstarben. Schlussfolgerung: Ein hoher ALCAM-Gehalt korreliert positiv mit dem Östrogenrezeptorstatus. Außerdem ist ALCAM ein prädiktiver Faktor für das Ansprechen einer taxanfreien Chemotherapie