Protein In Breast Cancer Patients Research Articles

Abstract 868: Novel pathogenic mutations in BRCA1 gene that may influence the interaction of BRCA1 protein with BARD1 protein in breast cancer patients

Abstract Breast cancer (BC) contributes to 30% of all women suffering from cancer in the US. Despite enhanced survival rates and recovery rates, pathogenic mutation discovery remains indispensable for early detection and prognosis. BRCA1 pathogenic mutations increase the chance of BC to 55% by the age of 70. A crucial mechanism of mutations is affecting protein-protein interaction. BRCA1 interacts with the BARD1 protein forming a complex that contributes to DNA repair and plays a vital role in tumor suppression. In this study, we aimed to investigate mutations that contribute to the dysfunction of BRCA1 protein through sequencing and computational analysis and their impact on the active site interacting with BRCA1 associated Ring Domain 1 (BRAD1) of BRCA1, and hence its beneficial role in BC prognosis. Herein, we represent a study on 48 BC patients sequenced using Devyser BRCA NGS kit, according to the manufacturer’s instructions. The reagent kit V2, 500 Cycles PE, on the Illumina MiSeq System. Using the Illumina MiSeq System to detect pathogenic mutations. Bioinformatics analysis was carried out for variant calling and different databases, such as ClinVar and BIC, were searched to identify the novel mutations. P2RANK software was used to identify and confirm the active site protein sequences. For BRCA1, we studied three missense mutations present in the ring domain of the protein interacting with BRAD1. We used PDBsum server to retrieve protein structures predicted by the Artificial Intelligence (AI)-based AlphaFold software. Structures were further confirmed using Ramachandran plot. We used Dynamut server to study the stability and structural variability due to mutations. Docking was carried out using HDock software and significant poses were sorted out based on their RMSD values. For BRCA1, Cys47Ser and Cys39Arg caused destabilization of the protein represented as Gibb’s energy by -2.257 kcal/mol and -0.318 kcal/mol respectively. On the other hand, Cys39Tyr caused the stabilization of protein by 0.247 kcal/mol. The effect of mutations on rigidity was determined in terms of vibrational entropy where Cys47Ser caused an increase of protein flexibility with the energy of 0.262 kcal.mol−1.K−1. Cys39Arg and Cys39Tyr decreased the flexibility of the protein and caused rigidity by -0.164 kcal.mol−1.K−1 and -0.371 kcal.mol−1.K−1 respectively. In, conclusion, investigating the three selected pathogenic mutations showed a clear impact on the structure of the protein favored different types of bonds in the active site that may alter the interaction between BRCA1 and BARD1. This study has a beneficial role to investigate the impact of BRCA1 mutations on the structural stability of the protein and thereby the risk of BC development Citation Format: Yahia Ahmad Yahia, Nasra F. Abdel Fattah, Marwa A. Abdel Wahed, Fatma S S Hafez, Sara HA Arwa, Samah A. Loutfy. Novel pathogenic mutations in BRCA1 gene that may influence the interaction of BRCA1 protein with BARD1 protein in breast cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 868.

Low HER2 expression in normal breast epithelium enables dedifferentiation and malignant transformation via chromatin opening.

Overexpression of the HER2 protein in breast cancer patients is a predictor of poor prognosis and resistance to therapies. We used an inducible breast cancer transformation system that allows investigation of early molecular changes. HER2 overexpression to similar levels as those observed in a subtype of HER2-positive breast cancer patients induced transformation of MCF10A cells and resulted in gross morphological changes, increased anchorage-independent growth of cells, and altered the transcriptional programme of genes associated with oncogenic transformation. Global phosphoproteomic analysis during HER2 induction predominantly detected an increase in protein phosphorylation. Intriguingly, this correlated with chromatin opening, as measured by ATAC-seq on acini isolated from 3D cell culture. HER2 overexpression resulted in opening of many distal regulatory regions and promoted reprogramming-associated heterogeneity. We found that a subset of cells acquired a dedifferentiated breast stem-like phenotype, making them likely candidates for malignant transformation. Our data show that this population of cells, which counterintuitively enriches for relatively low HER2 protein abundance and increased chromatin accessibility, possesses transformational drive, resulting in increased anchorage-independent growth in vitro compared to cells not displaying a stem-like phenotype.

Investigation of Interferon Gamma Activity Using Bioinformatics Methods.

Breast cancer grows from the breast tissue and is a severe health problem worldwide. Genetics is believed to be the primary cause of all cases of breast cancer via gene mutation. Bioinformatics methodology has been used to determine the sequences and structures of bioactive substances. This study aimed to analyze the function and structure of the Interferon Gamma (IFNγ) in healthy controls and patients with breast cancer using bioinformatics methods. Blood samples were collected from 75 patients with breast cancer and 25 healthy subjects as control samples. The results showed transition mutation (30%) and transversion mutation (70%) in patients with breast cancer. Moreover, missense mutations (84%) and silent mutations (16%) were detected by BLAST. In addition, the amino acid of the IFNγ protein consisting of alpha-helical, β-sheet, and coil of secondary structure was determined in this study using BioEdit. The results of the physicochemical properties of the IFNγ protein reflect the function, stability, molecular weight, isoelectric point, and instability index of the IFNγ protein using ProtParam. Moreover, the results of mutation affected the percentage of alpha-helix, β-turns, and coil in breast cancer patients compared to healthy groups with reference of NCBI using PSIpred program. Additionally, the PHYRE2 server and RasMol program showed a tertiary structure of the IFNγ protein in breast cancer patients. Furthermore, the STRING program revealed the poly IFNγ protein interacted with other proteins to perform its functions normally. From the recorded data in the current study, it was concluded that IFNγ is considered a marker for patients with breast cancer.

Abstract 12538: Effect of High Intensity Interval Training on High-sensitivity C-reactive Protein in Breast Cancer Patients Undergoing Anthracycline-based Chemotherapy

Introduction: Anthracycline-based chemotherapies are widely used chemotherapeutic agents for treatment of breast cancers, but these agents increase inflammation in the cardiovascular system. Circulating levels of high sensitivity C-reactive protein (hsCRP) have been validated to assess the degree of inflammation in adults. While exercise has been shown to reduce hsCRP in patients with coronary artery diseases, it is unclear whether high intensity interval training (HIIT) reduces hsCRP in breast cancer patients undergoing anthracycline-based chemotherapy. Hypothesis: We hypothesized that a group of participants that performed the 8-week HIIT intervention would demonstrate maintenance in hsCRP level compared to a group of participants that did not perform HIIT. Methods: Thirty breast cancer survivors were randomized to either the HIIT or control (CON) group. The HIIT group participated in 8 weeks of supervised exercise sessions 3 times/week. The CON group was asked to maintain their current activity levels. Fasting (≥ 12 hours) blood (∽30 cc) was obtained from the antecubital and hsCRP was measured by immunoturbidimetric assay. A paired t-test and 2x2 (group x time) repeated measures ANOVA were used to evaluate changes in hsCRP. Results: At baseline, there were no group differences in age (46.9±9.8 yrs), BMI (31.0±7.5 kg/m 2 ) and blood pressure (123.4 ± 16.8/72.3.9 ± 5.6 mmHg; p>0.05). Following 8 weeks, hsCRP did not significantly change (7.1±2.7 to 8.7±3.5 mg/L) in the HIIT group (p=0.21). However, there was a significant within-group change in the CON group (6.0±3.2 to 10.9.1±2.3 mg/L; p=0.02). There was no group x time interaction following 8 weeks (P=0.93). Conclusions: An 8-week HIIT intervention maintained circulating levels of hsCRP in breast cancer patients receiving anthracycline-based chemotherapy. Future large randomized controlled trials are warranted to investigate the impact of exercise-induced reductions in inflammation while undergoing anthracycline-based chemotherapy.

Evaluation of C-reactive protein in breast cancer by enzyme linked immunoassay technique.

To explore the serum levels of C-reactive protein in breast cancer patients, and to investigate the relationship between inflammation and progression of breast cancer. The case-control study was conducted at Bahria University Medical and Dental College, Karachi, from September 2015 to December 2018, and comprised breast cancer patients in group A and and an equal number of age-matched healthy women in control group B. C-reactive protein levels were evaluated in serum samples using enzyme-linked immunosorbent assay in both the groups and micro ribonucleic acid levels in serum were quantified using real time polymerase chain reaction. Data was analysed using SPSS 16. Of the 170 subjects, 85(50%) were in each of the two groups. C-reactive protein and micro ribonucleic acid expression were significantly different in group A (p<0.001). There was no correlation (r = 0.162, p>0.01) between the tumour markers in group B (p>0.05). Significantly raised C-reactive protein levels showed there was a link between inflammation and breast cancer.

S100A14 serum level and its correlation with prognostic factors in breast cancer

BackgroundBreast cancer is the most commonly occurring cancer in women worldwide. S100A14 is a novel important member of S100 proteins family. Its importance is due to its role in tumorigenesis and metastasis process. In this study, we aimed to determine serum levels of S100A14 protein in breast cancer patients and healthy individuals to know if it can be suggested as a new biomarker for breast cancer and to reveal whether it is correlated with cancer pathological features.MethodsThis cross-sectional study was performed in two groups: study group contains 46 breast cancer patients (29 metastatic and 17 non-metastatic) and control group contains 22 healthy women. Enzyme-linked immunoabsorbent assay was performed to determine S100A14 protein levels in samples. Pathological data were obtained for each patient. The data were statistically analyzed using Kruskal-Wallis H, Mann-Whitney U, and Spearman correlation tests.ResultsS100A14 serum levels were elevated in study group compared with control group (P < 0.05). S100A14 serum levels were significantly increased in distant breast cancer patients compared with regional breast cancer patients (P = 0.001). There was a strong positive correlation between serum S100A14 level and tumor grade (rs = 0.713, P < 0.001).ConclusionOur study indicated that S100A14 serum levels are elevated in breast cancer patients compared with control individuals. High S100A14 serum levels were correlated with poor tumor differentiation so it might have a prognostic significance for breast cancer tumors. The elevation of S100A14 levels in distant breast cancer patients suggests the ability of using serum S100A14 as a biomarker for detection of breast cancer metastasis.

Quantifying Serum Derived Differential Expressed and Low Molecular Weight Protein in Breast Cancer Patients.

Searching the biomarker from complex heterogeneous material for early detection of disease is a challenging task in the field of biomedical sciences. The study has been arranged to explore the proteomics serum derived profiling of the differential expressed and low molecular weight protein in breast cancer patient. Quantitative proteome was analyzed using the Nano LC/Mass and Bioinformatics tool. This quantification yields 239 total protein constituting 29% of differentially expressed protein, with 82% downregulated differential protein and 18% up-regulated differential protein. While 12% of total protein were found to be cancer inducing proteins. Gene Ontology (GO) described that the altered proteins with 0-60 kDa mass in nucleus, cytosol, ER, and mitochondria were abundant that chiefly controlled the RNA, DNA, ATP, Ca ion and receptor bindings. The study demonstrate that the organelle specific, low molecular weighted proteins are significantly important biomarker. That act as strong agents in the prognosis and diagnosis of breast cancer at early stage.

Expression of APOBEC3H Isoenzyme on the Level of Protein in Breast Cancer Patients

Breast Cancer is a disease that results from the alteration of the cellular genome in breast tissue and can lead to the second cause of cancer death in human so that it is an object of intensive research. APOBEC3H is a member of APOBEC3 sub-family of DNA cytosine deaminases, which have evolved as a crucial role of innate immune defense and have been contributing to cancer development throughout genetic mutations. Eighty subjects were conducted to this study, forty subjects for each breast cancer patients and healthy group. Blood samples were analyzed for this study. After clotting blood sera were used to determine the quantity of APOBEC3H protein by Elisa technique. This study demonstrates that APOBEC3H expression on the level of protein is significantly down-regulated in breast cancer patients compared to that of a healthy group. This study reveals that the APOBEC3H expression on the level of protein can be used as a potential biomarker for predicting breast cancer with the significant high value of AUC.

Synergistic inhibition of aggressive breast cancer cell migration and invasion by cytoplasmic delivery of anti-RhoC silencing RNA and presentation of EPPT1 peptide on “smart” particles

Overexpression of RhoC protein in breast cancer patients has been linked to increased cancer cell invasion, migration, and metastases. Suppressing RhoC expression in aggressive breast cancer cells using silencing RNA (siRNA) molecules is a viable strategy to inhibit the metastatic spread of breast cancer. In this report, we describe the synthesis of a series of asymmetric pH-sensitive, membrane-destabilizing polymers engineered to complex anti-RhoC siRNA molecules forming “smart” nanoparticles. Using β-CD as the particle core, polyethylene glycol (PEG) chains were conjugated to the primary face via non-cleavable bonds and amphiphilic polymers incorporating hydrophobic and cationic monomers were grafted to the secondary face via acid-labile linkages. We investigated the effect of PEG molecular weight (2 & 5 kDa) on transfection capacity and serum stability of the formed particles. We evaluated the efficacy of EPPT1 peptides presented on the free tips of the PEG brush to function as a targeting ligand against underglycosylated MUC1 (uMUC1) receptors overexpressed on the surface of metastatic breast cancer cells. Results show that “smart” nanoparticles successfully delivered anti-RhoC siRNA into the cytoplasm of aggressive SUM149 and MDA-MB-231 breast cancer cells, which resulted in a dose-dependent inhibition of cell migration and invasion. Further, EPPT1-targeted nanoparticles demonstrate a synergistic inhibition of cell migration and invasion imparted via RhoC knockdown and EPPT1-mediated signaling via the uMUC1 receptor.

Changes in physical activity behavior and C-reactive protein in breast cancer patients.

BackgroundC-reactive protein (CRP) concentrations are associated with morbidity and mortality in breast cancer patients and moderate-vigorous physical activity (MVPA) may help regulate CRP levels.PurposeThe purpose of this prospective study was to examine intraindividual and interindividual changes in MVPA on changes in CRP levels among early posttreatment breast cancer patients.MethodsDuring five data collections over the first year posttreatment for breast cancer, women (N = 138, Mage = 55.3, standard deviation = 11.1 years) completed a questionnaire, wore an accelerometer for seven consecutive days to measure MVPA, and provided blood samples to measure CRP concentrations. Intraindividual and interindividual associations between MVPA on CRP were tested using hierarchical linear modeling.ResultsBased on the intraindividual associations, lower concentrations of CRP were evident when participants engaged in more, as compared to their average, MVPA (β = −.02, p < .021). In addition, interindividual analyses showed that women who engaged in more MVPA across the study period had lower concentrations of CRP compared to women who engaged in less MVPA (β = −.24, p = .006).ConclusionsMVPA in breast cancer patients is consistently associated with lower CRP concentrations over time. Interventions may target individual MVPA early after diagnosis to mitigate CRP levels in breast cancer patients.

Correlation of Serum Levels of Urokinase Activation Plasminogen (uPA) and Its Inhibitor (PAI-1) with Hormonal and HER-2 Status in the Early Invasive Breast Cancer.

Introduction:Breast cancer is the most common malignant tumor in women. On the list of causes of death immediately after lung cancer. It is a heterogeneous disease, considering the differences in morphological, cytogenetic, molecular, clinical and therapeutic aspects, so that the prognosis in a patient with the same histological grade and pathological status may vary.Aim:In this paper we wanted to identify the correlation between the assay of the serum values of uPA-PAI-1 complexes and individual prognostic-predictive parameters, primarily with the status of estrogenic (Er), progesterogenic (PgR) and Her-2 receptors („human epidermal growth factor).Material and methods:The study was conducted at the Clinic for General and Abdominal Surgery, University Clinical Center of Sarajevo (CCUS), from September 2016 to April 2017. The study included 66 patients, ages 18 to 75, in whom by the needle biopsy preoperatively was pathohistologically verified primary invasive breast cancer.Results:Two thirds of the sample were classified as invasive ductal carcinoma, similar to the percentage (68.2%) of pT2 size, and almost half in the grade G3. Lymph node status was negative in 54.5% of respondents, and positive in 31.8% of respondents. Most patients had positive estrogenic (83.3%) and progesterone receptors (62.1%). Almost 80% was Her-2 negative. The blood vessel invasion was present in 56.1%, while the neural invasion was present in less than a third of the sample (30.3%). Median values of uPA-PAI-1 complexes were 1.4 (interquartile range 0.9); almost 70% of the sample was negative for the status analysis of uPA-PAI-1 complex (<1).Discussion:A statistically significant difference was determined in the mean values of uPA-PAI-1 complexes in subgroups according to menopausal status, tumor size, histological grade, histological type (invasive ductal carcinoma vs. invasive lobular cancer versus invasive ductal carcinoma vs. invasive lobular cancer), status axillary lymph nodes, Ki67 status (as binary variables), invasion of the blood vessels and neural invasion, as well as subgroups according to the status of expression of hormonal (estrogen and progesterone) receptors.Conclusion:There is a statistically significant difference in the mean values of the uPA-PAI-1 complex and Her-2 receptor expression. Generally, in perspective, this would be the role played by the uPA/PAI-1 complex in breast cancer, which is that the elevated complex values have a negative prognosis and effect on survival, similar to the negative Her-2 receptor status. Complex uPA/PAI-1 is not a specific serum protein in breast cancer patients and cannot be taken as an individual prognostic-predictive marker for mass pre- or post treatment screening and prediction. Unfortunately, none of the biomarkers are able to independently and fully identify patients of the unknown stage of the disease with better or worse prognosis or to identify cases of more aggressive tumor behavior of the same stage for timely inclusion of adjuvant therapy and reduction of the risk of metastatic disease. The decision on treatment and prognosis should be the result of a combination of all diagnostic, therapeutic, pathohistological and molecular-genetic variables.

Increased MAPK1/3 Phosphorylation in Luminal Breast Cancer Related with PIK3CA Hotspot Mutations and Prognosis

INTRODUCTION: While mutations in PIK3CA are most frequently (45%) detected in luminal breast cancer, downstream PI3K/AKT/mTOR pathway activation is predominantly observed in the basal subtype. The aim was to identify proteins activated in PIK3CA mutated luminal breast cancer and the clinical relevance of such a protein in breast cancer patients. MATERIALS AND METHODS: Expression levels of 171 signaling pathway (phospho-)proteins established by The Cancer Genome Atlas (TCGA) using reverse phase protein arrays (RPPA) were in silico examined in 361 breast cancers for their relation with PIK3CA status. MAPK1/3 phosphorylation was evaluated with immunohistochemistry on tissue microarrays (TMA) containing 721 primary breast cancer core biopsies to explore the relationship with metastasis-free survival. RESULTS: In silico analyses revealed increased phosphorylation of MAPK1/3, p38 and YAP, and decreased expression of p70S6K and 4E–BP1 in PIK3CA mutated compared to wild-type luminal breast cancer. Augmented MAPK1/3 phosphorylation was most significant, i.e. in luminal A for both PIK3CA exon 9 and 20 mutations and in luminal B for exon 9 mutations. In 290 adjuvant systemic therapy naïve lymph node negative (LNN) breast cancer patients with luminal cancer, high MAPK phosphorylation in nuclei (HR=0.49; 95% CI, 0.25–0.95; P=.036) and in tumor cells (HR=0.37; 95% CI, 0.18–0.79; P=.010) was related with favorable metastasis-free survival in multivariate analyses including traditional prognostic factors. CONCLUSION: Enhanced MAPK1/3 phosphorylation in luminal breast cancer is related to PIK3CA exon-specific mutations and correlated with favorable prognosis especially when located in the nuclei of tumor cells.

Abstract P5-08-51: SIAH2 protein expression is inversely correlated with the ER status and outcome to tamoxifen therapy in metastatic breast cancer patients

Abstract Introduction: In a previous study we observed a positive correlation between Seven in Absentia Homolog 2 (SIAH2) and Estrogen Receptor (ER) mRNA levels. Additionally, high SIAH2 mRNA levels were related to a favorable progression-free survival (PFS) after first-line tamoxifen. In contrast, others showed high SIAH2 protein levels in ER-negative breast cancer associated with an unfavorable relapse-free survival. In this study, we investigated the above discrepancy between SIAH2 protein and mRNA findings and evaluated the prognostic and predictive value of SIAH2 protein in breast cancer patients. Patients and methods: Tissue microarrays (TMAs) of formalin-fixed, paraffin-embedded primary breast tumors were immunohistochemically stained for SIAH2 protein. The TMAs contained core specimens of 759 patients with early disease and of 245 ER-positive patients with advanced disease treated with first-line tamoxifen. SIAH2 protein staining was scored for its intensity and proportion positive cells and subsequently evaluated for its relationship with metastasis-free survival (MFS) and PFS in uni- and multivariate analyses including traditional prognostic or predictive factors, respectively. Results: The proportion SIAH2-positive cells had a relationship with MFS and PFS, whereas staining intensity and a previous described score for SIAH2 combining intensity and proportion were not related with clinical outcome. Based on these results, tumors with more than 20% positive cells were considered as SIAH2-positive. In early disease, 267 patients (35%) had SIAH2-positive tumors, which were further characterized by decreased expression of ER at protein and mRNA levels (P &amp;lt;0.001 and P = 0.003, respectively). These SIAH2-positive tumors correlated with significant unfavorable MFS in lymph node negative, ER-positive breast cancer patients, but only in univariate analysis. In advanced disease, 86 patients (35%) had SIAH2-positive tumors which was associated with an unfavorable PFS after first-line tamoxifen in both uni- and multivariate analyses (HR = 1.45; 95% CI, 1.07 to 1.96; P = 0.015). Conclusions: SIAH2 protein expression is especially observed in ER-negative tumors and has no additional prognostic value in breast cancer. The proportion SIAH2-positive cells in ER-positive tumors can be used as biomarker to predict tamoxifen treatment failure in breast cancer patients with advanced disease. Future studies should establish if expression of certain microRNAs explain the observed discrepancy in SIAH2 mRNA and protein levels. Citation Format: van der Willik KD, Timmermans MM, Look MP, Reijm EA, van Deurzen CHM, den Bakker MA, Westenend PJ, Martens JWM, Berns EMJJ, Jansen MPHM. SIAH2 protein expression is inversely correlated with the ER status and outcome to tamoxifen therapy in metastatic breast cancer patients. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-08-51.

ALDH1A1 expression correlates with clinicopathologic features and poor prognosis of breast cancer patients: a systematic review and meta-analysis.

BackgroundAldehyde dehydrogenase 1 family member A1 (ALDH1A1) has been identified as a putative cancer stem cell (CSC) marker in breast cancer. However, the clinicopathological and prognostic significance of this protein in breast cancer patients remains controversial.MethodsThis meta-analysis was conducted to address the above issues using 15 publications covering 921 ALDH1A1+ cases and 2353 controls. The overall and subcategory analyses were performed to detect the association between ALDH1A1 expression and clinicopathological/prognostic parameters in breast cancer patients.ResultsThe overall analysis showed that higher expression of ALDH1A1 is associated with larger tumor size, higher histological grade, greater possibility of lymph node metastasis (LNM), higher level expression of epidermal growth factor receptor 2 (HER2), and lower level expression of estrogen receptor (ER)/progesterone receptor (PR). The prognosis of breast cancer patients with ALDH1A1+ tumors was poorer than that of the ALDH1A1- patients. Although the relationships between ALDH1A1 expression and some clinicopathological parameters (tumor size, LNM, and the expression of HER2) was not definitive to some degree when we performed a subcategory analysis, the predictive values of ALDH1A1 expression for histological grade and survival of breast cancer patients were significant regardless of the different cutoff values of ALDH1A1 expression, the different districts where the patients were located, the different clinical stages of the patients, the difference in antibodies used in the studies, and the surgery status.ConclusionsOur results indicate that ALDH1A1 is a biomarker to predict tumor progression and poor survival of breast cancer patients. This marker should be taken into consideration in the development of new diagnostic and therapeutic program for breast cancer.

The Clinical Significance of Serum Soluble Fas and p53 Protein in Breast Cancer Patients: Comparison with Serum CA 15-3

Serum sFas and p53 protein have been observed in breast cancer patients, but their clinical usefulness for diagnosis and therapy monitoring has not been clarified. The aim of this study was to compare the clinical utility of serum sFas and p53 protein with that of serum CA 15-3 as the most commonly used breast cancer tumor marker. Serum samples were taken from 35 normal healthy controls and 35 breast cancer patients before surgery, after 2 weeks of surgery and after six cycles of FAC chemotherapy. Serum sFas and p53 protein levels were measured using ELISA kits. Serum CA 15-3 levels were determined using IRMA kit. Mean Serum levels of sFas and CA 15-3 were significantly elevated while p53 protein was significantly declined in breast cancer patients than controls. Serum p53 protein showed the greatest significant area under the ROC curve (84.3%) followed by sFas (80.5%), then CA 15-3 (78%). The sensitivity, specificity and cut-off value for diagnosing breast cancer patients were 84.2%, 82.6% and 2.88 U/ml for p53 protein, 83.3%, 68.2% and 497.3 pg/ml for sFas and 45.8%, 100% and 23 U/ml for CA15-3. Surgical removal of breast resulted in a significant decline in serum sFas level with no effect on serum p53 protein and CA 15-3 levels. Six cycles of chemotherapy resulted in a significant elevation in serum sFas level with no effect on serum p53 protein and CA 15-3 levels. sFas was significantly correlated with tumor grade. It could be concluded that although serum p53 protein is superior to sFas and CA15-3 for diagnosis of breast cancer patients, only sFas is useful for monitoring the response of breast cancer patients to surgery and chemotherapy if the effect of systemic inflammatory reactions is excluded.

Expression of Human Endogenous Retrovirus Type K Envelope Protein is a Novel Candidate Prognostic Marker for Human Breast Cancer

We previously observed that the HERV type K (HERV-K) envelope (env) protein was expressed in the majority of human breast tumors from a U.S. cohort of women from Texas. We also made the preliminary observation that the expression of HERV-K env transcripts was associated with markers of disease progression. In this follow-up study, env protein expression was evaluated immunohistochemically in an additional 195 paraffin-embedded breast tumors from a second U.S. patient cohort (Baltimore, Maryland) and in 110 tumors from Chinese patients. Moreover, we compared env transcript expression between fresh-frozen normal and cancerous breast tissues. We observed that while env mRNA and protein expression was undetectable in normal breast tissue and in a subset of uninvolved normal-appearing tissue adjacent to the tumor epithelium, it was overexpressed in most tumors. Furthermore, env expression was associated with breast cancer progression. In Baltimore cohort women, HERV-K tumor positivity was significantly associated with disease stage and lymph node metastasis. In Chinese women, HERV-K env positivity was significantly associated with tumor size, TNM stage, and lymph node metastases, which is consistent with the observations in the U.S. cohort. We also found that Chinese breast cancer patients with a high expression of HERV-K had a decreased overall survival compared with patients who had either a moderate or low HERV-K expression in their tumors (P = 0.049, χ(2) log rank test). In conclusion, the HERV-K env gene is expressed in the majority of breast cancers from U.S. or Chinese women but not in normal breast tissue. High expression of HERV-K env protein in breast cancer patients is associated with markers of disease progression and poor disease outcome, indicating that HERV-K env protein is a novel candidate prognostic marker for breast cancer.

Spontaneous immunogenicity of ribosomal P0 protein in patients with benign and malignant breast lesions and delay of mammary tumor growth in P0‐vaccinated mice

A common carboxyl-terminal epitope (C-22 P0) of the ribosomal P proteins (P0, P1 and P2) was shown to elicit autoantibodies in systemic lupus erythematosus (SLE) and in head and neck cancer patients. In this report we provide evidence for the in vivo immunogenicity of the P0 protein in breast cancer patients. Using recombinant P proteins, we demonstrated that sera from breast carcinoma patients (8/75) displayed significant reactivity to P0 protein when compared with healthy donor sera (0/45). Four out of the eight sera showed simultaneous reactivity to all P proteins. Breast benign tumor (3/17) and mammary hyperplasia (3/17) patient sera also showed significant reactivity to P proteins, thus suggesting that the occurrence of P protein autoantibodies might reveal mammary cell cycle dysregulation. Patient sera reacting with all P proteins recognized C-22 P0. Anti-P0 autoantibodies did not correlate with prognostic parameters of breast carcinomas. High level expression of C-22 P0 was found in mammary carcinomas compared with normal adjacent epithelium and benign lesions. To determine the antitumor activity of P0 as an immunogen, BALB-neuT transgenic mice displaying age-related breast cancer progression were vaccinated using xenogeneic P0 at the stage of mammary atypical hyperplasia. P0 vaccination significantly delayed the onset of mouse mammary tumors that overexpressed C-22 P0. Sera from P0 vaccinated mice recognized C-22 P0. Evidence for immunity to the P0 protein, its overexpression in carcinomas and its peculiar surface localization on cancer cells, along with its antitumor activity as an immunogen might be relevant for the use of P0 protein in monitoring cancer progression and in planning immunotherapeutic strategies.

Human Endogenous Retrovirus K Triggers an Antigen-Specific Immune Response in Breast Cancer Patients

Recent evidence indicates that human cancer cells reactivate the expression of latent human endogenous retroviral (HERV) proteins. However, the extent to which cancer patients mount de novo immune responses against expressed HERV elements is unclear. In this study, we determined the extent of HERV-K env expression in human breast cancer (BC) and whether both humoral and cell-mediated immunity against HERV-K can be found in BC patients. We found HERV-K env protein expression in 88% of BC (n = 119) but not in normal breast (n = 76) tissues. ELISA screening assays detected significant titers of anti-HERV-K env IgG in a large proportion of BC patients. T-cell responses against HERV-K were also detected in peripheral blood mononuclear cells (PBMC) from BC patients stimulated with autologous dendritic cells pulsed with HERV-K env SU antigens. These responses included induction of T-cell proliferation (P = 0.0043), IFN-gamma production measured by enzyme-linked immunospot (P < 0.0001), and multiplex cytokine secretion (P = 0.0033). Multiplex cytokine analysis found a T-helper 1 cytokine response, including interleukin (IL)-2 (P = 0.0109), IL-6 (P = 0.0396), IL-8 (P = 0.0169), and IP-10 (P = 0.0045) secretion during in vitro stimulation of BC PBMC with HERV-K antigen. We also found HERV-K-specific CTLs that were capable of lysing target cells expressing HERV-K env protein in BC patients but not in normal female controls without cancer. These findings suggest that retroviral gene products are capable of acting as tumor-associated antigens activating both T-cell and B-cell responses in BC patients.

Expression of Bmi-1 protein in tumor tissues is associated with favorable prognosis in breast cancer patients

Abnormal expression of the cell cycle regulatory protein Bmi-1 has been studied in breast cancer, but the clinical relevance has not been fully elucidated. We studied the expression of Bmi-1 protein in breast cancer patients to define its clinical significance in breast cancer. Tissue microarrays were performed to evaluate the expression of Bmi-1 by immunohistochemistry in tumor tissues from 960 patients with stage I-III breast cancer. We assessed the relationship between the expression of Bmi-1 and pathologic prognostic indices as well as clinical long-term follow up outcome. Bmi-1 was expressed in 53.2% of breast cancer patients by immunohistochemistry, and the expression of Bmi-1 was significantly correlated with favorable prognostic indices at diagnosis. In univariate analysis, patients with Bmi-1 expression showed favorable relapse-free survival (88.6+/-2.7% vs. 72.3+/-4.3%, P=0.041) and favorable overall survival (93.5+/-2.2% vs. 82.6+/-3.5%, P<0.001) than patients without Bmi-1 expression. According to multivariate analyses, Bmi-1 expression was identified as independent prognostic factor for overall survival with a statistical significance (hazard ratio of Bmi-1 (-) patients compared to Bmi-1 (+) patients, 1.744; 95% CI, 1.013-3.003; P=0.045). This correlation of Bmi-1 expression with favorable overall survival was maintained in patients underwent uniform chemotherapy, regardless of undergoing adjuvant chemotherapy. In a subset analysis according to ER status, Bmi-1 expression associated with favorable overall survival only in ER-positive patients. Bmi-1 expression assessed with Immunohistochemistry may be associated with favorable overall survival in breast cancer patients, especially in patients with ER-positive breast cancer.

Prognostic value of Bcl-2 in breast cancer patients treated with neoadjuvant anthracycline based chemotherapy

We have analyzed the predictive/prognostic value of Bcl-2 protein in breast cancer patients treated with neoadjuvant chemotherapy. One hundred and ten patients were submitted to two different chemotherapeutic regimens: a) 5-fluorouracil, adriamycin or epirubicin, and cyclophosphamide (FAC/FEC) during 2–6 cycles before surgery and 3 or 4 additional cycles of FAC/FEC after surgery ( n = 40) and b) doxorubicin (D) 75 mg/m 2 or epirubicin (E) 120 mg/m 2 during 4 cycles before surgery, and 6 cycles of cyclophosphamide, methotrexate, and 5-fluorouracil (CMF) after surgery ( n = 70). Bcl-2 expression, evaluated by immunohistochemistry, did not change significantly after chemotherapy and was not related to clinical/pathological response. In FAC/FEC group, Bcl-2 positive expression after chemotherapy correlated with better disease free survival (DFS) and overall survival (OS) ( P = 0.008 and P = 0.001). In D/E group, Bcl-2 also correlated with better DFS and OS ( P = 0.03 and P = 0.054) in the post-chemotherapy biopsies. An unusual nuclear localization of Bax was observed in some biopsies, but this localization did not correlate with the tumor response or outcome of the patients. We found that a high Bcl-2 expression had no predictive value but had prognostic value in breast cancer patients treated with neoadjuvant anthracycline based chemotherapy.