Sporadic Breast Cancer Patients Research Articles

Abstract B82: High frequency of hypermethylation at the 14-3-3 sigma to gene silencing in breast cancer.

Abstract Background: Expression of 14-3-3 sigma is induced in response to DNA damage, and causes cells to arrest in G(2). Hypermethylation of CpG islands located in the promoter regions of tumor suppressor genes is now firmly established as an important mechanism for gene inactivation. Objetive: To study the relation of 14-3-3 sigma gene promoter hypermethylation and its transcription expression levels in sporadic breast carcinogenesis. Material and Methods:This is a prospective study we quantified methylation levels of promoter 14-3-3 gene in 107 women with breast cancer and 108 control subjects by Real Time QMS-PCR SYBR green and analyzed association with prognostics factor in breast cancer. Results: Median age was 58 years (32-88); 69% were postmenopausal women. Nodal involvement N0; 63%, N1;30%, N2;7%), tumor size (T1;58%, T2;35%, T3;4%, T4;4%) and grade G1; 20%, G2;37%, G3;30%). The methylation of 14-3-3σ were 60% of sporadic breast cancer patients and were 34% of normal breast (p=0.0047). The methylation of 14-3-3σ gene in serum was markedly related with T3-4 stage (p<0.05), nodal positive status (p<0.05) and poor outcome. With a median follow up 6 years we saw more probability of developing distance metastasis in patients with methylation 14-3-3σ (p>0.05). Conclusions: The recent identification of novel 14-3-3sigma-associated proteins by a targeted proteomics approach implies that 14-3-3sigma regulates diverse cellular processes, which may become deregulated after silencing of 14-3-3sigma expression in cancer cells. Therefore, it is possible that loss of σ expression contributes to malignant transformation by impairing the G2 cell cycle checkpoint function, thus allowing an accumulation of genetic defects. Perhaps in the detection of CpG methylation of 14-3-3σ may be used for diagnostic and prognostic purposes Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B82.

Clinicopathological significance and prognostic value of Xeroderma pigmentosum complementary group C (XPC) expression in sporadic breast cancer patients

Breast cancer is the most common type of cancer among women worldwide, and the incidence of breast cancer is increasing in the developing world. Estrogen exposure is a major risk factor for breast cancer, and estrogen oxidative metabolites have been implicated in chemical carcinogenesis. Xeroderma pigmentosum complementary group C (XPC) plays an important and multifaceted role in cell protection from oxidative DNA damage. Thus, XPC inactivation may be involved in the early stage of breast cancer. The aim of this study was to investigate the expression of XPC protein in sporadic breast cancer tissues and determine whether XPC expression influences breast cancer malignancy and clinical outcome. Fifteen cases of adjacent non-tumor breast tissue, 28 cases of fibroadenomas and 235 cases of breast carcinomas were examined by immunohistochemistry using polyclonal antibody to XPC. Both cytoplasmic and nuclear expression level of XPC were downregulated in breast carcinoma when compared to non-tumor tissues (P < 0.05). The nuclear expression level of XPC was significantly associated with expression of BCL2 (r = 0.231, P = 0.033) and p53 (r = 0.205, P = 0.011), and nuclear expression of XPC was significantly associated with patients' age (P = 0.024). Neither cytoplasmic nor nuclear expression level of XPC had impact on patients' survival in the whole samples. However, XPC expression was correlated with adverse survival in HER2-positive, but not HER2-negative, tumors, as demonstrated by Kaplan-Meier analysis. Our results suggested that the XPC protein is involved in the occurrence and progression of breast cancer.

P53-Dependent BRCA1 Nuclear Export Controls Cellular Susceptibility to DNA Damage

Subcellular localization regulates BRCA1 function, and BRCA1 is exported to the cytoplasm following DNA damage in a p53-dependent manner. Because more than 50% of solid tumors harbor p53 mutations, it is possible that genetically wild-type (wt) BRCA1 is functionally abnormal through compromised nuclear-cytoplasmic shuttling in sporadic breast cancer patients with dysfunctional p53. In this study, we have investigated the mechanisms of p53-dependent BRCA1 subcellular distribution and DNA damage-induced nuclear export, as well as the impact on the resulting cytotoxic response to therapy in human breast cancer. We first show that p53 mediates BRCA1 nuclear export via protein-protein binding, rather than by modulation of its transcription. Furthermore, it is the C-terminal (BRCT) region of BRCA1 that is critical for its interaction with p53, and p53 may promote BRCA1 nuclear export by interrupting the association of BRCA1 with BARD1. In sporadic breast cancer specimens, dysfunctional p53 strongly correlates with nuclear retention of sequence-verified wt BRCA1. This p53-dependent BRCA1 shuttling determines cellular susceptibility to DNA damage as augmentation of cytosolic BRCA1 significantly enhances cancer cell susceptibility to ionizing radiation. Taken together, our data suggest that p53 dysfunction compromises nuclear export of wt BRCA1 as a mechanism to increase cellular resistance to DNA damage in sporadic breast cancer. We propose that targeting nuclear BRCA1 to the cytoplasm may offer a unique strategy to sensitize p53-deficient sporadic breast cancers to DNA damage-based therapy.

Genetic Anticipation Is Associated with Telomere Shortening in Hereditary Breast Cancer

There is increasing evidence suggesting that short telomeres and subsequent genomic instability contribute to malignant transformation. Telomere shortening has been described as a mechanism to explain genetic anticipation in dyskeratosis congenita and Li-Fraumeni syndrome. Since genetic anticipation has been observed in familial breast cancer, we aimed to study telomere length in familial breast cancer patients and hypothesized that genetic defects causing this disease would affect telomere maintenance resulting in shortened telomeres. Here, we first investigated age anticipation in mother-daughter pairs with breast cancer in 623 breast cancer families, classified as BRCA1, BRCA2, and BRCAX. Moreover, we analyzed telomere length in DNA from peripheral blood leukocytes by quantitative PCR in a set of 198 hereditary breast cancer patients, and compared them with 267 control samples and 71 sporadic breast cancer patients. Changes in telomere length in mother-daughter pairs from breast cancer families and controls were also evaluated to address differences through generations. We demonstrated that short telomeres characterize hereditary but not sporadic breast cancer. We have defined a group of BRCAX families with short telomeres, suggesting that telomere maintenance genes might be susceptibility genes for breast cancer. Significantly, we described that progressive telomere shortening is associated with earlier onset of breast cancer in successive generations of affected families. Our results provide evidence that telomere shortening is associated with earlier age of cancer onset in successive generations, suggesting that it might be a mechanism of genetic anticipation in hereditary breast cancer.

The efficacy of taxane chemotherapy for metastatic breast cancer in BRCA1 and BRCA2 mutation carriers

We assessed the efficacy of taxane chemotherapy in BRCA1- and BRCA2-associated patients compared with sporadic metastatic breast cancer patients. Response rates (RRs) to and progression-free survival (PFS) after taxane chemotherapy of 35 BRCA1-associated and 13 BRCA2-associated metastatic breast cancer patients were compared with those outcomes in 95 matched (1:2) sporadic patients. Matching was performed for age at and year of diagnosis of primary breast cancer, year of metastatic disease, and line of therapy (first vs second or third). Among BRCA1-associated patients, the RR was worse (objective response [OR], 23% vs 38%; progressive disease [PD], 60% vs 19%; P < 0.001); and the median PFS shorter (2.2 vs 4.9 months; P = 0.04) compared with sporadic patients. In the subgroup of hormone receptor (HRec)-negative patients, BRCA1-associated patients (n = 20) had a worse RR (OR, 20% vs 42%, respectively; PD, 70% vs 26%, respectively; P = 0.03) and a shorter PFS (1.8 vs 3.8 months; P = 0.004) compared with sporadic patients (n = 19). These outcomes in HRec-positive patients were similar in BRCA1-associated (n = 11) and sporadic (n = 61) patients (OR, 36% vs 38%; PD, 28% vs 20%; median PFS, both 5.7 months). In BRCA2-associated patients, who were mainly HRec-positive, the OR was higher than in sporadic patients (89% vs 38%, respectively; P = 0.02), whereas the median PFS was not significantly different (7.1 vs 5.7 months, respectively). BRCA1-associated, HRec-negative metastatic breast cancer patients were less sensitive to taxane chemotherapy than sporadic HRec-negative patients. HRec-positive BRCA1- and BRCA2-associated patients had a sensitivity to taxane chemotherapy similar to that of sporadic patients.

Quantitative copy number analysis by multiplex ligation-dependent probe amplification (MLPA) of BRCA1-associated breast cancer regions identifies BRCAness, and as such treatment response.

10513 Background: Our group has previously employed array Comparative Genomic Hybridization (aCGH) to assess the genomic patterns of BRCA1-mutated breast cancers. We have shown that the so-called BRCA1-likeaCGH profile is also present in about half of all triple negative sporadic breast cancers and is predictive for benefit from bifunctional alkylating agents. As aCGH is a rather complex method, we translated the BRCA1aCGH profile to a Multiplex Ligation-dependent Probe Amplification (MLPA) assay, to identify both BRCA1-mutated breast cancers and sporadic cases with a BRCA1-likeaCGH profile. Methods: The most important genomic regions of the original aCGH based classifier (3q22-27, 5q12-14, 6p23-22, 12p13, 12q21-23, 13q31-34) were mapped to a set of 34 MLPA probes. The training set consisted of 39 BRCA1-likeaCGH breast cancers and 45 non-BRCA1-likeaCGH breast cancer patients, which had previously been analyzed by aCGH. Part of the sporadic tumors showing a BRCA1-likeaCGH profile had a low BRCA1 gene expression or BRCA1 promoter methylation. We trained a shrunken centroids classifier on the training set and validated in on an independent test set of 22 BRCA1-likeaCGH breast cancers and 19 non-BRCA1-likeaCGH breast cancer patients. Results: The training set of 84 patients could accurately be predicted by PAM (10-fold cross-validated accuracy, 94%). The classifier trained on the training set correctly predicted BRCA-like status of nearly all 41 breast tumors. Two out of 41 were misclassified, resulting in a specificity and sensitivity of both 95%. The assay performed equally well in identifying BRCA1 mutated as BRCA1-like patients. Conclusions: Since the MLPA assay can identify BRCA1-mutated and BRCA1-like sporadic breast cancer patients, this method could be both applied in clinical genetic testing, but also as a predictor of treatment benefit. BRCA1-like tumors are highly sensitive to chemotherapy with DNA damaging agents, and also to PARP-inhibitors. The MLPA assay is rapid and robust, can easily be multiplexed, and works well with DNA derived from paraffin-embedded tissues.

Prevalence of BRCA1/2 mutations in sporadic breast/ovarian cancer patients and identification of a novel de novo BRCA1 mutation in a patient diagnosed with late onset breast and ovarian cancer: implications for genetic testing

In order to adequately evaluate the clinical relevance of genetic testing in sporadic breast and ovarian cancer patients, we offered comprehensive BRCA1/2 mutation analysis in patients without a family history for the disease. We evaluated the complete coding and splice site regions of BRCA1/2 in 193 sporadic patients. In addition, a de novo mutation was further investigated with ultra deep sequencing and microsatellite marker analysis. In 17 patients (8.8%), a deleterious germline BRCA1/2 mutation was identified. The highest mutation detection ratio (3/7 = 42.9%) was obtained in sporadic patients diagnosed with breast and ovarian cancer after the age of 40. In 21 bilateral breast cancer patients, two mutations were identified (9.5%). Furthermore, 140 sporadic patients with unilateral breast cancer were investigated. Mutations were only identified in patients diagnosed with breast cancer before the age of 40 (12/128 = 9.4% vs. 0/12 with Dx > 40). No mutations were detected in 17 sporadic male breast cancer and 6 ovarian cancer patients. BRCA1 c.3494_3495delTT was identified in a patient diagnosed with breast and ovarian cancer at the age of 52 and 53, respectively, and was proven to have occurred de novo at the paternal allele. Our study shows that the mutation detection probability in specific patient subsets can be significant, therefore mutation analysis should be considered in sporadic patients. As a consequence, a family history for the disease and an early age of onset should not be used as the only criteria for mutation analysis of BRCA1/2. The relatively high mutation detection ratio suggests that the prevalence of BRCA1/2 may be underestimated, especially in sporadic patients who developed breast and ovarian cancer. In addition, although rare, the possibility of a de novo occurrence in a sporadic patient should be considered.

Abstract 1961: PKCΔ mediates glycodelin-induced differentiation of breast cancer cells

Abstract Glycodelin is a secreted lipocalin family glycoprotein, expressed mainly in differentiated epithelia in reproductive tissues. The expression of glycodelin in breast and endometrial cancer cells induces cell differentiation. We have found that transfection of glycodelin cDNA into MCF-7 breast cancer cells changes cellular morphology and gene expression towards less malignant direction. In pre-clinical xenograft mouse model glycodelin-producing cells formed significantly smaller tumors with lower proliferation rate than control cells. This is in keeping with the association of glycodelin with well-differentiated and less proliferative tumors in sporadic breast cancer patients. The aim of the present study is to elucidate the mechanism(s) mediating glycodelin-induced differentiation of breast cancer cells. The gene expression changes between glycodelin-producing and control cells were compared using a cDNA micro array. The cells were grown on Matrigel with and without protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA), PKCΔ inhibitor Rottlerin and various other kinase inhibitors. PKCΔ was also down-regulated with RNAi. The levels of PKCΔ were measured using Western Blotting. The gene expression changes induced by glycodelin were enriched in several pathways, including mitogen-activated protein kinase (MAPK) -pathway. Among various studied proteins of MAPK-pathway, we found that phosphorylated PKCΔ was down-regulated in glycodelin-producing cells. Further studies with activators and inhibitors indicated that addition of PKC activator PMA to cell culture medium induced dramatic morphological changes and increased motility of the control cells, while glycodelin-producing cells were unresponsive. The effect of PMA was abolished by Rottlerin, an inhibitor of PKCΔ, as well as by using siRNA for PKCΔ. These results suggest that the glycodelin-induced differentiation of breast cancer cells is mediated by PKCΔ, which in other studies has been proposed to be a potential target for cancer treatment. 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 1961. doi:10.1158/1538-7445.AM2011-1961

Human epidermal growth factor receptor 2, Na +/H + exchanger regulatory factor 1, and breast cancer susceptibility gene-1 as new biomarkers for familial breast cancers

The aim of this study is to evaluate the analysis of markers related with progression, to further characterize familial breast cancers. Here, we investigated the expression of breast cancer susceptibility gene-1, hypoxia-inducible factor-1α, vascular endothelial growth factor receptor 1, and Na+/H+ exchanger regulatory factor 1 in 187 microarrayed breast carcinomas from 94 familial and 93 sporadic breast cancer patients by immunohistochemical staining. Furthermore, the expression levels of these biomarkers were compared with triple-negative phenotype. Familiarity was significantly associated with younger age (P < .000), higher tumor grade (P = .038), negative estrogen receptor hormonal status (P = .036), and high proliferative activity (P = .029). The familial cancers were immunonegative for membranous Na+/H+ exchanger regulatory factor 1 expression compared with sporadic cancers (P = .001); notably, vascular endothelial growth factor receptor 1 staining correlated with cytoplasmic Na+/H+ exchanger regulatory factor 1 expression in familial tumors (P = .009). In multivariate analysis, the "new biomarkers," including negative human epidermal growth factor receptor 2 status (odds ratio, 4.538; 95% confidence interval, 1.756-11.728), negative membranous Na+/H+ exchanger regulatory factor 1 expression (odds ratio, 7.686; 95% confidence interval, 1.876-31.483) and positive nuclear breast cancer susceptibility gene-1 (odds ratio, 0.3982; 95% confidence interval, 0.169-0.936), significantly correlated with family history of breast cancer. We hypothesize that the evaluation of human epidermal growth factor receptor 2, Na+/H+ exchanger regulatory factor 1, and breast cancer susceptibility gene-1 could be clinically useful to identify familial breast tumors and to select patients candidate to breast cancer susceptibility genes 1/2 gene sequencing.

The TP53 gene polymorphisms and survival of sporadic breast cancer patients

The TP53 gene polymorphisms, Arg72Pro and PIN3 (+16 bp), can have prognostic and predictive value in different cancers including breast cancer. The aim of the present study is to investigate a potential association between different genotypes of these polymorphisms and clinicopathological variables with survival of breast cancer patients in Croatian population. Ninety-four women with sporadic breast cancer were retrospectively analyzed. Median follow-up period was 67.9 months. The effects of basic clinical and histopathological characteristics of tumor on survival were tested by Cox's proportional hazards regression analysis. The TNM stage was associated with overall survival by Kaplan-Meier analysis, univariate, and multivariate Cox's proportional hazards regression analysis, while grade was associated with survival by Kaplan-Meier analysis and univariate Cox's proportional hazards regression analysis. Different genotypes of the Arg72Pro and PIN3 (+16 bp) polymorphisms had no significant impact on survival in breast cancer patients. However, in subgroup of patients treated with chemotherapy without anthracycline, the A2A2 genotype of the PIN3 (+16 bp) polymorphism was associated with poorer overall survival than other genotypes by Kaplan-Meier analysis (P = 0.048). The TP53 polymorphisms, Arg72Pro and PIN3 (+16 bp), had no impact on survival in unselected sporadic breast cancer patients in Croatian population. However, the results support the role of the A2A2 genotype of the PIN3 (+16 bp) polymorphism as a marker for identification of patients that may benefit from anthracycline-containing chemotherapy.

Determining and Interpreting New Predictive Rules for Breast Cancer Familial Inheritance

DNA copy number alterations have been discovered to be key genetic events in development and progression of cancer. No clear data of familial and sporadic breast cancer are available. We focused on looking for an independent platform as a tool to identify the chromosomal profile in familial versus sporadic breast cancer patients. A total of 124 breast cancer patients were studied utilizing aCGH. The dataset was analyzed using Gaussian Mixture Models to determine the thresholds in order to assess gene copy number changes and to minimize the impact of noise on further data analyses. The identification of regions of consistent aberration across samples was carried out with statistical approaches and machine learning tools to draw profiles for familial and sporadic groups. Familial and sporadic cases resulted with a chromosome imbalance of 15% [false discovery rate (FDR): q=718E-5] and 18% (FDR: q=632E-13), respectively. The differential map evidenced two cytogenetic bands (8p23 and 11q13-11q14) significantly altered in familial versus sporadic cases (FDR: q=7E-4). The application of a new bioinformatics tool that discovers fuzzy classification rules (IFRAIS) let to individualize association of genes alterations that identify familial or sporadic cases. These results are comparable to those of the other systems used and are consistent from the biological point of view.

Sporadic breast cancer patients' germline DNA exhibit an AT‐rich microsatellite signature

Using a custom CGH-like oligonucleotide array to measure the global microsatellite content in the genomes of 72 cancer, cancer-free, and high risk patient and cell line samples (56 germline DNA and 16 in tumor or tumor cell line DNA) we found a unique, reproducible, and statistically significant pattern of 18 motif-specific microsatellite families (out of 962 possible 1-6 mer repeats) in breast cancer patient germline and tumor DNA, but not in germline DNA of cancer-free volunteer controls or in breast cancer patients with BRCA1/2 mutations. These high-similarity A/T rich repetitive motifs were also more pronounced in the germlines and tumors of colon cancer tumor patients (3/6 samples) and microsatellite unstable colon cancer cell lines; however, germline DNA of sporadic breast cancer patients exhibited the largest global content shift for those motifs with extreme AT/GC ratios. These results indicate that global microsatellite variability is complex, suggest the existence of a previously unknown genomic destabilization mechanism in breast cancer patients' germline DNA, and warrant further testing of such microsatellite variability as a predictor of future breast cancer development.

Influence of a family history of breast and/or ovarian cancer on breast cancer outcomes

Various published studies have been inconclusive in attempting to relate a family history of breast and/or ovarian cancer (BOC) to the survival of breast cancer patients. The aim of the study was to investigate the association of a family history of BOC with tumor characteristics, treatment response and the difference between the prognosis of familial breast cancer (FBC) patients and sporadic breast cancer (SBC) patients. Data on 348 operable FBC patients and 345 SBC patients were retrospectively analyzed. The overall survival (OS) and recurrence/metastasis-free survival (RFS) were compared for both groups. FBC cases were diagnosed at a relatively younger age (51.1±10.4 vs. 53.7±11.0 years, P=0.054) and presented a lower T stage (P=0.000) than the SBC cases. Patients with a family history of BOC had a significantly greater risk of recurrence/metastasis (P= 0.04) and a non-significantly increased risk of death (P=0.06) compared to the SBC patients. In a multivariate analysis, family history of BOC was an independent predictive factor for both recurrence/metastasis rate (P=0.01, HR=0.012, 95% CI 0.02-0.57) and mortality (P=0.044, HR=0.43, 95% CI 0.19-0.98) in the hormone receptor-positive population. Our results found that women diagnosed with FBC had an early onset of disease in the population studied, and the poor outcome of patients with a family history of BOC associated with survival was restricted to the hormone receptor-positive population.

Novel sequence variants and common recurrent polymorphisms of BRCA2 in Sri Lankan breast cancer patients and a family with BRCA1 mutations

We previously reported BRCA1 mutations and sequence variants in Sri Lankan breast cancer patients. Mutations and sequence variants of the BRCA2 gene were studied in 149 study participants from the same cohort. There were 55 familial and 54 sporadic breast cancer patients, 20 at-risk individuals and 20 healthy controls. Direct sequencing (exon 11) and sequencing of abnormal bands after screening with single-strand conformation polymorphism (remaining exons) were used to detect mutations and sequence variants. Twenty-three sequence variants were found in the BRCA2 gene. Two novel pathogenic frame-shift additions resulting in a premature stop codon (c.2403 insA/exon 11, c.2667 insT/exon 11) were identified. Possibly pathogenic two novel missense mutations (c.1191 A>C/exon 10, c.5695 A>C/exon 11) one novel intronic variant (IVS15-21 insTT), four novel silent mutations (c.969 C>T/exon 9, c.1353 C>T/exon 10, c.2766 A>C/exon 11 and c.7452 A>G/exon 14) and one novel missense mutation (c.971 C>G/exon 9) were observed. One previously reported possibly pathogenic intronic variant (IVS81 G>C) and several previously reported silent mutations, missense mutations, and one 5' UTR polymorphism were detected. Pathogenic and possibly pathogenic mutations were more frequent in the BRCA2 gene among Sri Lankan familial breast cancer patients when compared to our previous findings for the BRCA1 gene.

Abstract P6-04-02: Profiling of BRCA1 Mutated Breast Tumours Using a Breast Cancer Specific Microarray To Identify a Profile of BRCAl-Deficiency

Abstract Background The BRCA 1 tumour suppressor gene is mutated in a significant proportion of hereditary breast cancer cases. Additionally, downregulation of BRCA1 mRNA and protein expression is reported in approximately one third of sporadic breast cancers. BRCA1 is strongly implicated in the maintenance of genomic stability by its involvement in multiple cellular pathways including: DNA damage signalling, DNA repair, cell cycle regulation, protein ubiquitination, chromatin remodelling, transcriptional regulation and apoptosis. To date, gene expression profiling has identified: (1) at least five breast cancer subtypes and (2) that BRCA1 mutant tumours segregate with basal-like breast cancers. These studies also provide evidence that breast cancers with germline mutations in BRCA1 are different from non BRCA1-related tumours. The main aim of this study is to investigate the underlying biology of BRCA1-mutated breast cancer. Methods Extensive gene expression profiling and data analysis were performed on a cohort of 70 FFPE (Formalin Fixed Paraffin Embedded) derived BRCA 1 mutated breast tumours and matched sporadic controls using the Almac Breast Cancer DSATM research tool. Functional analysis was performed with DAVID and METACORE. Validation of gene targets was performed by qRT-PCR and Western blotting. Results A list of differentially expressed transcripts has been derived from the comparison of these BRCA1 mutant breast tumours and matched sporadic controls. Functional analysis of this gene list has identified the key genes and molecular pathways that are deregulated in these tumours. BRCA1 deficiency was associated with deregulation of pathways involved in: (1) immune response, (2) metastasis and invasion, (3) cytoskeletal remodelling, (4) spindle assembly and chromosome separation, (5) apoptosis and survival. Validation of the key genes underlying this BRCA1-deficient breast cancer profile has been performed. Conclusions This approach has revealed a set of transcripts that could potentially be used to identify both hereditary and sporadic breast cancer patients with BRCA1- deficiency. The ability to perform gene expression profiling from FFPE derived breast tissue could also have significant clinical application. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-04-02.

Abstract P3-04-07: Loss of Expression of the 14-3-3 Sigma by CpG Island Methylation and Relations with Prognostic Factor in Breast Cancer

Abstract Background: Expression of 14-3-3 σ is a tumor suppressor gene induced in response to DNA damage, and has been implicated in G2/M cell cycle arrest by p53. Hypermethylation of CpG islands located in the promoter regions of tumor suppressor genes is now firmly established as an important mechanism for gene inactivation. Objetive: To correlation methylation levels of promoter 14-3-3σ with association prognostic factors in breast cancer.Material and Methods:This is a prospective study we quantified methylation levels of promoter 14-3-3 σ gene in 107 women with breast cancer and 108 control subjects by Real Time QMS-PCR SYBR green and analyzed association with prognostics factor in breast cancer. Results: Median age was 58 years (32-88); 69% were postmenopausal women.Nodal involvement N0; 63%,N1;30%,N2;7%), tumor size (T1;58%,T2;35%,T3;4%,T4;4%) and grade G1; 20%,G2;37%,G3;30%). The methylation of 14-3-3σ were 60% of sporadic breast cancer patients and were 34% of normal breast (p=0.0047).The methylation of 14-3-3σ gene in serum was markedly related with T3-4 stage (P&amp;lt;0.05),nodal positive status (P&amp;lt;0.05) and poor outcome. With a median follow up 6 years we saw more probability of developing distance metastasis in patients with methylation 14-3-3σ (P&amp;gt;0.05).Conclusions:Hypermethylation of the 14-3-3σ a promoter is an early and frequent event in breast neoplastic transformation, leading to the suggestion that silencing of 14-3-3σ may be an important event in tumor progression and particularly in breast carcinogenesis.Therefore, it is possible that loss of σ expression contributes to malignant transformation by impairing the G2 cell cycle checkpoint function, thus allowing an accumulation of genetic defects. Perhaps in the detection of CpG methylation of 14-3-3σ may be used for diagnostic and prognostic purposes Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-04-07.

Abstract PD07-07: Indicators of Homologous Recombination Deficiency in Breast Cancer and Association with Response to Neoadjuvant Chemotherapy

Abstract Background: Tumors with homologous recombination (HR) deficiency are highly sensitive to DNA double strand break (DSB) inducing agents, such as alkylating agents and poly (ADP-ribose) polymerase (PARP)-inhibitors. BRCA1 or BRCA2- mutated tumors, which are HR deficient, have characteristic DNA gains and losses that can be assessed by an array Comparative Genomic Hybridization (aCGH) classifier, one for BRCA1 mutations and one for BRCA2 mutations. We have studied these aCGH profiles together with several other HR deficiency indicators in sporadic breast cancers and we have correlated their presence to neoadjuvant chemotherapy response. Material and Methods: A total of 163 HER2-negative pre-treatment biopsies were examined, procured from sporadic breast cancer patients scheduled to receive neoadjuvant therapy with doxorubicin and cyclophosphamide. Triple negative (TN) and estrogen receptor positive (ER+/HER2-) tumors were analyzed separately. aCGH was performed to assess BRCA1-like and BRCA2-like profiles. In addition, BRCA1 promoter methylation, BRCA1 mRNA expression and amplification of the BRCA2-inhibiting gene EMSY were analyzed. Response to neoadjuvant treatment was assessed by measuring pathological complete remission (pCR) and near pCR at the time of surgery. Results: Inactivation of BRCA1 was frequent in TN tumors: 57% of these tumors showed a BRCA1-like profile at aCGH. BRCA 1 promoter methylation and reduced BRCA1 mRNA expression were observed in 25% and 36% of the TN tumors, respectively. The BRCA1-like aCGH profile was not clearly associated with a better neoadjuvant treatment response (58% vs. 48%, p=0.47). In ER+ tumors, a BRCA2-like aCGH profile and the amplification of the BRCA2 inhibiting gene EMSY were frequently observed (43% and 13% respectively). A BRCA2-like aCGH profile was associated with a significantly higher response rate (35% vs. 14%, p=0.014). EMSY amplification and a BRCA2-like aCGH profile occurred together in only one case, suggesting mutual exclusivity. EMSY was not associated with treatment response, questioning the role of EMSY in HR deficiency. Conclusion: Alterations associated with BRCA1 inactivation are present in about half of the TN breast cancers, but were not predictive of chemotherapy response. In ER+/HER2- tumors, the BRCA2-like aCGH profile predicts sensitivity to DSB-inducing chemotherapy, and possibly as well to new targeted agents, such as the PARP inhibitors. After validation in independent series the aCGH classifiers may lead to a diagnostic test that could assist in neoadjuvant treatment selection. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD07-07.

Abstract P2-10-04: Characterization of Mutations and Sequence Variations in Breast Cancer Susceptibility Gene 2 (BRCA2) in a Group of Breast Cancer Patients in Sri Lanka

Abstract Background: Germline mutations in tumour suppressor genes BRCA1 and BRCA2 confer susceptibility to breast and ovarian cancers. Breast cancer is a leading cause of death in women aged 40-60 years in Sri Lanka. Incidence though low in Sri Lanka, compared to the developed world has almost doubled during the last 15 years. BRCA1 mutations and sequence variants in a group of Sri Lankan breast cancer patients have been characterized recently. Here we report mutations and sequence variants of BRCA2 using the same cohort. Methods: A total of 129 patients comprising of 75 familial breast cancer patients (mean age at diagnosis is 44.64 ± 12.7 years) and 54 sporadic breast cancer patients (mean age at diagnosis 49.13 ± 11 years) were analyzed for BRCA2 sequence variants and mutations. 20 healthy controls were used to compare the results of the patients. Genomic DNA from blood samples was isolated and all exons except exon 11 were screened using Single Strand Conformation Polymorphism (SSCP). Samples in which abnormal bands were detected were confirmed by direct sequencing using MegaBACE 1000 automated DNA Sequencer. Exon 11 will be subjected to direct sequencing without SSCP Technique. Representative samples from exons which did not show abnormal bands were also subjected to direct sequencing. Results: To date twelve sequence variants have been found in this study. One reported sequence variant: c.203G&amp;gt;A/exon2 (44 familial and 39 sporadic cases), one novel silent mutation: c.969C&amp;gt;T/exon9, one novel missense mutation: c.971C&amp;gt;G/exon9, and one reported intronic variant: IVS8-1G&amp;gt;C/exon9 (in one familial case each) were detected. In exon 10, three reported missense mutations: c.1093A&amp;gt;C (10 familial cases), c.1342A&amp;gt;T/C (58 familial cases) and c.1352C&amp;gt;T (4 familial cases), two novel silent mutations: c.1191A&amp;gt;C (5 familial cases) and c.1353C&amp;gt;T (1 familial case) and one reported silent mutation: c.1593A&amp;gt;G (5 familial cases) were characterized. In exon 14, one novel silent mutation: c.7452A&amp;gt;G (20 familial cases and 28 sporadic cases) and one reported silent mutation: c.7470A&amp;gt;G (one familial case) were identified. Discussion: Among the BRCA2 exons screened to date, seven six reported sequence variants and five novel mutations have been detected. Out of them one pathogenic mutation (IVS8 1G&amp;gt;C/exon9) found in one familial case and in the same patient two other novel missense and novel silent mutations were detected (c.971C&amp;gt;G/exon9 and c.969C&amp;gt;T/exon9). Studies are in progress to screen the remaining exons of BRCA2 gene in this cohort of breast cancer patients. Data obtained will help in developing an economical screening test for BRCA2 mutations in Sri Lanka. Supported by Sida/Secretariat for Research Cooperation Grant for Molecular Biology and Biotechnology Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P2-10-04.

Two functional variations in 5′-UTR of hoGG1 gene associated with the risk of breast cancer in Chinese

8-Hydroxy-2'-deoxyguanine (8-OHdG) is produced by the oxidative stress-induced damage in DNA, which could pair with adenine (A) during DNA replication, leading to G-T transversion mutations. Glycosylase hOGG1 can recognize and excise oxidized guanines from duplex DNA. This work aims to investigate the relationship between the functional variations in 5-untranslated region (5'-UTR) of hOGG1 gene and the risk of breast cancer. Genotypes were analyzed in 518 sporadic breast cancer patients and 777 health controls. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated by logistic regression. Risk-stratified subgroup analysis was performed to reveal the associations between the detected variations and the risk of characteristic breast cancer. In addition, immunohistochemistry was carried out to assess the functional effect of these variations on hOGG1gene expression. Five variations in 5'-UTR of hOGG1 gene are found in this study. Three of them, c.-18G>T, c.-23A>G, and c.-53G>C, are known single nucleotide polymorphisms, the other two, c.-45G>A and c.-63G>C, are rare variations. The frequency of c.-18G/T and c.-53G/C was significantly higher in breast cancer patients than those in healthy controls (P = 0.03, OR 2.01, 95% CI 1.04-3.90; and P = 0.01, OR 2.43, 95% CI 1.17-5.04, respectively). Both variations were especially prevalent in premenopausal status, and in the triple (estrogen receptor, progesterone receptor, and human epidermal growth factor Receptor 2) negative subgroups, respectively. Moreover, the variation of c.-18G>T could cause a reduced expression of hOGG1 gene.

Investigation of DNA damage response and apoptotic gene methylation pattern in sporadic breast tumors using high throughput quantitative DNA methylation analysis technology

Background-Sporadic breast cancer like many other cancers is proposed to be a manifestation of abnormal genetic and epigenetic changes. For the past decade our laboratory has identified genes involved in DNA damage response (DDR), apoptosis and immunesurvelliance pathways to influence sporadic breast cancer risk in north Indian population. Further to enhance our knowledge at the epigenetic level, we performed DNA methylation study involving 17 gene promoter regions belonging to DNA damage response (DDR) and death receptor apoptotic pathway in 162 paired normal and cancerous breast tissues from 81 sporadic breast cancer patients, using a high throughput quantitative DNA methylation analysis technology.Results-The study identified five genes with statistically significant difference between normal and tumor tissues. Hypermethylation of DR5 (P = 0.001), DCR1 (P = 0.00001), DCR2 (P = 0.0000000005) and BRCA2 (P = 0.007) and hypomethylation of DR4 (P = 0.011) in sporadic breast tumor tissues suggested a weak/aberrant activation of the DDR/apoptotic pathway in breast tumorigenesis. Negative correlation was observed between methylation status and transcript expression levels for TRAIL, DR4, CASP8, ATM, CHEK2, BRCA1 and BRCA2 CpG sites. Categorization of the gene methylation with respect to the clinicopathological parameters showed an increase in aberrant methylation pattern in advanced tumors. These uncharacteristic methylation patterns corresponded with decreased death receptor apoptosis (P = 0.047) and DNA damage repair potential (P = 0.004) in advanced tumors. The observation of BRCA2 -26 G/A 5'UTR polymorphism concomitant with the presence of methylation in the promoter region was novel and emerged as a strong candidate for susceptibility to sporadic breast tumors.Conclusion-Our study indicates that methylation of DDR-apoptotic gene promoters in sporadic breast cancer is not a random phenomenon. Progressive epigenetic alterations in advancing tumors result in aberrant DDR-apoptotic pathway thereby promoting tumor development. We propose, since pathological epigenetic changes of the DDR-apoptotic genes are reversible modifications, these could further be targeted for therapeutic interventions.