Adjacent Normal Breast Tissue Research Articles

Expression and clinical significance of Clusterin protein in invasive ductal breast cancer

Objective To investigate the expression and the clinical significance of Clusterin protein in invasive ductal breast cancer.Methods The expression of Clusterin protein in 70 cases of invasive ductal breast cancer,20 cases of breast hyperplasia,and 10 cases of adjacent normal breast tissue was examined with SP immunohistochemistry (IHC),and their correlation with some clinicopathological parameters were studied.Results Clusterin expression was detected in 71.4％of breast cancer tissues,which was significantly higher than that in benign lesions (20.0 ％) and adjacent normal tissues (0) (x2 =17.143,P ＜ 0.05; x2 =19.048,P ＜ 0.05).Clusterin expression in breast cancer was relevant to the histological grade (x2 =5.233,P ＜ 0.05),lymph node metastasis (x2 =4.677,P ＜ 0.05),and the progression in the clinical stage (x2 =4.667,P ＜ 0.05),but irrelevant to the age (x2 =0.024,P ＞ 0.05) and tumor size (x2 =0.406,P ＞ 0.05).Clusterin expression was negatively correlated to the expression of ER and PR in breast cancer tissues (r =-0.362,P ＜ 0.05 and r =-0.290,P ＜ 0.05,respectively),but uncorrelated with C-erbB-2 expression (r =0.129,P ＞ 0.05).Conclusion Clusterin overexpressed in breast cancer tissues suggests an important role of clusterin in invasive ductal breast cancer carcinogenesis and development through inhibition of cell apoptosis. It may become new clinical diagnosis marker for breast cancer and novel target for breast cancer therapy. Key words: Carcinoma, ductal, breast; Clusterin; Immunohistochemistry

Expression pattern of ATM and cyclin D1 in ductal carcinoma, normal adjacent and normal breast tissues of Iranian breast cancer patients

ATM protein kinase plays a critical role in maintaining genome integrity by activating a biochemical chain reaction that in turn leads to cell cycle checkpoint activation and repair of DNA damage. Cyclin D1 acts in regulating the G1 phase of the cell cycle. Experimental and clinical studies suggest them to be involved in transformation and tumour progression. To elucidate the role of ATM and cyclin D1 expression in sporadic breast cancer, we investigated the possible link between their RNA expression levels in ductal carcinoma and normal adjacent versus normal breast tissues measured by Taqman real-time PCR in 119 breast tissues. Results showed that cyclin D1 over-expressed in 51.4% of breast tumours, whereas ATM expression was down regulated in 55% of breast tumours compared to both normal adjacent and normal controls (P ≤ 0.01). Cyclin D1 expression in adjacent normal and normal tissues was not significantly differed, whereas ATM expression in normal adjacent was lower than normal control (P ≤ 0.01). Over-expression of cyclin D1 correlated with ER(+) and/or PR(+) (oestrogen/progesterone receptor) status, whereas it mostly under-expressed in HER2(+) (human epidermal growth factor 2) tumours. ATM under-expression was more observed in triple-negative tumours (ER(-), PR(-) and HER2(-)). Our results indicated that reduced expression of the ATM and aberrant cyclin D1 expressions may contribute to the development and/or malignant progression of breast carcinomas also the latter could be involved in the regulation of hormone sensitivity associated with ER and PR.

G-protein coupled receptor-associated sorting protein 1 (GASP-1), a potential biomarker in breast cancer

An innovative “2-D high performance liquid electrophoresis” (2-D HPLE) technology was used to identify serum biomarkers associated with the early stage of breast cancer in addition to other more advanced stages. 2-D HPLE is a newly developed electrophoretic technology that separates 100s of serum albumin complexes on a polyvinyl membrane based on their surface charges. Association of cancer proteins or their fragments (biomarkers) with pre-existing albumin complexes in the blood of cancer patients results in altered mobility on the membrane. Using 2-D HPLE we identified that a specific fragment of G-protein coupled receptor-associated sorting protein 1 (GASP-1) was present in the sera of patients with early stage disease but absent in sera of normal patients. GASP-1 has been shown to modulate lysosomal sorting and functional down-regulation of a variety of G-protein coupled receptors in neuronal cells. However, no reports have linked GASP-1 to breast cancer pathogenesis. GASP-1 was detected in tumor extracts of 7 cases of Stage 2 and Stage 3 breast cancers, but not in adjacent normal tissue as revealed by western blot analysis using an antibody developed against a GASP-1 peptide identified by our 2-D HPLE technology. Using this antibody, we immunohistochemically detected over-expression of GASP-1 in all of 107 cases of archived ductal breast carcinoma tumor samples, while normal adjacent breast tissue from 12 cases of ductal carcinoma showed little or no staining. Additionally, all 10 cases of metastatic breast carcinoma present in lymph nodes were positive. Strong positive GASP-1 staining was observed in all tumor tissue including ductal carcinoma in situ (DCIS) and invasive ductal carcinoma. Additionally, we observed a wide spectrum of enhanced staining of premalignant ductal epithelial cells present in benign ducts and those found in atypical ductal hyperplasia (ADH). These studies identify GASP-1 as a potential new serum and tumor biomarker for breast cancer and suggest that GASP-1 may be a novel target for the development of breast cancer therapeutics.

Phosphorylation of Caspase-7 by p21-activated Protein Kinase (PAK) 2 Inhibits Chemotherapeutic Drug-induced Apoptosis of Breast Cancer Cell Lines

p21-activated kinase (PAK) 2, a member of the PAK family of serine/threonine protein kinases, plays an important role in physiological processes such as motility, survival, mitosis, and apoptosis. However, the role of PAK2 in resistance to chemotherapy is unclear. Here we report that PAK2 is highly expressed in human breast cancer cell lines and human breast invasive carcinoma tissue compared with a human non-tumorigenic mammary epithelial cell line and adjacent normal breast tissue, respectively. Interestingly, we found that PAK2 can bind with caspase-7 and phosphorylate caspase-7 at the Ser-30, Thr-173, and Ser-239 sites. Functionally, the phosphorylation of caspase-7 decreases its activity, thereby inhibiting cellular apoptosis. Our data indicate that highly expressed PAK2 mediates chemotherapeutic resistance in human breast invasive ductal carcinoma by negatively regulating caspase-7 activity.

Significance of STAT3 Activation in Breast Cancer

Signal transducer and activator of transcription 3 (STAT3) is a latent transcription factor whose activation plays immense role in cell proliferation, survival, differentiation and development. Activation of STAT3 relies on phosphorylation at tyrosine residues promoting dimerization and subsequent nuclear translocation and activation of transcription. This study explores the relation between STAT3 and activated STAT3 (p-STAT3) in malignancy of breast. A high expression of STAT3 was found in breast cancer compared to adjacent normal breast tissues. A significant correlation between STAT3 and p-STAT3 was also observed. We also examined possible associations of these biomarkers with various clinicopathological parameters of breast cancer patients. The results revealed that both STAT3 and p-STAT3 are strongly associated with some of the major risk factors of breast cancer. Our findings implicate that targeting STAT3 signalling pathway could be an effective therapeutic approach for breast cancer.

Abstract 1960: Phosphorylation of caspase-7 by p21-activated protein kinase (PAK)2 inhibits staurosporine-induced apoptosis of MCF-7 breast cancer cells

Abstract P21-activated kinase (PAK) 2, a member of PAK family of serine/threonine kinases, plays an important role in physiological processes such as motility, survival, mitosis, and apoptosis. However, the role of PAK2 in chemotherapy resistance, as well as cell proliferation, is not clear. In this report, we showed that the protein expression level of PAK2 is higher in human breast cancer cell lines and human breast invasive carcinoma tissues compared with adjacent normal breast tissues. Importantly, we found that PAK2 can bind with caspase-7 and phosphorylate caspase-7 at Ser30, Thr173 and Ser239. Phosphorylation of caspase-7 decreases its activity, thereby inhibiting cellular apoptosis. Knocking down PAK2 expression can restore caspase-7 activity and promote apoptosis of MCF-7 breast cancer cells. Our data suggests that the highly expressed PAK2 mediates chemotherapy resistance in human breast invasive ductal carcinoma and that PAK2 is a novel target of chemotherapy for breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1960. doi:10.1158/1538-7445.AM2011-1960

Abstract 5066: Molecular profiles of breast cancer in Barbados

Abstract Background: Breast cancer is a complex disease with varying incidence and mortality rates across populations. This study is the first investigation of molecular subtypes of breast tumors in a Barbados, West Indies population which is predominantly African in origin. Methods: To identify molecular subtypes of breast cancer we collected 260 formalin-fixed and paraffin-embedded (FFPE) tissue blocks from the Pathology Laboratory, Queen Elizabeth Hospital, St. Michael, Barbados. Pathologic features, including diagnosis, grade, tumor size, and axillary lymph node metastasis, were abstracted from pathology reports. Histology diagnosis, grading of invasive breast cancer and carcinoma in situ were performed using protocols of the College of American Pathologists and World Health Organization. Tissue microarrays were constructed from these 260 confirmed breast cancer samples and adjacent normal breast tissue at the University of Chicago. Immunohistochemical (IHC) assays were performed with commercial antibodies. To identify breast cancer subtypes, the tissues were evaluated for the expression of ER, PR, HER2, CK5/6 and EGFR. Vimentin staining served as a control for tissue fixation quality in archival tumors. Allred scores for ER and PR were calculated. HER2 was evaluated by IHC according to ASCO/CAP guidelines. EGFR was evaluated according to PharmDX recommendations. Vimentin and CK 5/6 were evaluated according to Dabbs, 2006. Breast cancer subtypes were defined as luminal A (ER+ and/or PR+, HER2-), luminal B (ER+ and/or PR+, HER2+), basal-like (ER-, PR-, HER2-, CK5/6+, and/or EGFR+), HER2+/ER- (HER2+, ER-, PR-), and unclassified (negative for all five markers). Results: The mean age of the breast cancer patients was 57 years old (SD=15 years). There were 63.7% patients with ER positive tumors, 50.6% with PR positive tumors, and 21.2% with HER2 positive tumors. The distribution of breast cancer subtypes was luminal A (52.5%), luminal B (12.7%), HER2+/ER- (8.5%), basal-like (24.6%), and unclassified (1.7%). The majority of patients had high grade tumors (52.3% grade II and 39.1% grade III). The luminal A and B tumors contained ductal and lobular types and several special types of breast carcinomas such as tubular, mucinous, cribriform, signet ring cells and acinic cell types. The basal-like tumors were more likely to be medullary carcinomas. The basal-like (70.2%) and HER2+/ER- (65.0%) tumors were more likely to be high-grade tumors than the luminal A (20.2%) and luminal B (40.0%). Conclusions: We found that the proportion of estrogen receptor positive tumors in Barbados was similar to that observed in African Americans, lower than that of Caucasians and Asians but higher than Nigerian. There was a relatively high proportion of basal-like breast cancer in this cohort of African descent. Further epidemiology studies are warranted to identify reasons for this similarity, including possibly the increasing Western influence on the Barbadian lifestyle. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5066. doi:10.1158/1538-7445.AM2011-5066

Abstract 3840: Rap1 GTPase activating protein (Rap1GAP) is downregulated in DCIS and invasive ductal carcinoma

Abstract Introduction: Rap1 and Rap2, both small GTPases of the Ras family, serve as crucial regulators in cell polarity, adhesion, migration, and lumen formation via crosstalk with cytoskeletal proteins. Rap1 GTPase activating protein (Rap1GAP), which negatively regulates Rap1 and Rap2 activity, has found to be downregulated in multiple cancers, including oropharyngeal squamous cell carcinoma, melanoma, papillary and follicular thyroid carcinoma, and colorectal carcinoma but has not yet been studied in breast cancer. In order to determine if Rap1GAP downregulation plays a role in breast cancer formation, we decided to expand our prior work to study its expression in breast cancer tissue. Methods: Twenty-one formalin-fixed paraffin-embedded human tissue blocks were selected on the basis that they contained normal breast tissue and either adjacent breast ductal carcinoma in situ (DCIS) or invasive ductal carcinoma. Using immunohistochemistry, we assessed the expression of Rap1GAP. We also assayed two human breast cancer cell lines with different levels of invasiveness, MCF7 (non-invasive) and MDA-MB-231 (invasive), to determine different levels of Rap1GAP expression and subsequent changes in Rap1 and Rap2 expression and activity. Results: Rap1GAP expression in both DCIS and invasive ductal carcinoma were found to be decreased compared to adjacent normal breast tissue. However, Rap1GAP expression was not statistically different between DCIS and invasive carcinoma lesions. Rap1GAP was found to be expressed highly in MCF7 but not in MDA-MB-231 cells. Moreover, only MDA-MB-231 cells exhibited high basal Rap2 activity. Activated Rap1 (RAP1-GTP) was not detected in either line. Conclusion: Our findings suggest that loss of Rap1GAP occurs as an early event in breast cancer tumorigenesis with a significant decrease in the transition from normal to DCIS, with a possible further decrement in the DCIS to invasive transition. Interestingly, while Rap1 and Rap2 were expressed in non-invasive and invasive cell lines, loss of Rap1GAP in the invasive cell line was associated with increase in active Rap2, but not active Rap1. These findings suggest that as in other cancers, loss of Rap1GAP may contribute to the invasive phenotype and thus further investigation is warranted. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3840. doi:10.1158/1538-7445.AM2011-3840

Expressions and Significance of Ezrin and AKT2 in Human Breast Cancer Tissue

Objective To investigate the expressions and clinical significance of Ezrin and AKT2 in human breast carcinoma.Methods The expression of Ezrin and AKT2 in 56 cases of breast carcinoma and 30 cases of adjacent nontumor normal breast tissue was examined by SP immunohistochemistry method.The relationships between these two factors and the clinicopathological factors were evaluated.Results(1) The positive expression of Ezrin and AKT2 in 56 cases of breast carcinoma was 62.50% and 76.79% respectively.There was a significant different between carcinoma and normal breast tissue(P0.05).(2) The overexpression of Ezrin in breast carcinoma had no relationships with the size of primary tumor,age and pathological type;while had some relationship with histological classification,clinical TNM staging and lymph node metastasis(P0.05).The expression of AKT2 was correlated with lymph node metastasis(P0.05);while no relationship with age,size of primary tumor,histological classification,clinical TNM staging and pathological type(P0.05).(3) Ezrin and AKT2 were significantly and directy correlated with each other in invasive ductal carcinoma.Conclusion The overexpression of Ezrin and AKT2 may play an important role in the carcinogenesis and progression of breast carcinoma.They may be considered as the indications of biological behavior and prognosis of breast carcinoma.

The Association of Infrared Imaging Findings of the Breast with Hormone Receptor and Human Epidermal Growth Factor Receptor 2 Status of Breast Cancer

Evidence on breast infrared (IR) imaging and its association with estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2) statuses of breast cancers is limited. The aim of this study was to investigate the association of IR imaging findings and ER, PR, and HER2 status in breast cancers. A total of 163 women with 171 pathologically proven breast cancers underwent IR imaging of the breast before surgery. Five IR signs were used to score the lesions: IR1, the temperature difference (ΔT) of the lesion site from that of the contralateral mirror image site; IR2, ΔT of the lesion site from that of the adjacent normal breast tissue in the same breast; IR3, abnormal vascular morphologic patterns; IR4, focal bulge or edge sign with back heat at the lesion site; and IR5, asymmetric thermographic pattern between the lesion site and the contralateral breast. The association of different IR signs with ER, PR, and HER2 status was evaluated using Fisher's exact test. IR1 was inversely associated with ER (P = .010) and PR status (P = .039). IR2 was inversely related to PR status (P = .020). IR5 was inversely associated with ER (P = .037) and PR (P = .022) status. No IR sign was associated with HER2 status. Triple-negative (ER-negative, PR-negative, and HER2-negative) cancers tended to show higher IR1 scores compared to other types of cancers (P = .029). Breast IR findings were associated with ER and PR status of breast cancers. Triple-negative cancers more frequently featured higher IR1 scores than other types of cancers.

Expression of BMI-1 and Mel-18 in breast tissue - a diagnostic marker in patients with breast cancer

BackgroundPolycomb Group (PcG) proteins are epigenetic silencers involved in maintaining cellular identity, and their deregulation can result in cancer. Expression of Mel-18 and Bmi-1 has been studied in tumor tissue, but not in adjacent non-cancerous breast epithelium. Our study compares the expression of the two genes in normal breast epithelium of cancer patients and relates it to the level of expression in the corresponding tumors as well as in breast epithelium of healthy women.MethodsA total of 79 tumors, of which 71 malignant tumors of the breast, 6 fibroadenomas, and 2 DCIS were studied and compared to the reduction mammoplastic specimens of 11 healthy women. In addition there was available adjacent cancer free tissue for 23 of the malignant tumors. The tissue samples were stored in RNAlater, RNA was isolated to create expression microarray profile. These two genes were then studied more closely first on mRNA transcription level by microarrays (Agilent 44 K) and quantitative RT-PCR (TaqMan) and then on protein expression level using immunohistochemistry.ResultsBmi-1 mRNA is significantly up-regulated in adjacent normal breast tissue in breast cancer patients compared to normal breast tissue from noncancerous patients. Conversely, mRNA transcription level of Mel-18 is lower in normal breast from patients operated for breast cancer compared to breast tissue from mammoplasty. When protein expression of these two genes was evaluated, we observed that most of the epithelial cells were positive for Bmi-1 in both groups of tissue samples, although the expression intensity was stronger in normal tissue from cancer patients compared to mammoplasty tissue samples. Protein expression of Mel-18 showed inversely stronger intensity in tissue samples from mammoplasty compared to normal breast tissue from patients operated for breast cancer.ConclusionBmi-1 mRNA level is consistently increased and Mel-18 mRNA level is consistently decreased in adjacent normal breast tissue of cancer patients as compared to normal breast tissue in women having had reduction mammoplasties. Bmi-1/Mel-18 ratio can be potentially used as a tool for stratifying women at risk of developing malignancy.

Expression of SM22α and its relationship with lymph node metastasis in breast cancer

to analyze the relationship between the expression of SM22α and the lymph node (LN) metastasis of breast cancer and to investigate its molecular mechanisms. reverse transcription-polymerase chain reaction (RT-PCR) was performed to detect the expression of SM22α in breast cancer tissue and adjacent normal breast tissue. RT-PCR and Western blot were employed to investigate the SM22α mRNA and protein level in tissues of breast fibroadenoma, breast cancer without LN metastasis and breast cancer with LN metastasis. RT-PCR and zymography were used to detect the MMP2 and MMP9 expression and activity and TIMP1 expression level in breast fibroadenoma, breast cancer samples without LN metastasis and those with LN metastasis respectively. the expression level of SM22α mRNA in breast cancer was significantly lower than that in breast fibroadenoma or adjacent normal breast tissue (5.1% ± 2.4% vs 15.1% ± 5.3% vs 30.1% ± 5.1%, P < 0.01). The protein and mRNA expression level of SM22α in breast cancer samples with LN metastasis were significant lower than those of breast cancer without LN metastasis (6.2% ± 3.1% vs 10.1% ± 4.1%, P < 0.01). Both the expression and activity of MMP2 and MMP9 in breast cancer samples with LN metastasis were significant higher than those without LN metastasis (P < 0.01). A strong negative correlation was found between SM22α protein level and MMP2 activity (r = -0.848; n = 27; P < 0.01) or MMP9 activity (r = -0.916; n = 27; P < 0.01) in breast cancer tissue. a down-regulation of SM22α in breast cancer is correlated with LN metastasis. SM22α may inhibit the LN metastasis through a negative regulation of MMP2 and MMP9 in breast cancer.

PLCD1 is a functional tumor suppressor inducing G2/M arrest and frequently methylated in breast cancer

Chromosome 3p harbors multiple tumor-suppressor genes. PLCD1, located at 3p22, encodes an enzyme that mediates regulatory signaling of energy metabolism, calcium homeostasis and intracellular movement. We investigated the epigenetic alterations of PLCD1 and its tumor suppressor function in breast cancer. Frequent downregulation/silencing of PLCD1 was shown in most breast cancer cell lines by using semi-quantitative PCR. Promoter methylation of PLCD1 was detected in 78% (7/9) of cell lines and 52% (13/25) of primary tumors by Methylation-specific PCR (MSP), but not in any tumor adjacent breast tissues and normal breast tissues, which was further confirmed by bisulfite genomic sequencing (BGS). The silencing of PLCD1 could be reversed by pharmacological demethylation, indicating a methylation-mediated mechanism. Ectopic expression of PLCD1 in silenced breast cancer cells significantly inhibited their colony formation. In addition, PLCD1 inhibited tumor cell migration and induced cell cycle G2/M arrest. Thus, this study for the first time demonstrates the frequent inactivation of PLCD1 by promoter methylation and its tumor inhibitory function in breast cancer. Tumor-specific methylation of PLCD1 might serve as a biomarker for possible early detection and prognosis prediction of breast cancer.

Significant elevation of CLDN16 and HAPLN3 gene expression in human breast cancer

Cancer development involves the destruction of tight junctions, deprivation of cell polarity, and increased cell mobility. Claudin 16 (CLDN16) is a tight junction protein and plays important roles in the maintenance of cell polarity, cellular arrangement, adhesion, paracellular transport, and ionic permeability of various epithelia. A novel link protein, HAPLN3, functions in hyaluronic acid binding and cell adhesion. Both genes are hypothesized to be related to cancer development and metastasis. The purpose of this study was to estimate the roles of the genes CLDN16 and HAPLN3 in breast cancer. A total of 146 samples were collected from breast cancer tissues and their adjacent normal breast tissues. Reverse transcription and real-time polymerase chain reaction were used to estimate gene expression levels. There were significantly increased gene expression of CLDN16 (p<0.0001) and HAPLN3 (p<0.0001) among breast cancer tissues compared with normal tissues, irrespective of clinical pathological parameters. The absolute increased gene expression level of CLDN16 was significantly negatively correlated with estrogen (r=-0.46; p<0.0001) and progesterone receptor (r=-0.384; p=0.001) staining density. However, a significantly positive correlation (r=0.24; p=0.04) between the absolute increased HAPLN3 gene level and human epidermal receptor 2 staining density was found. There was no significant association between overall survival and the two gene expression levels. The gene up-expression of both CLDN16 and HAPLN3 was suggested to be involved in the development of breast cancer and to be a biomarker and target treatment for breast cancer.

Angiogenin Regulation by Estradiol in Breast Tissue: Tamoxifen Inhibits Angiogenin Nuclear Translocation and Antiangiogenin Therapy Reduces Breast Cancer Growth In vivo

Angiogenin, a 14.2-kDa polypeptide member of the RNase A superfamily, has potent angiogenic effects. Nuclear accumulation of angiogenin is essential for its angiogenic activity. Increased angiogenin expression has been associated with the transition of normal breast tissue into invasive breast carcinoma. In this article, we investigated whether estradiol (E(2)) affected angiogenin in breast tissue. We used microdialysis for sampling of extracellular angiogenin in vivo. In vitro cultures of whole normal breast tissue, breast cancer cells, and endothelial cells were used. We show that extracellular angiogenin correlated significantly with E(2) in normal human breast tissue in vivo and that exposure of normal breast tissue biopsies to E(2) stimulated angiogenin secretion. In breast cancer patients, the in vivo angiogenin levels were significantly higher in tumors compared with the adjacent normal breast tissue. In estrogen receptor-positive breast cancer cells, E(2) increased and tamoxifen decreased angiogenin secretion. Moreover, E(2)-induced angiogenin derived from cancer cells significantly increased endothelial cell proliferation. Tamoxifen reversed this increase as well as inhibited nuclear translocation of angiogenin. In vivo, in experimental breast cancer, tamoxifen decreased angiogenin levels and decreased angiogenesis. Additionally, treating tumor-bearing mice with an antiangiogenin antibody resulted in tumor stasis, suggesting a role for angiogenin in estrogen-dependent breast cancer growth. Our results suggest previously unknown mechanisms by which estrogen and antiestrogen regulate angiogenesis in normal human breast tissue and breast cancer. This may be important for estrogen-driven breast cancer progression and a molecular target for therapeutic interventions.

Molecular profiles of breast cancer in Ilorin, Nigeria.

1602 Background: Breast cancer is a complex disease with diversity between tumors and within tumor. Major differences exist in incidence and mortality rates across populations, making it imperative to investigate molecular pathogenesis in various populations. Methods: To identify molecular subtypes of breast cancer we collected Formalin-fixed and Paraffin-Embedded (FFPE) tissue blocks from Ilorin. Pathologic features and patients' data were extracted from histology reports. Tissue microarrays were constructed from FFPE tumor samples and adjacent normal breast tissue at the University of Chicago. Immunohistochemical assays were performed using a DAKO immunostainer (DAKO, Carpinteria, CA) with antibodies and antigen unmasking. Breast cancer subtypes were defined as luminal A (ER+ and/or PR+, HER2-), luminal B (ER+ and/or PR+, HER2+), basal-like (ER-, PR-, HER2-, CK5/6+, and/or EGFR+), HER2+/ER- (HER2+, ER-, PR-), and unclassified (negative for all five markers). Results: From 2003 to 2007, 203 histologicaly confirmed breast cancer patients were enrolled, including 5 males and 5 bilateral breast cancers. Median age at diagnosis was 46 yrs. Majority of patients (59%) presented with large tumors (≥ 5 cm) as median time from symptoms onset to cancer diagnosis was 6 months (interquartile range 3-12 months). Proportion of ER+, PR+ and HER2+ tumors were 27%, 16%, and 30%, respectively. Commonest breast cancer subtype was basal-like (25%), followed by unclassified (24%), luminal A (20%), HER2+/ER- (19%), and luminal B (11%). Patients with ER+ tumors had longer duration of symptoms (median 8 months) than patients with ER- tumors (5 months) but ER+ patients had smaller tumors (median 5 cm) than ER- patients (6 cm, p = 0.02). Conclusions: This study replicates previous findings that triple negative breast cancer was over-represented in African population in a different geographic region of North Central Nigeria as found in the Southwest earlier on. Patients seek medical help after many months of cancer symptoms, suggesting lack of awareness of breast cancer in the population. Study also showed that ER- breast cancer grows faster than ER+ breast cancer, underscoring urgent need to develop effective breast cancer awareness and early detection programs in Africa. No significant financial relationships to disclose.

Correlation of telomere length shortening with promoter methylation profile of p16/Rb and p53/p21 pathways in breast cancer

Unregulated cell growth, a major hallmark of cancer, is coupled with telomere shortening. Measurement of telomere length could provide important information on cell replication and proliferation state in cancer tissues. Telomere shortening and its potential correlation with downregulation of cell-cycle regulatory elements were studied by the examination of relative telomere length and methylation status of the TP53, P21 and P16 promoters in tissues from breast cancer patients. Telomere length was measured in 104 samples (52 tumors and paired adjacent normal breast tissues) by quantitative PCR. Methylation profile of selected genes was analyzed in all samples using a matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI–TOF MS). Our results demonstrated a significant shortening of tumor telomere regions compared with paired adjacent normal tissues (P<0.001). Similarly, telomere lengths were significantly shorter in advanced stage cases and in those with higher histological grades (P<0.05). Telomere shortening in cancer tissues was correlated with a different level of hypermethylation in the TP53, P21 and P16 promoters (r=−0.33, P=0.001; r=−0.70, P<0.0001 and r=−0.71, P<0.0001, respectively). The results suggested that inactivation of p16/Rb and/or p53/p21 pathways by hypermethylation may be linked to critical telomere shortening, leading to genome instability and ultimately to malignant transformation. Thus, telomere shortening and promoter hypermethylation of related genes both might serve as breast cancer biomarkers.

Effect of Low-Dose Tamoxifen on Steroid Receptor Coactivator 3/Amplified in Breast Cancer 1 in Normal and Malignant Human Breast Tissue

Nuclear receptor coactivator expression and activity may partly explain the complex agonist/antagonist effects of tamoxifen at clinical level. In a preoperative trial, dose reduction from 20 to 1 mg tamoxifen was associated with retained antiproliferative effect on breast cancer. Here, we assessed the gene expression of the steroid receptor coactivators SRC-1, SRC-2/transcription intermediary factor 2, and SRC-3/amplified in breast cancer 1 (AIB1) and the growth factor receptor HER-2/neu under three tamoxifen dose regimens. Surgical specimens from estrogen receptor-positive breast cancer and adjacent normal breast tissue from 64 patients treated 4 weeks preoperatively with 20, 5, or 1 mg/d tamoxifen and 28 nontreated breast cancer controls were analyzed for coactivator and HER-2/neu mRNA expression using real-time reverse transcription-PCR. The gene expression levels were related to immunohistochemical expression of Ki67, serum levels of insulin-like growth factor I and sex hormone binding globulin, other prognostic factors, and clinical outcome. The coactivators and HER-2/neu mRNA levels were higher in malignant compared with normal tissue (P < 0.001). Tamoxifen significantly increased the expression of coactivators in normal and malignant tissue irrespective of dose, especially for SRC-3/AIB1 (P < 0.001 tamoxifen-treated versus nontreated subjects). SRC-3/AIB1 and HER-2/neu mRNA levels were positively correlated (P = 0.016), but the coactivators could not explain the variability of Ki67, insulin-like growth factor I, and sex hormone binding. Although not significant, SRC-3/AIB1 tended to be higher in subjects with poor clinical outcome and unfavorable prognostic factors. Increased coactivator mRNA levels seem to be an early response to tamoxifen without dose-response relationship in the 1- to 20-mg range. Clinical and molecular effects of low-dose tamoxifen should be further explored.

PTEN mutation spectrum in breast cancers and breast hyperplasia

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is one of the most frequently mutated human tumor suppressor genes. The present study aims to investigate the role of PTEN mutation in breast carcinogenesis by analyzing PTEN mutation spectrum and the protein expression in breast cancers, adjacent hyperplastic lesions, benign breast lesions and normal breast tissues. All 9 exons of PTEN gene were amplified by PCR with DNA extracted from 50 of human breast cancers and corresponding adjacent breast hyperplasia tissues, adjacent normal breast tissues, as well as 50 breast benign lesions residing in or around Yunnan, China, respectively. PCR products were then sequenced for mutation screening. And we also proved the effect of mutations on the expression of PTEN protein by immunohistochemistry. PTEN mutations were detected in 11 of 50 (22%) breast cancers and 4 of 50 (8%) adjacent ductal hyperplasia, all of which were atypical ductal hyperplasia and same PTEN mutation were detected in the corresponding cancer tissues. No PTEN mutation was detected in all adjacent normal breast tissues and 50 cases of breast benign lesions. The mutation sites concentrated at exon 3, 4, 5 and 7; no mutation was detected in exon 1, 2, 6, 8, or 9 and splicing sites of all introns. The hottest mutation spots were exon 5 with missense mutations. Immunohistochemical analysis showed that 24 of 50 (48%) breast cancers and 6 of 50 (12%) adjacent breast hyperplasia demonstrated negative immuno-staining of PTEN (loss of PTEN protein expression). All the 4 adjacent breast tissues harbored PTEN mutations and 9 of 11 breast cancers with PTEN mutation were loss of PTEN expression. Statistical analysis revealed that PTEN gene mutations were correlated with the PTEN expression. The incidence of PTEN mutations is relatively high in patients with sporadic breast cancer in the region of Yunnan, China and exists at the early stage of breast cancer development. The PTEN mutations have significant effect on the expression silencing of PTEN protein indicating the important role of PTEN mutation in carcinogenesis of breast cancers.

Discovery and verification of protein differences between Er positive/Her2/neu negative breast tumor tissue and matched adjacent normal breast tissue

This study was designed to quantify and identify differences in protein levels between tumor and adjacent normal breast tissue from the same breast in 18 women with stage I/II ER positive/Her2/neu negative invasive breast cancer. Eighteen separate difference gel electrophoresis (DIGE) gels were run (1 gel per patient). Relative quantification was based on DIGE analysis. After excision and tryptic digestion, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and peptide mass mapping were used to identify protein spots. Two hundred and forty-three spots were differentially abundant between normal and cancer tissues. Fifty spots were identified: 41 were over abundant and nine were less abundant in cancers than in normal breast tissue. Western blotting provided independent confirmation for three of the most biologically and statistically interesting proteins. All 18 gels were replicated by another technician and 32% of the differentially abundant proteins were verified by the duplicate analysis. Follow-up studies are now examining these proteins as biomarkers in blood.