Neoadjuvant chemotherapy and bevacizumab for HER2-negative breast cancer.

BACKGROUND: Bevacizumab, a monoclonal antibody against vascular endothelial growth factor A, has shown clinical efficacy in patients with human epidermal growth factor receptor 2 (HER2)-negative metastatic breast cancer. We evaluated the efficacy, measured according to the rate of pathological complete response (absence of invasive and intraductal disease in the breast and the axillary lymph nodes), and the safety of adding bevacizumab to neoadjuvant chemotherapy in patients with early-stage breast cancer. METHODS: We randomly assigned 1948 patients with a median tumor size of 40 mm on palpation to receive neoadjuvant epirubicin and cyclophosphamide followed by docetaxel, with or without concomitant bevacizumab. Patients with untreated HER2-negative breast cancer were eligible if they had large tumors, hormone-receptor-negative disease, or hormone-receptor-positive disease with palpable nodes or positive findings on sentinel-node biopsy, and no increased cardiovascular or bleeding risk. RESULTS: Overall, the rates of pathological complete response were 14.9% with epirubicin and cyclophosphamide followed by docetaxel and 18.4% with epirubicin and cyclophosphamide followed by docetaxel plus bevacizumab (odds ratio with addition of bevacizumab, 1.29; 95% confidence interval, 1.02 to 1.65; P=0.04); the corresponding rates of pathological complete response were 27.9% and 39.3% among 663 patients with triple-negative tumors (P=0.003) and 7.8% and 7.7% among 1262 patients with hormone-receptor-positive tumors (P=1.00). Breast-conserving surgery was possible in 66.6% of the patients in both groups. The addition of bevacizumab, as compared with neoadjuvant therapy alone, was associated with a higher incidence of grade 3 or 4 toxic effects (febrile neutropenia, mucositis, the hand-foot syndrome, infection, and hypertension) but with a similar incidence of surgical complications. CONCLUSIONS: The addition of bevacizumab to neoadjuvant chemotherapy significantly increased the rate of pathological complete response among patients with HER2-negative early-stage breast cancer. Efficacy was restricted primarily to patients with triple-negative tumors, in whom the pathological complete response is considered to be a reliable predictor of long-term outcome. (Funded by Sanofi-Aventis and Roche, Germany; ClinicalTrials.gov number, NCT00567554.).

Differential expression of histone deacetylases HDAC1, 2 and 3 in human breast cancer--overexpression of HDAC2 and HDAC3 is associated with clinicopathological indicators of disease progression.

BACKGROUND: In breast cancer, the role of epigenetic alterations including modifications of the acetylation status of histones in carcinogenesis has been an important research focus during the last years. An increased deacetylation of histones leads to increased cell proliferation, cell migration, angiogenesis and invasion. Class 1 histone deacetylases (HDAC) seem to be most important during carcinogenesis. METHODS: The immunhistochemical expression of HDAC1, 2 and 3 was analyzed on tissue microarrays (TMAs) from 238 patients with primary breast cancer. We analyzed the nuclear staining intensity (negative, weak, moderate, strong) as well as the percentage of positive tumor cells and calculated the immunoreactivity score (0-12). Expression was correlated with clinicopathological parameters and patient survival. RESULTS: In this cohort, we found a differential positive expression of HDAC1, HDAC2 and HDAC3. HDAC2 and HDAC3 expression was significantly higher in less differentiated tumors: HDAC2 (n=207), p<0.001 and HDAC3 (n=220), p<0.001 and correlated with negative hormone receptor status: HDAC2 (n=206), p=0.02 and HDAC3 (n=219), p=0.04. Additionally, a high HDAC2 expression was significantly associated with an overexpression of HER2 (n=203, p=0.005) and the presence of nodal metastasis (n=200, p=0.04).HDAC1 was highly expressed in hormone receptor positive tumors (n=203; p<0.001). CONCLUSION: As a conclusion, our results show that the class-1 HDAC isoenzymes 1, 2 and 3 are differentially expressed in breast cancer. HDAC2 and HDAC3 are strongly expressed in subgroups of tumor with features of a more aggressive tumor type.

Integration of metabolomics and expression of glycerol-3-phosphate acyltransferase (GPAM) in breast cancer-link to patient survival, hormone receptor status, and metabolic profiling.

Changes in lipid metabolism are an important but not well-characterized hallmark of cancer. On the basis of our recent findings of lipidomic changes in breast cancer, we investigated glycerol-3-phosphate acyltransferase (GPAM), a key enzyme in the lipid biosynthesis of triacylglycerols and phospholipids. GPAM protein expression was evaluated and linked to metabolomic and lipidomic profiles in a cohort of human breast carcinomas. In addition, GPAM mRNA expression was analyzed using the GeneSapiens in silico transcriptiomics database. High cytoplasmic GPAM expression was associated with hormone receptor negative status (p = 0.013). On the protein (p = 0.048) and mRNA (p = 0.001) levels, increased GPAM expression was associated with a better overall survival. Metabolomic analysis by GC-MS showed that sn-glycerol-3-phosphate, the substrate of GPAM, was elevated in breast cancer compared to normal breast tissue. LC-MS based lipidomic analysis identified significantly higher levels of phospholipids, especially phosphatidylcholines in GPAM protein positive tumors. In conclusion, our results suggest that GPAM is expressed in human breast cancer with associated changes in the cellular metabolism, in particular an increased synthesis of phospholipids, the major structural component of cellular membranes.

Remodeling of central metabolism in invasive breast cancer compared to normal breast tissue - a GC-TOFMS based metabolomics study.

BACKGROUND: Changes in energy metabolism of the cells are common to many kinds of tumors and are considered a hallmark of cancer. Gas chromatography followed by time-of-flight mass spectrometry (GC-TOFMS) is a well-suited technique to investigate the small molecules in the central metabolic pathways. However, the metabolic changes between invasive carcinoma and normal breast tissues were not investigated in a large cohort of breast cancer samples so far. RESULTS: A cohort of 271 breast cancer and 98 normal tissue samples was investigated using GC-TOFMS-based metabolomics. A total number of 468 metabolite peaks could be detected; out of these 368 (79%) were significantly changed between cancer and normal tissues (p<0.05 in training and validation set). Furthermore, 13 tumor and 7 normal tissue markers were identified that separated cancer from normal tissues with a sensitivity and a specificity of >80%. Two-metabolite classifiers, constructed as ratios of the tumor and normal tissues markers, separated cancer from normal tissues with high sensitivity and specificity. Specifically, the cytidine-5-monophosphate / pentadecanoic acid metabolic ratio was the most significant discriminator between cancer and normal tissues and allowed detection of cancer with a sensitivity of 94.8% and a specificity of 93.9%. CONCLUSIONS: For the first time, a comprehensive metabolic map of breast cancer was constructed by GC-TOF analysis of a large cohort of breast cancer and normal tissues. Furthermore, our results demonstrate that spectrometry-based approaches have the potential to contribute to the analysis of biopsies or clinical tissue samples complementary to histopathology.

Decentral gene expression analysis: analytical validation of the Endopredict genomic multianalyte breast cancer prognosis test.

BACKGROUND: EndoPredict (EP) is a clinically validated multianalyte gene expression test to predict distant metastasis in ER-positive, HER2-negative breast cancer treated with endocrine therapy alone. The test is based on the combined analysis of 12 genes in formalin-fixed, paraffin-embedded (FFPE) tissue by reverse transcription-quantitative real-time PCR (RT-qPCR). Recently, it was shown that EP is feasible for reliable decentralized assessment of gene expression. The aim of this study was the analytical validation of the performance characteristics of the assay and its verification in a molecular-pathological routine laboratory. METHODS: Gene expression values to calculate the EP score were assayed by one-step RT-qPCR using RNA from FFPE tumor tissue. Limit of blank, limit of detection, linear range, and PCR efficiency were assessed for each of the 12 PCR assays using serial samples dilutions. Different breast cancer samples were used to evaluate RNA input range, precision and inter-laboratory variability. RESULTS: PCR assays were linear up to Cq values between 35.1 and 37.2. Amplification efficiencies ranged from 75% to 101%. The RNA input range without considerable change of the EP score was between 0.16 and 18.5 ng/µl. Analysis of precision (variation of day, day time, instrument, operator, reagent lots) resulted in a total noise (standard deviation) of 0.16 EP score units on a scale from 0 to 15. The major part of the total noise (SD 0.14) was caused by the replicate-to-replicate noise of the PCR assays (repeatability) and was not associated with different operating conditions (reproducibility). Performance characteristics established in the manufacturer's laboratory were verified in a routine molecular pathology laboratory. Comparison of 10 tumor samples analyzed in two different laboratories showed a Pearson coefficient of 0.995 and a mean deviation of 0.15 score units. CONCLUSIONS: The EP test showed reproducible performance characteristics with good precision and negligible laboratory-to-laboratory variation. This study provides further evidence that the EP test is suitable for decentralized testing in specialized molecular pathological laboratories instead of a reference laboratory. This is a unique feature and a technical advance in comparison with existing RNA-based prognostic multigene expression tests.

Androgen receptor expression in primary breast cancer and its predictive and prognostic value in patients treated with neoadjuvant chemotherapy.

The androgen receptor (AR) has been shown to be of potential prognostic importance in retrospective cohorts. We evaluated immunohistochemical AR expression on a tissue microarray of 673 core biopsies from primary breast cancer patients treated with neoadjuvant docetaxel/doxorubicin/cyclophosphamide (TAC) chemotherapy in the prospective GeparTrio phase-III trial. AR was detected in 53.2% of tumours. Lowest AR expression was detected in triple-negative breast cancers (TNBC) with 21.2%. Highest AR expression was observed in Luminal A-like tumours with 67%. In AR-positive tumours, pathological complete response (pCR) rate was 12.8% compared to 25.4% in AR-negative tumours (P < 0.0001). In multivariate analysis, AR independently predicted pCR (OR 1.86; 95% CI [1.16-2.79] P = 0.0086). Overall patients with an AR-positive tumour had a significant better disease-free (DFS) (AR-positive 78.9% vs. AR-negative 72.5%; log-rank P = 0.0329) and overall survival (OS) (88.8% vs. 82.7%; log-rank P = 0.0234) than those with AR-negative tumours. Stratified analysis revealed that in the TNBC subgroup, but not in the other subgroups defined by ER, PgR and HER2, AR expression predicted a better DFS (AR-positive 85.7% vs. AR-negative 65.5% log-rank P = 0.0544) and OS (95.2% vs. 76.2%; log-rank P = 0.0355). Within the non-pCR subgroup, AR positivity selected a group with a significant better DFS (P = 0.045) and OS (0.021) but not within the pCR group. Patients with an AR-negative tumour have a higher chance of achieving a pCR than those with an AR-positive one. But, patients with AR-positive tumours have a better survival especially if they did not achieve a pCR.

Prognostic significance of Dicer expression in ovarian cancer-link to global microRNA changes and oestrogen receptor expression.

MicroRNA (miRNA) deregulation is a hallmark of human cancer. However, the mechanisms underlying miRNA alteration and the specific role of proteins involved in miRNA processing remains to be elucidated. Dicer is a key enzyme in the miRNA processing pathway that is essential for the production of mature miRNAs from their precursors. We tested the hypothesis that Dicer has biological and clinical relevance in ovarian cancer, using a range of methods including in vitro manipulation of Dicer expression. We observed down-regulation of Dicer in a subgroup of ovarian carcinomas, and found that decreased Dicer expression correlates significantly with reduced patient survival in serous cancers and advanced disease stages. Moreover, microarray and functional analysis suggest that reduced Dicer expression is connected with a global down-regulation of the microRNAome and with gene expression changes, particularly reduced expression of oestrogen receptor (ER) mRNA and protein in tumour tissue and in cell culture. Our data suggest a common mechanism for miRNAs changes by alterations in the basic machinery controlling miRNA biogenesis, of which Dicer is a central enzyme. These alterations of miRNA processing are of prognostic value and may play a role in the molecular pathogenesis of ovarian carcinoma and, possibly, other tumours. Knowledge of these molecular pathways may help toward new targeted therapeutic approaches for ovarian cancer.

Expression of classical NF-kappaB pathway effectors in human ovarian carcinoma.

AIMS: Functional studies have demonstrated that nuclear factor (NF)-kappaB promotes tumour progression in ovarian cancer cells. However, surprisingly little is known of the expression of effectors of the NF-kappaB pathway in human ovarian cancer in vivo. METHODS AND RESULTS: Immunohistochemistry and in situ hybridization revealed that in a cohort of 85 primary ovarian carcinomas, total p65 expression was inversely correlated to nuclear and cytoplasmic phospho-IkappaBalpha (P = 0.002 and P = 0.05, respectively), and IkappaBalpha mRNA expression (P = 0.032). In contrast, phospho-p65 expression was paralleled by the expression of nuclear (P = 0.027) and cytoplasmic phospho-IkappaBalpha (P = 0.01). Total p65 expression was an adverse prognostic factor for overall survival (P = 0.018). In contrast, total IkappaBalpha and phosphorylated nuclear and cytoplasmic IkappaBalpha expression were favourable prognostic markers (P = 0.001, P = 0.031, P = 0.001, respectively). Cytoplasmic phospho-IkappaBalpha expression remained a significant prognostic factor on multivariate analysis (P = 0.010). In cultured, stimulated OVCAR-3 ovarian cancer cells the cytoplasmic retranslocation of p65 was delayed by inhibition of the nuclear membrane transporter chromosomal region maintenance/exportin1 protein (CRM1). A positive association of p65 and CRM1 expression was demonstrated in ovarian cancer tissue (P < 0.0001). CONCLUSIONS: Total and phosphorylated IkappaBalpha protein expression might serve as markers for NF-kappaB activation in human ovarian carcinoma. Cytoplasmic localization of p65 may be related to deregulated nucleocytoplasmic transport in carcinomas overexpressing CRM1.

A prognostic gene expression index in ovarian cancer - validation across different independent data sets.

Ovarian carcinoma has the highest mortality rate among gynaecological malignancies. In this project, we investigated the hypothesis that molecular markers are able to predict outcome of ovarian cancer independently of classical clinical predictors, and that these molecular markers can be validated using independent data sets. We applied a semi-supervised method for prediction of patient survival. Microarrays from a cohort of 80 ovarian carcinomas (TOC cohort) were used for the development of a predictive model, which was then evaluated in an entirely independent cohort of 118 carcinomas (Duke cohort). A 300-gene ovarian prognostic index (OPI) was generated and validated in a leave-one-out approach in the TOC cohort (Kaplan-Meier analysis, p = 0.0087). In a second validation step, the prognostic power of the OPI was confirmed in an independent data set (Duke cohort, p = 0.0063). In multivariate analysis, the OPI was independent of the post-operative residual tumour, the main clinico-pathological prognostic parameter with an adjusted hazard ratio of 6.4 (TOC cohort, CI 1.8-23.5, p = 0.0049) and 1.9 (Duke cohort, CI 1.2-3.0, p = 0.0068). We constructed a combined score of molecular data (OPI) and clinical parameters (residual tumour), which was able to define patient groups with highly significant differences in survival. The integrated analysis of gene expression data as well as residual tumour can be used for optimized assessment of the prognosis of platinum-taxol-treated ovarian cancer. As traditional treatment options are limited, this analysis may be able to optimize clinical management and to identify those patients who would be candidates for new therapeutic strategies.

Identification of biology-based breast cancer types with distinct predictive and prognostic features: role of steroid hormone and HER2 receptor expression in patients treated with neoadjuvant anthracycline/taxane-based chemotherapy.

INTRODUCTION: Reliable predictive and prognostic markers for routine diagnostic purposes are needed for breast cancer patients treated with neoadjuvant chemotherapy. We evaluated protein biomarkers in a cohort of 116 participants of the GeparDuo study on anthracycline/taxane-based neoadjuvant chemotherapy for operable breast cancer to test for associations with pathological complete response (pCR) and disease-free survival (DFS). Particularly, we evaluated if interactions between hormone receptor (HR) and human epidermal growth factor receptor 2 (HER2) expression might lead to a different clinical behavior of HR+/HER2+ co-expressing and HR+/HER2- tumors and whether subgroups of triple negative tumors might be identified by the help of Ki67 labeling index, cytokeratin 5/6 (CK5/6), as well as cyclooxygenase-2 (COX-2), and Y-box binding protein 1 (YB-1) expression. METHODS: Expression analysis was performed using immunohistochemistry and silver-enhanced in situ hybridization on tissue microarrays (TMAs) of pretherapeutic core biopsies. RESULTS: pCR rates were significantly different between the biology-based tumor types (P = 0.044) with HR+/HER2+ and HR-/HER2- tumors having higher pCR rates than HR+/HER2- tumors. Ki67 labeling index, confirmed as significant predictor of pCR in the whole cohort (P = 0.001), identified HR-/HER- (triple negative) carcinomas with a higher chance for a pCR (P = 0.006). Biology-based tumor type (P = 0.046 for HR+/HER2+ vs. HR+/HER2-), Ki67 labeling index (P = 0.028), and treatment arm (P = 0.036) were independent predictors of pCR in a multivariate model. DFS was different in the biology-based tumor types (P < 0.0001) with HR+/HER2- and HR+/HER2+ tumors having the best prognosis and HR-/HER2+ tumors showing the worst outcome. Biology-based tumor type was an independent prognostic factor for DFS in multivariate analysis (P < 0.001). CONCLUSIONS: Our data demonstrate that a biology-based breast cancer classification using estrogen receptor (ER), progesterone receptor (PgR), and HER2 bears independent predictive and prognostic potential. The HR+/HER2+ co-expressing carcinomas emerged as a group of tumors with a good response rate to neoadjuvant chemotherapy and a favorable prognosis. HR+/HER2- tumors had a good prognosis irrespective of a pCR, whereas patients with HR-/HER- and HR-/HER+ tumors, especially if they had not achieved a pCR, had an unfavorable prognosis and are in need of additional treatment options.

Topoisomerase IIalpha mRNA and protein expression in ovarian carcinoma: correlation with clinicopathological factors and prognosis.

Topoisomerase IIalpha (Top IIalpha) is a nuclear enzyme that plays a central role in DNA metabolism, and is a molecular target for a variety of chemotherapeutic agents. Top IIalpha has recently gained attention as a biomarker for therapy response and patient survival. In this study, we attempted to assess the feasibility of measuring Top IIalpha gene expression in RNA, isolated from archival formalin-fixed paraffin-embedded tissue specimens, which are used routinely in pathology laboratories. We have employed a new technique on the basis of magnetic particles' separation and purification of nucleic acids, and evaluated both protein and mRNA expressions from the same routinely processed tissue blocks. We investigated the expression of Top IIalpha mRNA and protein by real-time reverse transcription polymerase chain reaction and immunohistochemistry, in a cohort of 133 primary ovarian carcinomas, and evaluated the association between Top IIalpha expression and clincopathological variables as well as patient outcome. Elevated Top IIalpha mRNA expression was observed in high-grade tumors (P=0.003) and advanced stage disease (P=0.011). In univariate Kaplan-Meier analysis, patients with higher expression of Top IIalpha nuclear protein had a significantly decreased overall survival (P=0.045). Interestingly, we detected cytoplasmic protein expression of Top IIalpha in a subset of samples. Cytoplasmic expression of Top IIalpha was associated with the expression of chromosomal region maintenance/exportin 1 (CRM1)-a nuclear export protein (P=0.008). Our study suggests that Top IIalpha overexpression is involved in the progression of ovarian cancer in a subset of the patients. Our results encourage the further evaluation of the prognostic and predictive values of Top IIalpha expression in ovarian carcinoma, which might help to assess the patients' risk profile, and the planning of an individualized therapy.

Expression of multidrug resistance-associated protein 1 in invasive ovarian carcinoma: implication for prognosis.

AIMS: Multidrug resistance is a major impediment in chemotherapeutic treatment of ovarian carcinoma patients. The aim of this study was to investigate the expression of multidrug resistance-associated protein 1 (MRP1) and to assess the possible associations with clinicopathological variables and patient outcome in primary ovarian carcinoma. METHODS AND RESULTS: Tumour specimens from 129 patients were obtained before chemotherapy and analysed by immunohistochemistry on tissue microarrays, and by real-time reverse transcriptase-polymerase chain reaction on RNA extracted from formalin-fixed paraffin-embedded tissue specimens using a new technique. Significantly increased MRP1 protein expression was observed in high-grade tumours (P = 0.005) and advanced International Federation of Gynaecology and Obstetrics stages (P = 0.036). On univariate Kaplan-Meier analysis, patients with higher expression of MRP1 protein had significantly decreased overall survival (P = 0.006). On multivariate Cox regression analysis, MRP1 protein expression retained its significance as an independent negative prognostic marker for overall survival (hazard ratio = 6.52, P = 0.003). Furthermore, MRP1 expression correlated with topoisomerase IIalpha expression both at mRNA and protein level (P < 0.001 and P = 0.023, respectively). CONCLUSION: In summary, in patients with primary ovarian cancer, overexpression of MRP1 is an adverse marker for patient outcome and cancer aggressiveness. Our data provide a translational basis for further clinical studies on the predictive value of MRP1 expression for response to chemotherapy.

High class I HDAC activity and expression are associated with RelA/p65 activation in pancreatic cancer in vitro and in vivo.

BACKGROUND: The strong association between aberrant HDAC activity and the occurrence of cancer has led to the development of a variety of HDAC inhibitors (HDIs), which emerge as promising new targeted anticancer therapeutics. METHODS: Due to the pivotal role of RelA/p65 in the tumorigenesis of pancreatic neoplasia we examined the expression of class I HDACs 1, 2 and 3 in a large cohort of human pancreatic carcinomas and correlated our findings with RelA/p65 expression status. Furthermore, we investigated the impact of the HDIs SAHA and VPA on RelA/p65 activity in pancreatic cancer cell culture models. RESULTS: Class I HDACs were strongly expressed in a subset of pancreatic adenocarcinomas and high expression was significantly correlated with increased nuclear translocation of RelA/p65 (p = 0.024). The link of HDAC activity and RelA/p65 in this tumor entity was confirmed in vitro, where RelA/p65 nuclear translocation as well as RelA/p65 DNA binding activity could be markedly diminished by HDI treatment. CONCLUSION: The RelA/p65 inhibitory effects of SAHA and VPA in vitro and the close relationship of class I HDACs and RelA/p65 in vivo suggest that treatment with HDIs could serve as a promising approach to suppress NF-kappaB activity which in turn may lead to enhanced apoptosis and chemosensitization of pancreatic cancers.

IMP3 expression in human ovarian cancer is associated with improved survival.

The insulin-like growth factor-II mRNA-binding protein IMP3 plays an important role in embryogenesis and recent reports suggest an involvement in tumorigenesis. Although IMP3 expression has been well studied in mouse and human fetal and adult gonads, its role in ovarian cancer is unknown. We investigated the expression of IMP3 at protein and mRNA levels in a cohort of primary ovarian carcinomas and in 11 ovarian cancer cell lines. Western blot analysis revealed an expression of IMP3 in all ovarian cancer cell lines and immunohistochemistry demonstrated a positive cytoplasmic staining in 32 of 68 carcinomas (47%). In contrast, epithelium of borderline tumors, as well as, benign ovarian lesions and normal ovaries exhibited only weak or no IMP3 expression. In univariate Kaplan-Meier analysis, IMP3 protein expression was significantly associated with better overall survival (P=0.048). To confirm these findings, we further determined IMP3 mRNA expression in 43 ovarian cancer specimens by real time quantitative reverse transcription-polymerase chain reaction. A significant correlation between protein and mRNA levels (r=0.414, P=0.006), as well as a correlation of IMP3 mRNA expression with patient overall survival (P=0.044), was observed. Our results demonstrate that IMP3 is expressed in a subpopulation of ovarian cancer and a marker of good prognosis.

Polo-like kinase isoforms in breast cancer: expression patterns and prognostic implications.

Polo-like kinase (PLK) family members are known to be functionally involved in mitotic signaling and in cytoskeletal reorganization in both normal and malignant cells. In this study, expression of PLK1 and PLK3 was determined immunohistochemically in tissue specimens of 135 breast carcinomas, and expression was correlated with clinicopathological parameters and patient prognosis. Strong PLK isoform overexpression was observed in 42.2% (PLK1) and 47.4% (PLK3) of breast carcinomas when compared with non-transformed breast tissue. A positive correlation of PLK isoform expression with tumor grade, vascular invasion, erbB2/HER-2 expression and markers of proliferative activity was evident. Additionally, an inverse correlation of PLK isoform expression and estrogen receptor status was observed. Overexpression of PLK3 but not of PLK1 was significantly linked to reduced median overall (P<0.001) and relapse-free (P=0.021) survival time. PLK3 expression remained an independent prognostic factor for overall (RR=3.2, P=0.002) and relapse-free (RR=2.9, P=0.049) survival in multivariate survival analysis. These results suggest PLK3 as a novel independent prognostic marker in breast cancer and hint toward a role for PLK isoform overexpression in disease progression. Therefore, in vivo inhibition of PLK family members might represent a rewarding approach in the development of new anticancer drugs for this tumor entity.

Expression of the ELAV-like protein HuR is associated with higher tumor grade and increased cyclooxygenase-2 expression in human breast carcinoma.

PURPOSE: The human ELAV (embryonic lethal abnormal vision)-like protein HuR stabilizes a certain group of cellular mRNAs that contain AU-rich elements in their 3'-untranslated region. Cell culture studies have shown that the mRNA of cyclooxygenase (COX)-2 can be stabilized by HuR. EXPERIMENTAL DESIGN: To investigate a possible contribution of dysregulation of mRNA stability to the progression of cancer and to overexpression of COX-2, we studied expression of HuR in 208 primary breast carcinomas by immunohistochemistry. RESULTS: There were two different staining patterns of HuR in tumor tissue of breast carcinomas: nuclear expression was seen in 61% of cases; and an additional cytoplasmic expression was seen in 30% of cases. Expression of HuR was significantly associated with increased COX-2 expression; this association was particularly significant for cytoplasmic HuR expression (P < 0.0005). We further observed a significant association of cytoplasmic (P = 0.002) or nuclear HuR (P = 0.027) expression with increased tumor grade. Only 13% of the grade 1 carcinomas showed cytoplasmic expression of HuR, compared with 46% of the grade 3 carcinomas. There was no significant correlation between HuR expression and other clinicopathological parameters such as histological type, tumor size, or nodal status as well as patient survival. CONCLUSIONS: Our results suggest that overexpression of HuR in tumor tissue may be part of a regulatory pathway that controls the mRNA stability of several important targets in tumor biology, such as COX-2. Based on our results, additional studies are necessary to investigate whether HuR might be a potential target for molecular tumor therapy.

Quantitative imaging of the tissue contrast agent [Gd(DTPA)]²â» in articular cartilage by laser ablation inductively coupled plasma mass spectrometry.

Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) is an emerging analytical technique in the generation of quantitative images of MR contrast agent distribution in thin tissue sections of articular cartilage. An analytical protocol is described that includes sample preparation by cryo-cutting of tissue sections, mass spectrometric measurements by LA-ICP-MS and quantification of gadolinium images by one-point calibration, standard addition method (employing matrix-matched laboratory standards) and isotope dilution analysis using highly enriched stable Gd-155 isotope (abundance 92 vs 14.8% in the [Gd(DTPA)]²â» contrast agent). The tissue contrast agent concentrations of [Gd(DTPA)]²â» in cartilage measured in this work are in agreement with findings obtained by magnetic resonance imaging and other analytical methodologies. The LA-ICP-MS imaging data also confirm the observation that the spatial distribution of [Gd(DTPA)]²â» in the near-equilibrium state is highly inhomogeneous across cartilage thickness with the highest concentration measured in superficial cartilage and a strong decrease toward the subchondral bone. In the present work, it is shown for the first time that LA-ICP-MS can be applied to validate the results from quantitative gadolinium-enhanced MRI technique of articular cartilage.

Comparative metabolomics of estrogen receptor positive and estrogen receptor negative breast cancer: alterations in glutamine and beta-alanine metabolism.

Molecular subtyping of breast cancer is necessary for therapy selection and mandatory for all breast cancer patients. Metabolic alterations are considered a hallmark of cancer and several metabolic drugs are currently being investigated in clinical trials. However, the dependence of metabolic alterations on breast cancer subtypes has not been investigated on -omics scale. Thus, 204 estrogen receptor positive (ER+) and 67 estrogen receptor negative (ER-) breast cancer tissues were investigated using GC-TOFMS based metabolomics. 19 metabolites were detected as altered in a predefined training set (2/3 of tumors) and could be validated in a predefined validation set (1/3 of tumors). The metabolite changes included increases in beta-alanine, 2-hydroyglutarate, glutamate, xanthine and decreases in glutamine in the ER- subtype. Beta-alanine demonstrated the strongest change between ER- and ER+ breast cancer (fold change=2.4, p=1.5E-20). In a correlation analysis with genome-wide expression data in a subcohort of 154 tumors, we found a strong negative correlation (Spearman R=-0.62) between beta-alanine and 4-aminobutyrate aminotransferase (ABAT). Immunohistological analysis confirmed down-regulation of the ABAT protein in ER- breast cancer. In a Kaplan-Meier analysis of a large external expression data set, the ABAT transcript was demonstrated to be a positive prognostic marker for breast cancer (HR=0.6, p=3.2E-15). BIOLOGICAL SIGNIFICANCE: It is well-known for more than a decade that breast cancer exhibits distinct gene expression patterns depending on the molecular subtype defined by estrogen receptor (ER) and HER2 status. Here, we show that breast cancer exhibits distinct metabolomics patterns depending on ER status. Our observation supports the current view of ER+ breast cancer and ER- breast as different diseases requiring different treatment strategies. Metabolic drugs for cancer including glutaminase inhibitors are currently under development and tested in clinical trials. We found glutamate enriched and glutamine reduced in ER- breast cancer compared to ER+ breast cancer and compared to normal breast tissues. Thus, metabolomics analysis highlights the ER- subtype as a preferential target for glutaminase inhibitors. For the first time, we report on a regulation of beta-alanine catabolism in cancer. In breast cancer, ABAT transcript expression was variable and correlated with ER status. Low ABAT transcript expression was associated with low ABAT protein expression and high beta-alanine concentration. In a large external microarray cohort, low ABAT expression shortened recurrence-free survival in breast cancer, ER+ breast cancer and ER- breast cancer.

The EndoPredict Gene-Expression Assay in Clinical Practice - Performance and Impact on Clinical Decisions.

The validated EndoPredict assay is a novel tool to predict the risk of metastases of patients with estrogen receptor positive, HER2 negative breast cancer treated with endocrine therapy alone. It has been designed to integrate genomic and clinical information and includes clinico-pathological factors such as tumor size and nodal status. The test is feasible in a decentral setting in molecular pathology laboratories. In this project, we investigated the performance of this test in clinical practice, and performed a retrospective evaluation of its impact on treatment decisions in breast cancer. During one year, EndoPredict assays from 167 patients could be successfully performed. For retrospective evaluation of treatment decisions, a questionnaire was sent to the clinical partner. Regarding the molecular EP class, samples from 56 patients (33.5%) had a low-risk, whereas 111 patients (66.5%) showed a high-risk gene profile. After integration of the clinicopathological factors the combined clinical and molecular score (EPclin) resulted in a low-risk group of 77 patients (46.4%), while 89 (53.6%) had a high risk EPclin score. The EPclin-based estimated median 10-year-risk for metastases with endocrine therapy alone was 11% for the whole cohort. The median handling time averaged three days (range: 0 to 11 days), 59.3% of the tests could be performed in three or less than three days. Comparison of pre- and post-test therapy decisions showed a change of therapy in 37.7% of patients. 16 patients (12.3%) had a change to an additional chemotherapy while 25.4% of patients (n = 33) changed to an endocrine therapy alone. In 73 patients (56.2%) no change of therapy resulted. In 6.1% of patients (n = 8), the patients did not agree to the recommendation of the tumor board. Our results show that the EndoPredict assay could be routinely performed in decentral molecular pathology laboratories and the results markedly change treatment decisions.