Serum Of Breast Cancer Patients Research Articles

Epigenetics Differences between Estrogen Receptor (ERS1) and Her2/Neu Status in Breast Cancer Patients.

Abstract Background: The CpG island methylator phenotype is associated with distinct clinicophatological characteristics as Estrogen Receptor (ESR1) positive and amplification of HER2 in breast cancer.Objetive: To evaluate epigenetics differences in tumor-related genes to ERS1 and HER2/neu status in primary breast cancer.Material and Methods: We quantified methylation levels of promoter of 5 genes (ERS1, RAR- β, 14-3-3 sigma, APC, E-Cadherin) which are to confer growth adventage to cells, in 107 women with breast cancer and 108 control subjects. Real Time QMS-PCR SYBR green (methylation-specific PCR) was used to analyze the hypermethylation. Tumours were classified as phenotype basal, luminal A, Luminal B and phenotype HER2+.Results: Ours analyses revealed low or absent methylation ESR1and 14-3-3σ in healthy controls and significant differences between breast cancer patients (pts) and healthy controls in relative serum levels of methylated gene promoters ESR1 (p=0.0112) and 14-3-3s (p=0.0047). Presence of methylated ESR1 in serum of breast cancer patients was associated with ER-negative phenotype (p=0.0179). Of the available cases, 60 pts (56%) were Luminal A, 10 pts (9.3%) Luminal B, 13 pts (12%) Basal like and 9 pts (8.4%) HER2+. We observed that methylated ERS1 was preferably associated with phenotype Basal Like and worse interval progression free and survival global though p>0.05 and the amplification HER2+ was correlation with significant more frequent methylation gene (p<0.05). Thet hypermethylation of normal ERS1 and 14-3-3σ combined differentiated between breast cancer patients and healthy controls (p=0.0001) with a sensitivity of 81% (95% CI: 72–88%) and specificity of 88% (95% CI: 78–94%).Conclusions: This study identifies the presence of variations in global levels of methylation promoters genes in healthy controls and breast cancer with different phenotype classes and shows that these differences have clinical significance. These showed that frequent methylation had a strong association with molecular phenotype of breast cancer and perhaps in the future can explain therapy resistance related to RE and HER2/neu status in breast cancer patients. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 1151.

CpG hypomethylation of MDR1 gene in tumor and serum of invasive ductal breast carcinoma patients

Objectives: Multidrug resistance 1 ( MDR1) gene encodes P-glycoprotein (P-gp), a transmembrane calcium-dependent efflux pump, implicated in drug resistance. In this prospective study, methylation status of MDR1 promoter and its correlation with clinicopathological parameters were evaluated in tumor and serum of breast cancer patients. Design and methods: Methylation-specific PCR was carried out to investigate the promoter methylation status of MDR1 in tumor and serum of 100 patients with invasive ductal carcinomas of breast (IDCs). The effect of promoter methylation on protein expression was evaluated by immunohistochemistry. Results: MDR1 was hypomethylated in 47% tumors and 44% paired sera of IDC patients and correlated significantly with increased tumor size and advanced tumor stage. Promoter hypomethylation of MDR1 in serum DNA showed 98% specificity and 50% sensitivity. Conclusions: Hypomethylation of MDR1 promoter in IDCs accounted for P-gp overexpression and aggressive biologic behavior in a subset of patients. Detection of these epigenetic changes in circulating DNA may not only enhance insight into the biological behavior of the primary tumor of an individual but may also provide valuable information regarding prognosis that can be readily monitored throughout the disease course.

Anti-p53 antibodies in serum: relationship to tumor biology and prognosis of breast cancer patients

The aim of this study was to analyze the concentration of anti-p53 antibodies in the serum of breast cancer patients and to correlate these results with various clinical, pathological and biochemical parameters. We also wanted to assess the prognostic significance of these antibodies in our patients. Sera from 61 patients with breast cancer and 20 individuals without malignancies were analyzed using enzyme-linked immunoadsorbent assay. High levels of anti-p53 antibodies were detected in twenty-one (35%) breast cancer patients and one control (5%). The difference was statistically significant. We observed an inverse relationship between the anti-p53 antibodies and the age of the patients. We found significant association of anti-p53 antibodies with tumor size, histological grade of the tumors and the number of axillary lymph nodes involved. The levels of anti-p53 antibodies were higher in patients with negative estrogen and progesterone receptors in comparison with patients with positive steroid receptors, but the difference was not statistically significant. No relation was observed between anti-p53 antibodies neither with the Cathepsin D levels in the cytosol nor with the HER-2/neu extracellular domain in the serum. Patients with primary tumors and higher levels of anti-p53 antibodies had shorter 5-year survival than patients with lower levels of anti-p53 antibodies. Our results support the role of anti-p53 antibodies as a biomarker of less favorable phenotype as well as a prognostic factor for patients with breast cancer.

Vascular Endothelial Growth Factor in the Serum of Breast Cancer Patients

Here we present the results of comparative immunoenzyme assay of the initial serum levels of VEGF in breast cancer patients (stages T1N0M0 and T2N0M0) and apparently healthy women (controls). It was found that VEGF concentrations in the serum of patients with breast cancer stages T1N0M0 and T2N0M0 significantly surpassed the control levels. Increased levels of VEGF surpassing the threshold values were more often observed in patients with T2N0M0 breast cancer compared to patients with T1N0M0 tumor. At the same time, this marker cannot be used in the diagnostics of this disease because in only 21.4% patients serum level of VEGF surpassed the upper boundary for this growth factor observed in the serum of control women. Serum concentration of VEGF in patients with stages T1N0M0 and T2N0M0 breast cancer did not depend on patient's age and reproductive function and receptor status of the primary tumor (estrogen and progesterone receptors), but was closely associated with tumor histogenesis and differentiation degree. Significantly higher levels of VEGF were observed in patients with lobular infiltrative breast carcinoma compared to patients with ductal tumors and in patients with low-differentiated tumors compared to highly and moderately differentiated tumors. High initial concentrations of VEGF (>300 pg/ml) were more often detected in patients with T2N0M0 breast cancer developing relapses within the first 3 years of follow-up compared to patients without relapses during the corresponding period (p=0.001). These findings suggest that serum level of VEGF in patients with T2N0M0 breast cancer before treatment can be used as an additional marker in parallel with standard clinical and morphological signs of the disease for more precise prognosis of early relapse (during the first 3 years of follow-up).

Hyaluronan-CD44 Interaction with Protein Kinase Cϵ Promotes Oncogenic Signaling by the Stem Cell Marker Nanog and the Production of MicroRNA-21, Leading to Down-regulation of the Tumor Suppressor Protein PDCD4, Anti-apoptosis, and Chemotherapy Resistance in Breast Tumor Cells

Multidrug resistance and disease relapse is a challenging clinical problem in the treatment of breast cancer. In this study, we investigated the hyaluronan (HA)-induced interaction between CD44 (a primary HA receptor) and protein kinase Cepsilon (PKCepsilon), which regulates a number of human breast tumor cell functions. Our results indicate that HA binding to CD44 promotes PKCepsilon activation, which, in turn, increases the phosphorylation of the stem cell marker, Nanog, in the breast tumor cell line MCF-7. Phosphorylated Nanog is then translocated from the cytosol to the nucleus and becomes associated with RNase III DROSHA and the RNA helicase p68. This process leads to microRNA-21 (miR-21) production and a tumor suppressor protein (e.g. PDCD4 (program cell death 4)) reduction. All of these events contribute to up-regulation of inhibitors of apoptosis proteins (IAPs) and MDR1 (multidrug-resistant protein), resulting in anti-apoptosis and chemotherapy resistance. Transfection of MCF-7 cells with PKCepsilon or Nanog-specific small interfering RNAs effectively blocks HA-mediated PKCepsilon-Nanog signaling events, abrogates miR-21 production, and increases PDCD4 expression/eIF4A binding. Subsequently, this PKCepsilon-Nanog signaling inhibition causes IAP/MDR1 down-regulation, apoptosis, and chemosensitivity. To further evaluate the role of miR-21 in oncogenesis and chemoresistance, MCF-7 cells were also transfected with a specific anti-miR-21 inhibitor in order to silence miR-21 expression and inhibit its target functions. Our results indicate that anti-miR-21 inhibitor not only enhances PDCD4 expression/eIF4A binding but also blocks HA-CD44-mediated tumor cell behaviors. Thus, this newly discovered HA-CD44 signaling pathway should provide important drug targets for sensitizing tumor cell apoptosis and overcoming chemotherapy resistance in breast cancer cells.

Serum protein profiling for diagnosis of breast cancer using SELDI-TOF MS

In search for novel markers for breast cancer, we aimed to identify and validate novel serum protein profiles specific for breast cancer, and assess the influence of clinical (subjects age) and pre-analytical (sample storage duration) variables on the constructed classifiers. To this end, sera of breast cancer patients (n=152) and healthy controls (n=129), randomly divided into a training and test set, were analysed by surface-enhanced laser desorption/ionisation time-of-flight mass spectrometry (SELDI-TOF MS). In the training set, 14 peak clusters were found to differ significantly in expression between cases and controls. None of the peak clusters were influenced by subjects age and sample storage duration. Ten peak clusters were also found significantly discriminative in the test set. Peak clusters were structurally identified as C3a des-arginine anaphylatoxin, (tentative) inter-alpha-trypsin inhibitor heavy chain 4 fragments and a fibrinogen fragment. Logistic regression analyses on the training set yielded a classification model with a moderate performance on the test set, corresponding to those reported in previously performed validation studies. Most likely originating from the highly heterogeneous nature of breast cancer, selection of breast cancer subgroups for comparison with healthy controls is expected to improve results of future diagnostic SELDI-TOF MS studies.

Use of surface-enhanced laser desorption/ionisation time-of-flight mass spectrometry (SELDI-TOF-MS) to detect breast cancer markers in serum

e22133 Background: Breast cancer is one of the most frequent and deadly cancers worldwide. Although the survival of patients has increased over the last decades, many patients die from metastatic relapse. Progresses in screening or early diagnosis will improve survival of breast cancer. Breast cancer has never had any good serum tumor markers. Therefore, we developed and evaluated a proteomics approach to search for new biomarkers in serum of breast cancer patients. Methods: Blood samples of 50 women with breast cancer (CA) and 50 healthy women (CTRL), matched to the age, were drawn prior to surgery. We used SELDI-TOF-MS for protein profiling with three different active surfaces of the protein chips: cationic exchanger (CM-10), hydrophobic surface (H50) and a strong anion exchange surface (Q10) with different binding properties. Data were analyzed by multivariate statistical techniques and artificial neural networks. Results: SELDI-TOF- MS could discriminate between serum of breast cancer patients and healthy women. We could generate a statistic significant (p<0.001) panel with 15 biomarkers resulting of multiple peaks with different molecular weights. The diagnostic pattern could differentiate CA from CRTL with specificity of 77% and sensitivity of 85% in serum. Conclusions: In this study we could exemplify SELDI-TOF-MS as a potential screening method to detect breast cancer patients by serum analysis. The protein chip technology could greatly facilitate the discovery of new and better biomarkers in breast cancer patients. This promising approach provides a high sensitivity and specificity by a less invasive method similar to mammography that is used in screening programs. No significant financial relationships to disclose.

Association of serum levels of the growth factor GP88 with disease progression in breast cancer patients

e22021 Background: The autocrine growth factor GP88 is an important player in breast cancer. GP88 is expressed in human BC tumors in correlation with their tumorigenicity. Increased GP88 expression was associated with anti-estrogen therapy resistance in ER+ cells and Herceptin resistance in Her-2 overexpressing breast tumors. Inhibition of GP88 expression inhibited tumor incidence and growth in nude mice. Immunohistochemical studies have shown that GP88 is expressed in invasive ductal carcinomas (IDC) and that high GP88 expression correlated with increased recurrence and mortality. Since GP88 is found in serum, we hypothesized that GP88 was elevated in the sera of breast cancer patients compared to healthy individuals and that GP88 serum level increases with disease progression. Methods: An IRB approved prospective study was established at the University of Maryland Breast Clinic to determine the serum level of GP88 in breast cancer patients (BC pts). Approximately 5 ml of blood was drawn every three months. GP88 serum concentration was determined in triplicate by human GP88 enzyme immunoassay. 190 BC pts were accrued. Sera from healthy volunteers (HV) were obtained to establish GP88 baseline. BC patient characteristics: Caucasian- 91, African American-92, Asian-6; median age, 51 (range 29- 86), stage I - 48, II - 52, III - 26, IV - 63. Results: Median serum GP88 level was 28.7 ng/ml (range 16.6–38.2) in HV, 40.7 ng/ml (range 6.4–100) in early stage (stage 1 -3) BC pts (p- value = 0.007) and 45.3 ng/ml (range 9.8 to 158.4) in stage 4 BC patients (p- value= 0.0007). Statistically significant increase in serum GP88 level was found in early stages as well as in metastatic disease when compared to HV. In addition, patients that were initially diagnosed with early stage disease but recurred showed a 5 to 10 fold increase in their GP88 serum levels. Conclusions: GP88 serum level is significantly higher in the sera of BC than HV subjects. Moreover, GP88 serum level increased in association with disease recurrence and progression. This study identifies GP88 as a measurable biomarker for disease progression not only at the tissue but also at the serum level. These results are also interesting since GP88 is also a therapeutic target of malignant progression of breast carcinoma. No significant financial relationships to disclose.

Value and Limitations of Measuring HER-2 Extracellular Domain in the Serum of Breast Cancer Patients

The human epidermal receptor-2 (HER-2) is overexpressed or amplified in 15% to 25% of breast cancers. Determination of HER-2 tumor status offers clinically useful information, as it selects patients who may benefit from treatment with trastuzumab, the monoclonal antibody against HER-2. Currently approved methods for HER-2 testing include immunohistochemistry or fluorescent in situ hybridization using tumor tissue. A fragment of HER-2 composed of its extracellular domain (ECD) can also be detected in the serum of some patients with breast cancer. As an easily accessible tumor marker, it could offer additional useful prognostic or predictive information. This review will briefly address the biology of the circulating HER-2 ECD and discuss the evidence to support the role, if any, for measuring HER-2 ECD levels in women with breast cancer. In particular, we focus on the value and limitations of serum ECD in both early and advanced breast cancer in the following clinical contexts: as a marker of HER-2 tumor tissue status; clinical implications of raised levels in women who have a tumor not overexpressing HER-2; as a prognostic indicator and as a predictor of response to treatment; and as a monitoring tool for early recurrence. On the basis of our review of the literature, we conclude that there is currently insufficient evidence to support the use of serum HER-2 ECD in the routine management of individual patients with breast cancer. This conclusion is in agreement with the 2007 American Society of Clinical Oncology guidelines on the use of biomarkers in breast cancer.

Elevated levels of hydroxylated phosphocholine lipids in the blood serum of breast cancer patients

The difference in serum phospholipid content between stage-IV breast cancer patients and disease-free individuals was studied by employing a combination of chemometric statistical analysis tools and mass spectrometry. Chloroform-extracted serum samples were profiled for their lipid class composition and structure using precursor ion, neutral loss, and product ion tandem mass spectrometric (MS/MS) scanning experiments. Changes in the relative abundance of phospholipids in serum as a consequence of cancer progression, measured through electrospray ionization (ESI) mass spectrometry of flow-injected serum samples collected from 25 disease-free individuals and 50 patients diagnosed with stage-IV breast cancer, were statistically evaluated using principal component analysis (PCA), analysis of variance (ANOVA) and receiver operating characteristic (ROC) analysis. Lipids whose abundance changed significantly as a consequence of cancer progression were structurally characterized using product ion spectra, and independently quantified using precursor ion scan experiments against an internal standard of known concentration. Phosphocholine lipids that displayed a statistically significant change as a consequence of cancer progression were found to contain an oxidized fatty acid moiety as determined by MS3 experiments.

Prognostic significance of GSTP1 and MGMT hypermethylation in invasive ductal breast carcinoma patients.

Abstract Abstract #4053 Purpose: Methylation-mediated suppression of detoxification, DNA repair and tumor suppressor genes has been implicated in cancer development, by shifting cells from the normal cellular cycle to a state of high proliferation that favors tumor development and progression. The current study was designed to analyze the methylation status of these genes in sera of breast cancer patients and determine the correlation of promoter hypermethylation of these genes with disease prognosis and patient survival.
 Experimental design: To test the hypothesis that promoter methylation of GSTP1 and MGMT is associated with disease prognosis, the methylation status of these genes was analyzed in invasive ductal carcinoma tissues and corresponding sera of breast cancer patients and correlated with disease free survival of these patients.
 Results: Promoter methylation of GSTP1 and MGMT was observed in 25% and 38% of breast tumor tissues, respectively and in 23% and 35% of the corresponding sera of breast cancer patients. There was significant association between methylation of GSTP1 and advanced tumor stage. Patients harboring methylated GSTP1 were more likely to have disease progression and reduced overall survival compared with patients who did not have the methylated gene.
 Conclusion: GSTP1 methylation in tumor and sera may serve as a poor prognostic marker in patients with invasive ductal carcinoma. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4053.

Elevated serum levels of the growth factor GP88 are found in breast cancer patients when compared to healthy individuals.

Abstract Abstract #2006 Background: GP88 is an autocrine growth factor that plays a critical role in breast tumorigenesis. GP88 is expressed in human BC tumors in a positive correlation with their tumorigenicity. Increased GP88 expression is associated with resistance to anti-estrogen therapy in ER + cells and with herceptin resistance in Her-2 overexpressing breast tumors. Inhibition of GP88 expression in human breast adenocarcinoma inhibited tumor incidence and growth in nude mice. Immunohistochemical studies have shown that GP88 was expressed in invasive ductal carcinomas in correlation with the expression of poor prognosis markers whereas normal tissues and benign lesions were negative. High GP88 expression in tumor biopsies was accompanied by decreased disease-free survival. Since GP88 can be secreted, we have hypothesized that GP88 could be secreted in the circulation and found in serum. We examined whether GP88 could be found in the circulation and whether GP88 could be elevated in the sera of breast cancer patients when compared to healthy individuals.
 Methods: An IRB approved blood sampling study was conducted at the University of Maryland Breast Clinic to determine the serum level of GP88 in healthy volunteers (HV) and breast cancer patients (BC pts). Serum GP88 concentration was determined in triplicate by quantitative enzyme immunoassay. 189 BC pts were accrued. In addition, sera from 18 HV were obtained to establish a GP88 baseline in healthy volunteers. BC patient characteristics: Race: Caucasian- 91, African American-92, Asian-6; median age, 51 (range 29-86), stage I – 48, II - 52, III – 26, IV - 63.
 Results: Circulating GP88 was measurable in the serum. Median level of GP88 was 28.7 ng/ml (range 16.6-38.2) in HV; 40.7 ng/ml (range 6.4-100) in early stage (stage 1 –3) BC pts (p- value = 0.007) and 45.3 ng/ml (range 9.8 to 158.4) in stage 4 metastatic BC patients (p-value= 0.0007). Statistically significant increase in circulating GP88 level was found in early stages as well as in metastatic disease. Correlation studies with BC prognostic factors such as stage, tumor size, lymph node involvement, tumor grade and presence of ER and HER-2 will be presented.
 Conclusion: GP88 can be detected in the sera of HV and BC pts. Comparison between the two groups of subjects indicates that GP88 level is significantly higher in the sera of BC pts. These studies are important as they identify as a measurable circulating biomarker GP88 that is also a therapeutic target of malignant transformation or malignant progression of breast carcinoma (BC). Future studies will examine whether there is any correlation between the serum level of GP88 and therapeutic response to systemic therapy in breast cancer patients.
 This study was supported by grant from MIPS, the Avon Foundation and 1R43 CA 124179-01A1 from the National Institutes of Health. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2006.

Using custom protein microarrays to identify autoantibody biomarkers for the early detection of breast cancer.

Abstract Abstract #2003 Background: Cancer patients make antibodies to tumor-derived proteins that are potential biomarkers for early detection. To detect autoantibodies to tumor antigens in patient sera, we have adapted novel high-density custom protein microarrays (NAPPA) expressing 6,500 candidate tumor antigens for biomarker detection. These arrays are probed with sera from patients with early stage breast cancer and healthy women. Using this approach, we identified antibodies in the sera of breast cancer patients.
 Methods: 6,500 full-length human antigens were expressed using mammalian reticulocyte lysate and captured onto NAPPA protein microarrays. Protein expression (>90%) was confirmed with anti-GST antibodies. Patient sera were added, and bound IgG detected with secondary antibodies. Serum samples were obtained from 103 patients with stages I-III breast cancer, and 103 age-matched control women, all undergoing routine mammography.
 Results: Using high-density protein microarrays, sera from breast cancer patients (n=53) and healthy donor sera (n=53) were screened for autoantibodies to 6,500 protein antigens. Antigens were selected for further analysis if the 95th percentile of signal of cases and controls were significantly different (p<0.05) and if the number of cases with signals above the 95th percentile of controls was larger than the number expected due to random chance (p<0.05). Known tumor antigens, such as p53, were detected, as well as novel antigens such as DCC1, Rab7L and USP44. In total, 768 unique antigens were selected for further analysis with an independent set of breast cancer patient (n=50) and healthy donor sera (n=50).
 Conclusions: Custom in-situ protein microarrays can be used to detect serum tumor antigen-specific antibodies and enables the rapid, simultaneous detection of immunogenic tumor antigens from patient sera. These autoantibodies are being evaluated as potential biomarkers for the early diagnosis of breast cancer. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2003.

Circulating microRNAs in breast cancer and healthy subjects

BackgroundIt has been demonstrated that extracellular mRNA can be detected in the circulation. Our hypothesis was that circulating miRNAs are also present and differentially expressed in the serum of breast cancer patients compared to controls.FindingsWe measured miRNA in the serum of samples with and without the addition of miRNA prior to analysis. To test our RNA extraction efficiency, we spiked-in serial dilutions of single-strand C elegens miR-39 (cel-miR-39) and human miR-145 (has-miR-145) into goat serum and a 10 year old human serum specimen. We next analyzed miR-16, -145, and -155 in archived serum specimens from 21 participants, 13 of whom did and 8 of whom did not have breast cancer. We were able to detect the miRNAs from all the serum samples to which the miRNAs had been added. We were also able to detect endogenous miR-16, -145, and -155 in all serum samples. While the expression of all three miRNAs was similar in samples from healthy women compared to those with breast cancer, women with progesterone receptor (PR, p = 0.016) positive tumors had higher miR-155 expression than tumors that were negative for these receptors.Conclusion1) RNA species can be detected in archived serum; 2) miR-155 may be differentially expressed in the serum of women with hormone sensitive compared to women with hormone insensitive breast cancer. Screening serum for miRNAs that predict the presence of breast cancer is feasible, and may be useful for breast cancer detection.

HspBP1 levels are elevated in breast tumor tissue and inversely related to tumor aggressiveness

HspBP1 is a co-chaperone that binds to and regulates the chaperone Hsp70 (Hsp70 is used to refer to HSPA1A and HSPA1B). Hsp70 is known to be elevated in breast tumor tissue, therefore the purpose of these studies was to quantify the expression of HspBP1 in primary breast tumors and in serum of these patients with a follow-up analysis after 6 to 7 years. Levels of HspBP1, Hsp70, and anti-HspBP1 antibodies in sera of breast cancer patients and healthy individuals were measured by enzyme-linked immunosorbent assay. Expression of HspBP1 was quantified from biopsies of tumor and normal breast tissue by Western blot analysis. The data obtained were analyzed for association with tumor aggressiveness markers and with patient outcome. The levels of HspBP1 and Hsp70 were significantly higher in sera of patients compared to sera of healthy individuals. HspBP1 antibodies did not differ significantly between groups. HspBP1 levels were significantly higher in tumor (14.46 ng/microg protein, n = 51) compared to normal adjacent tissue (3.17 ng/microg protein, n = 41, p < 0.001). Expression of HspBP1 was significantly lower in patients with lymph node metastasis and positive for estrogen receptors. HspBP1 levels were also significantly lower in patients with a higher incidence of metastasis and death following a 6 to 7-year follow-up. The HspBP1/Hsp70 molar ratio was not associated with the prognostic markers analyzed. Our results indicate that low HspBP1 expression could be a candidate tumor aggressiveness marker.

Multigene Methylation in Serum of Sporadic Chinese Female Breast Cancer Patients as a Prognostic Biomarker

Objective: DNA methylation is a common molecular alteration in human neoplasia and can be detected easily in the bloodstream of patients. Here, we investigated whether DNA methylation in sera is of prognostic significance in breast cancer patients. Methods: Methylation status of BRCA1, p16 and 14-3-3σ was examined by methylation-specific PCR assay in the sera of sporadic breast cancer patients and healthy serum controls. Results: The panel gene methylation frequencies were 29% of sporadic breast cancers for p16, 32% for BRCA1 and 82% for 14-3-3σ; all were significantly associated with grades and estrogen receptor status. Only p16 methylation was associated with histological type. p16 and BRCA1 methylation were associated with progesterone receptor status, while 14-3-3σ was significantly associated with lymph node metastases. Seventy percent of patients with p16 methylation showed elevated serum CEA levels; of the breast cancer patients with BRCA1 methylation, 75.8% showed elevated serum CEA levels and 69.7% showed elevated serum CA15.3 levels. When analyzing all investigated patients, multivariate analysis showed methylated BRCA1 and/or p16 serum DNA to be independently associated with poor outcome, with a relative risk of death of 6.0. Conclusions: Epigenetic markers in sera, especially BRCA1/p16, may be more promising targets for the diagnosis of sporadic breast cancer than previous prognostic markers.

Development of novel peptide inhibitor of Lipoxygenase based on biochemical and BIAcore evidences

Lipoxygenase (LOX) are enzymes implicated in a broad range of inflammatory diseases, cancer, asthma and atherosclerosis. These diverse biological properties lead to the interesting target for the inhibition of this metabolic pathway of LOX. The drugs available in the market against LOX reported to have various side effects. To develop potent and selective therapeutic agents against LOX, it is essential to have the knowledge of its active site. Due to the lack of structural data of human LOX, researchers are using soybean LOX (sLOX) because of their availability and similarities in the active site structure. Based on the crystal structure of sLOX-3 and its complex with known inhibitors, we have designed a tripeptide, FWY which strongly inhibits sLOX-3 activity. The inhibition by peptide has been tested with purified sLOX-3 and with LOX present in blood serum of breast cancer patients in the presence of substrate linoleic acid and arachidonic acid respectively. The dissociation constant ( K D) of the peptide with sLOX-3 as determined by Surface Plasmon Resonance (SPR) was 3.59 × 10 − 9 M. The kinetic constant ( K i) and IC 50, as determined biochemical methods were 7.41 × 10 − 8 M and 0.15 × 10 − 6 M respectively.

Collagen IV levels are elevated in the serum of patients with primary breast cancer compared to healthy volunteers

Collagen IV is a major component of the vascular basement membrane and may be a marker of angiogenesis. Serum levels of this protein are elevated in some human cancers. Our objectives were to compare collagen IV levels in the serum of breast cancer patients and healthy women and to examine changes during preoperative chemotherapy. Sera from 51 patients with stage II–III breast cancer and 55 healthy controls were analysed. Collagen IV level was measured by a commercially available sandwich enzyme link immunoassay. Baseline serum levels were compared between cancer patients and healthy women and paired pre- and post-chemotherapy measurements were also performed in 39 patients who received preoperative chemotherapy and were correlated with response to therapy. The median serum collagen IV concentration was significantly higher in cancer patients (166 μg l−1) than in healthy women (115 μg l−1), P<0.001. Chemotherapy induced a significant further increase in serum collagen IV (167 μg l−1 prechemo vs 206 μg l−1 postchemo, P=0.001). There were no correlations between baseline collagen IV levels and response to therapy, age, clinical stage or HER2 status. In conclusion, patients with breast cancer have elevated levels of collagen IV compared to healthy women and collagen IV levels increase further during chemotherapy.

English

&nbsp; The levels of heavy and essential metals in the serum of breast cancer patients were determined, in order to find out which of them could be of importance in the treatment and prognosis of the cancer. Serum copper, zinc, iron, magnesium, manganese, chromium, cadmium, lead and selenium were measured using atomic absorption spectrophotometer in 29 newly diagnosed breast cancer patients and compared with 30 healthy control subjects. The mean serum copper level was significantly lower in patients with breast cancer compared with controls. The mean serum zinc and manganese levels were insignificantly lowered in with breast cancer compared to controls. However, the levels of magnesium, iron, chromium, cadmium lead and selenium were not significantly raised in breast cancer patients compared with the controls. The exact mechanism responsible for the alterations in trace metals in patients with breast cancer is largely unclear and requires further evaluation. However, the serum copper level may be of value in determining the prognosis of these patients. &nbsp; Key word:&nbsp;Breast cancer, essential trace metals, prognosis.

Quantitative detection of methylated ESR1 and 14-3-3-σ gene promoters in serum as candidate biomarkers for diagnosis of breast cancer and evaluation of treatment efficacy

The aim of the present study was to investigate the association between gene hypermethylation and main clinicopathological features of breast cancer, including diagnosis and treatment response. A sensitive SYBR green methylation-specific PCR technique was used to analyze the utility of circulating DNA with CpG island hypermethylation of ESR1, APC, RARB, 14-3-3-σ, and E-cad gene promoter regions as breast cancer biomarkers. Analyses were conducted of preoperative sera from 106 women with breast cancer, 34 with benign breast disease and 74 with no evidence of breast disease and of post-treatment sera from 60 of the breast cancer patients. Mean serum values of methylated ESR1 and 14-3-3-σ gene promoters significantly differed between breast cancer patients and healthy controls (P = 0.0112 for ESR1 and P = 0.0047 for 14-3-3-σ). When their results were combined, it was found that hypermethylation of these two genes differentiated between breast cancer patients and healthy controls (P < 0.0001) with a sensitivity of 81% (95% confidence interval: 72-88%) and specificity of 88% (95% CI: 78-94%). Presence of methylated ESR1 in serum of breast cancer patients was associated with the ER negative phenotype (P = 0.0179). Serum hypermethylation at ESR1 and 14-3-3-σ loci was observed in cancer patients, in situ carcinoma and benign breast disease. No significant differences in methylated ERS1 or 14-3-3-σ values were observed between pre-surgery and post-treatment measurements. Preliminary clinical applications of this approach have revealed several shortcomings, including a frequent presence of methylated 14-3-3-σ in sera from women with breast benign disease. These findings cast some doubts on the utility for early cancer diagnosis of highly sensitive techniques to identify hypermethylation of specific gene promoters in DNA extracted from serum. Although numerous issues remain to be resolved, the quantitative measurement of circulating methylated DNA remains a promising tool for cancer risk assessment.