Invasive Breast Carcinoma Patients Research Articles

Adjust cut-off values of immunohistochemistry models to predict risk of distant recurrence in invasive breast carcinoma patients

BackgroundMultigene assays are recommended for hormone receptor-positive invasive breast carcinoma to determine the risk of recurrence, but they are highly expensive. We investigated the prognostic values of immunohistochemistry (IHC)-based prognostic models as an alternative to multigene assays. MethodsThe risk categories estimated by the IHC-based prognostic models were correlated to those estimated by the multigene assays in 71 cases and the follow-up results in 642 consecutive cases of HER2− luminal-type early breast cancer. Cut-off values of IHC-based models were adjusted based on survival outcome to reveal maximum Harrell C index or based on the maximum positive likelihood ratio correlated to multigene assay. ResultsAll investigated IHC-based models could predict the risk of distant recurrence, but their cut-off values required adjustment. Using distant recurrence-free survival (DRFS) to refine the cut-off values could improve the prognostic values. Adjusting the cut-off values using the results of multigene assays, the positive predictive values of an estimate of low risk or low recurrence score (≤ 21) were higher than 90%. On average, 23% of cases got different results of risk assessment after adjustment. Although cut-off values adjusted by multigene assay were not identical to those refined by survival, the adjusted values (17.1 and 23.8) and the refined values (17.5 and 24.5) of the best model (Magee Eq. 1) were close. Among all the evaluated models, Magee equation 2 was the only one without Ki67, and its prognostic values were the lowest. Using 20% as cut-off for Ki67 as suggested by St. Gallen consensus, we could confidently define luminal A cancer. ConclusionIt is necessary to adjust the cut-off values of IHC-based prognostic models to fit the purpose. If the estimated risk is clearly high or low, it may be reasonable to omit multigene assays when cost is a consideration.

Abstract 3927: The presence of COX-2 and tumor-associated macrophages as a prognostic marker for invasive breast carcinoma patients

Abstract Inflammation is an important mediator of tumor progression. The objective of this study was to assess whether the tissue localization of COX-2 and tumor-associated macrophages were associated with clinicopathological features of invasive carcinoma, including collagen deposition and patient survival outcome. A tumor microarray (TMA) of 371 biopsy specimens from patients with invasive breast carcinoma was analyzed for expression of high levels of COX-2, and the macrophage markers CD68 and CD163 in either the tumor nest (TN) or the tumor-associated stroma (TS). The study population for this TMA was female patients, 18 to 80 years of age with a median follow up of 8.4 years. Survival tables were calculated according to the Kaplan-Meier method. We found that elevated collagen deposition was associated with high stromal expression of COX-2 (P < 0.0001); however, the amount of collagen deposition was not a predictor for survival outcome. In order to better analyze patient data, samples were divided into quartiles based on their levels of COX-2 expression and/or macrophage infiltration. Tumor nests with high COX-2 expression had worse patient outcome (P = 0.011). One possible mechanism for this is COX-2 dependent recruitment of macrophages to the tumor microenvironment, supported by our finding of high infiltration of CD68+ macrophages in the TS, and CD163+ macrophages in both TN and TS. This recruitment of macrophages was associated with worse overall survival (OS). Furthermore, patient survival was worsened even more if macrophages expressed COX-2, suggesting positive amplification of COX-2 signaling via macrophage recruitment. This notion is further established by the finding of high presence of CD163+ macrophages in the TN as an independent prognostic factor as revealed by multivariate analysis (P < 0.0001, HR = 4.67). These results suggest that in invasive carcinoma the localization of inflammatory markers within the tumor are biomarkers for patient survival outcome. Therefore, we propose that these patients may benefit from therapy with a selective COX-2 inhibitor such as celecoxib. Citation Format: Karla Esbona, Sandeep Saha, Yanyao Yi, Menggang Yu, Kari Wisinski, Lee G. Wilke, Patricia J. Keely. The presence of COX-2 and tumor-associated macrophages as a prognostic marker for invasive breast carcinoma patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3927.

Activated-PAK4 predicts worse prognosis in breast cancer and promotes tumorigenesis through activation of PI3K/AKT signaling.

The p21-activated kinase 4 (PAK4) is sufficient to transform noncancerous mammary epithelial cells and to form tumors in the mammary glands of mice. The accumulated information suggests that PAK4 might be an oncogenic protein in breast cancer. In this study, we sought to identify the role for PAK4 in breast cancer progression. Immunohistochemical study revealed that high PAK4 expression is associated with larger tumor size, lymph node metastasis, and advanced stage cancer in 93 invasive breast carcinoma patients. Moreover, high PAK4 expression was significantly associated with poor overall and disease-free survival. PAK4 remained an independent adverse prognosticator after univariate and multivariate analysis. Ectopic expression of wild-type PAK4 in MDA-MB-231 cells activated PI3K/AKT signaling and resulted in the enhancement of the cell proliferation, migration, and invasion, whereas PAK4-induced effects were blocked by the PAK4 kinase inhibitor PF- 3758309, PAK4 siRNAs or the PI3K inhibitor LY294002. Furthermore, a kinase-active PAK4 (S474E) strongly induced PI3K/AKT activation, and promoted proliferation, migration and invasion in breast cancer cells. A kinase-inactive PAK4 KD (K350A/K351A) did partially upregulate PI3K/AKT, and promoted invasive phenotype. Taken together, these findings suggest that PAK4-activated PI3K/AKT signaling is both kinase-dependent and -independent, which contributes to breast cancer progression. Thus, our results imply that dual inhibition of PAK4 and PI3K/AKT signaling might be a potential therapeutic approach for breast cancer therapy.

Clinical role of HER2 gene amplification and chromosome 17: a study on 154 IHC-equivocal cases of invasive breast carcinoma patients.

Accurate evaluation of human epidermal growth factor receptor 2 (HER2) status is quite crucial for invasive breast tumor patients in order to select anti-HER2 therapy for effective clinical outcomes. Immunohistochemistry (IHC) assay is routinely used to evaluate the HER2 oncoprotein overexpression but is unable to explain the chromosomal and genetic alterations and has been considered as a hot issue in IHC-equivocal cases. We investigated these molecular aberrations in correlation with prognostic factors. A cohort of 154 IHC-equivocal (+2) cases was selected and retrospectively analyzed by dual-probe fluorescence in situ hybridization (FISH) assay by using locus-specific HER2 and centromere enumeration probes (CEP17) for the identification of HER2 proto-oncogene amplification and chromosomal copy number per cell, respectively. The data were analyzed by SPSS 16.0 version using chi-square test (p < 0.05). We identified 36 out of 154 cases (23.4%) showing HER2 gene amplification (average HER2 gene copies per cell >4 or <4 with HER2/CEP17 ratio >2) in concordance with HER2 oncoprotein overexpression, and significant correlation was observed with prognostic parameters including histological type, tumor grade II to III, histology and pathological type, lymphatic invasion, ductal carcinoma in situ (DCIS), and estrogen-positive and progesterone-negative receptors. Of the 154 cases, 18 cases (11.7%) showed polysomy 17 with CEP17 probe signals per cell ≥3 and 22 cases (14.3%) presented monosomy 17 (CEP17 probe signals per cell ≤1). Our data indicate that the use of anti-HER2 therapy should not be suggested unless true evaluation of HER2 protein expression is made regarding gene amplification essentially in IHC-ambiguous invasive breast tumors.

PD-L1 mediated the differentiation of tumor-infiltrating CD19+ B lymphocytes and T cells in Invasive breast cancer

ABSTRACTAccumulating evidence suggests that B cells play important roles in inhibiting the immune response in autoimmune disorders and human tumors as well as murine tumor models. In an effort to explore the role of B cells in human breast cancer etiology, we examined the presence of CD19+ B lymphocytes in 134 cases of invasive breast carcinoma (IBCa) and 31 breast fibroadenoma, and assessed its relationship with PD-L1 (programmed death-ligand 1) expression in breast cancer. We found that the density of CD19+ B lymphocytes was higher in IBCa compared with fibroadenoma, and significantly associated with increasing tumor grade, negative estrogen status. Similar findings were observed for the expression of IL-10 in IBCa. Meanwhile, CD19+ B lymphocytes were shown to be highly coincident with PD-L1 and IL-10 in IBCa. We further demonstrated that CD19+ B cells can differentiate into CD19+CD24+CD38+ B cells when co-cultured with PD-L1hi MDA-MB231 cells. In addition, the percentage of CD19+CD24+CD38+ B cells was higher in breast tissue and peripheral blood cells of IBCa patients than that of benign tumor and health individuals. And CD19+CD24+CD38+ B cells were found to be IL-10 secreting B cells. Finally, we showed that CD19+ B cells from IBCa patients but not healthy individuals induced formation of CD4+CD25+Foxp3+ T cells when co-cultured with T cells from IBCa patients and healthy subjects (80.4% and 30.8% respectively). The induction of CD4+CD25+Foxp3+ T cells by CD19+ B cells was further shown to be mediated by PD-L1. Together, these results are suggestive of a role for CD19+ B lymphocytes in immune suppression and tumor evasion via PD-L1 in breast cancer.

Back to Basics: Traditional Nottingham Grade Mitotic Counts Alone are Significant in Predicting Survival in Invasive Breast Carcinoma.

Newer multigene molecular profiling assays for breast carcinoma rely heavily on the quantification of genes of proliferation, whereas traditional histological grading reports the mitotic count. The mitotic activity of invasive breast carcinomas may be undervalued; therefore, an evaluation of the prognostic significance of mitotic score in predicting prognosis was performed. Retrospective analysis of a single institutional cohort of newly diagnosed estrogen receptor positive (ER+), HER2 negative (HER2-) unilateral invasive breast carcinomas was performed. Mitotic scores from the 3-part Nottingham combined histological grade were compared with clinical parameters. Mitoses were counted on Olympus BX50 microscopes and assigned scores of 1-3 based on observed mitoses. A total of 1292 ER+, HER2- invasive breast carcinoma patients were identified, with a median follow-up time of 2.6 years (range 0-14 years). Higher mitotic score was significantly associated with younger age, larger tumor size, angiolymphatic invasion, node-positive disease, higher stage, and the use of hormonal and cytotoxic chemotherapy. Mitotic score was significant in modeling time to local/regional recurrence (p = 0.02), recurrence-free survival/RFS (p < 0.001), and overall survival/OS (p = 0.01) with higher mitotic scores associated with worse outcomes. Higher mitotic score correlated significantly with intermediate/high risk Oncotype Dx recurrence scores (p = 0.009). First-generation molecular profiling assays for estrogen receptor positive invasive breast carcinomas derive much of their predictive power from quantifying genes of proliferation into a single score. Sometimes overlooked in the profusion of molecular data, the time-tested, mitotic count in the Nottingham combined histological grade is a good single-parameter predictor of survival.

Protein expression and methylation of MGMT, a DNA repair gene and their correlation with clinicopathological parameters in invasive ductal carcinoma of the breast.

Epigenetic mechanisms such as DNA methylation are being increasingly recognized to play an important role in cancer and may serve as a cancer biomarker. The aim of this study was to evaluate the promoter methylation status of MGMT (O6-methylguanine-DNA methyltransferase) and a possible correlation with the expression of MGMT and standard clinicopathological parameters in invasive ductal breast carcinoma patients (IDC) of Kashmir. Methylation-specific PCR was carried out to investigate the promoter methylation status of MGMT in breast tumors paired with the corresponding normal tissue samples from 128 breast cancer patients. The effect of promoter methylation on protein expression in the primary breast cancer and adjacent normal tissues was evaluated by immunohistochemistry (n = 128) and western blotting (n = 30). The frequency of tumor hypermethylation was 39.8 % and a significant difference in methylation frequency among breast tumors were found (p < 0.001) when compared with the corresponding normal tissue. Immunohistochemical analysis showed no detectable expression of MGMT in 68/128 (53.1 %) tumors. MGMT promoter methylation mediated gene silencing was associated with loss of its protein expression (rs = -0.285, p = 0.001, OR = 3.38, 95 % CI = 1.59-7.17). A significant correlation was seen between loss of MGMT and lymph node involvement (p = 0.030), tumor grade (p < 0.0001), loss of estrogen receptors (ER; p = 0.021) and progesterone receptors (PR) (p = 0.016). Also, MGMT methylation was found to be associated with tumor grade (p = 0.011), tumor stage (p = 0.009), and loss of ER (p = 0.003) and PR receptors (p = 0.009). To our knowledge, our findings, for the first time, in Kashmiri population, indicate that MGMT is aberrantly methylated in breast cancer and promoter hypermethylation could be attributed to silencing of MGMT gene expression in breast cancer. Our data suggests that MGMT promoter hypermethylation could have a potential function as molecular biomarker of breast oncogenesis. Also, based on their predictive value of response to therapy, the immunohistochemical evaluation and interpretation of MGMT may also help in future to establish therapeutic strategies for patients with breast cancer.

Tumoral load quantification of positive sentinel lymph nodes in breast cancer to predict more than two involved nodes

AimOne-Step Nucleic Acid Amplification (OSNA) can detect isolated tumour loads in axillary lymph nodes of breast cancer patients. We investigated the predictability of the non-sentinel lymph node (SLN) metastatic involvement (MI) based on the OSNA SLN assessment in surgical invasive breast cancer. MethodsWe studied surgical breast invasive carcinoma patients, not taking neoadjuvant chemotherapy, having SLN positive by OSNA and having received axillary lymphadenectomy. Age, basic histopathological, immunohistochemical, SLN biopsy and lymphadenectomy data were compared between patients with or without MI of more than 2 non-SLN in both univariate and multivariate analyses. The discriminating capacity of the multivariate model was characterized by the ROC AUC. Results726 patients from 23 centers in Spain aged 55.3 ± 12.2 years were analysed. The univariate analysis comparing patients with or without MI of more than 2 non-SLN detected statistically significant differences in primary tumour size, multifocality, presence of lymphovascular infiltration, positive proliferation index with ki67, immunophenotype and logTTL (Tumour Total Load). The multivariate logistic analyses (OR (95% CI)) confirmed multifocality (2.16 (1.13–4.13), p = 0.019), lymphovascular infiltration (4.36 (2.43–7.82), p < 0.001) and logTTL (1.22 (1.10–1.35), p < 0.001) as independent predictors, and exhibit an AUC (95% CI) of 0.78 (0.72–0.83) with an overall fit (Hosmer–Lemeshow test) of 0.359. A change in the slope of both sensitivity and specificity is observed at about 10,000 copies/μL, without relevant changes in the Negative Predictive Values. ConclusionsUsing OSNA technique, the MI of more than 2 non-SLN can be reliably predicted.

Long-term use of metformin and the molecular subtype in invasive breast carcinoma patients - a retrospective study of clinical and tumor characteristics.

BackgroundMetformin may exhibit inhibitory effects on cancer cells by inhibiting mTOR signaling pathway. The aim of our retrospective study was to examine if patients with breast carcinoma (BC) and diabetes mellitus (DM) receiving metformin have a lower stage of carcinoma in comparison to patients not receiving metformin, and if the use of metformin correlates with the molecular subtype of BC.MethodsA chart review of 253 patients with invasive BC and DM (128 on metformin and 125 not on metformin) was performed. Control group consisted of 320 consecutive patients with invasive BC without DM. BC subtypes were classified by immunohistochemical surrogates as luminal A (estrogen receptor [ER] + and/or progesterone receptor [PR]+, HER-2-), luminal B (ER + and/or PR+, HER-2+), HER-2 (ER-, PR-, HER-2+), triple-negative/basal (ER-, PR-, HER-2-).ResultsPatients on metformin had a lower proportion of T3 or T4 tumors than patients who were not receiving metformin (16% vs. 26%; p = 0.035). No statistical difference was found between the two study groups in N stage. Patients with DM on metformin, with DM not on metformin and the control group had different molecular subtypes of BC (p = 0.01): the luminal A subtype was found in 78%, 83% and 71%, the luminal B in 12.6%, 9% and 11%, HER-2 in 0.8%, 1.6% and 8%, and the triple-negative/basal-like subtype in 8.6%, 6.4% and 10%, respectively.ConclusionOur data indicate that long-term use of metformin use correlates with molecular subtype of BC in diabetics on metformin in comparison to diabetics not on metformin and patients without DM. However, most likely, different distribution of the molecular subtypes of BC in these three groups of patients was caused by other risk factors for breast carcinoma, such as age of patients or obesity.

Relationship between Preoperative Serum CA15-3 and CEA Levels and Clinicopathological Parameters in Breast Cancer

CEA and CA 15.3 serum tumor markers are currently used in clinical practice for monitoring therapy. The aim of this study was to evaluate serum level of these markers among healthy females and invasive breast carcinoma (IBC) patients and to determine any relationships with clinicopathological factors. 60 Iranian females were enrolled in this study, 30 healthy and 30 diagnosed with breast cancer who had not received any preoperative chemotherapy or hormone therapy. Enzyme linked immunosorbent assays were used for the quantitative determination of the cancer associated antigens, CEA and MUC1 (CA 15- 3). The serological levels of CEA and CA 15-3 (5.0033 ± 0.49 μg/L and 178.1667 ± 15.11 U/ml) in the breast cancer patients were significantly higher (p=0.00) than the serum levels of normal controls (1.1237 ± 0.11 μg/L and 21.13 ± 3.058 U/ml). Regarding the CEA marker, a significant correlation with grade of tumor was shown. Furthermore, there was a low correlation between CA 15-3 and CEA marker with correlation coefficient r=0.08. Collectively, markedly high levels of CEA and CA 15-3 were found in our patients, pointing to their use as additional tools after clinical diagnosis.

Concordance between Core Needle Biopsy and Surgical Excision Specimens for Tumour Hormone Receptor Profiling According to the 2011 St. Gallen Classification, in Clinical Practice

To focus on and compare the tumor hormone receptor profiles on core needle biopsy (CNB) and subsequent surgical excision specimens in a large clinical series of invasive breast carcinoma patients, with regard to guidelines proposed at the St. Gallen International Expert Consensus on the Primary Therapy of Early Breast Cancer 2011. A total of 993 consecutive patients who had hormone receptors (HR) assays performed on both CNB and subsequent surgical excision specimens of invasive carcinomas were included (1,110 tumors). Concordant tumor HR profiles between CNB and surgical excision specimens were noted in 1,085 of 1,110 tumors (97.75%). Among 138 tumors considered negative on CNB (both HR assays <1%), 10 cases (7.2%) displayed an HR profile positive on surgical excision specimen. Discrepancies between CNBs and surgical excision specimens are very seldom noted. HR assay evaluation on surgical excision specimens should only be considered in patients when both HR assays are negative on CNBs.

Genomic Instability: A Stronger Prognostic Marker Than Proliferation for Early Stage Luminal Breast Carcinomas

BackgroundThe accurate prognosis definition to tailor treatment for early luminal invasive breast carcinoma patients remains challenging.Materials and MethodsTwo hundred fourteen early luminal breast carcinomas were genotyped with single nucleotide polymorphisms (SNPs) array to determine the number of chromosomal breakpoints as a marker of genomic instability. Proliferation was assessed by KI67 (immunohistochemistry) and genomic grade index (transcriptomic analysis). IHC3 (IHC4 score for HER2 negative tumors) was also determined.ResultsIn the training set (109 cases), the optimal cut-off was 34 breakpoints with a specificity of 0.94 and a sensitivity of 0.57 (Area under the curve (AUC): 0.81[0.71; 0.91]). In the validation set (105 cases), the outcome of patients with > 34 breakpoints (11 events / 22 patients) was poorer (logrank test p < 0.001; Relative Risk (RR): 3.7 [1.73; 7.92]), than that of patients with < 34 breakpoints (19 events / 83 patients).Whereas genomic grade and KI67 had a significant prognostic value in univariate analysis in contrast to IHC3 that failed to have a statistical significant prognostic value in this series, the number of breakpoints remained the only significant parameter predictive of outcome (RR: 3.47, Confidence Interval (CI [1.29; 9.31], p = 0.014)) in multivariate analysis .ConclusionGenomic instability, defined herein as a high number of chromosomal breakpoints, in early stage luminal breast carcinoma is a stronger prognostic marker than proliferation.

Association of osteopontin and cyclooxygenase-2 expression with breast cancer subtypes and their use as potential biomarkers

Breast cancer is one of the most common malignant tumors among females worldwide and remains a leading cause of cancer-related mortality. Due to the heterogeneous clinical nature of breast cancer, it is necessary to identify new biomarkers that are associated with tumor growth, angiogenesis and metastasis. Osteopontin (OPN) and cyclooxygenase-2 (COX-2) are known to be overexpressed in invasive breast cancer and their overexpression is associated with aggressive histological and clinical features. The present study assessed OPN and COX-2 expression in various subtypes of breast cancer. The expression of OPN and COX-2 was analyzed using immunohistochemistry (IHC) in a cohort of 67 invasive ductal breast carcinoma patients. The statistical analysis was performed using standard statistical software SPSS version 18.0. The associations between OPN and COX-2 and the human epidermal growth factor receptor type 2 (HER2)-overexpressing and non-HER2-overexpressing subtypes were evaluated using the Mann-Whitney U test. The mean OPN level was significantly higher in the HER2-overexpressing subtype compared with the non-HER2-overexpressing subtype. Furthermore, the mean COX-2 expression levels were higher in the HER2-overexpressing subtype compared with the luminal A, luminal B or triple-negative groups. It is well known that carcinomas overexpressing HER2/neu have a worse prognosis than luminal tumors. Hence, it may be hypothesized that an elevated expression of OPN and COX-2 in a HER2-overexpressing subtype may contribute to a more aggressive behavior and be used as diagnostic and prognostic markers in breast cancer.

Abstract B40: Methylation signatures specific to triple negative breast cancer subtypes

Abstract Objective: We identify methylation signatures specific to triple negative breast cancer (TNBC) subtypes using publicly available data, and examine their association with expression. Materials and Methods: DNA methylation, RNAseq expression and clinical data from breast invasive carcinoma patients were obtained from The Cancer Genome Atlas (TCGA) Data portal. The classification of each patient sample as TNBC was defined according to a combination of FISH and/or IHC results for ER, PR and HER2 negativity. Each TNBC specimen was classified into one of the following molecular subtypes by applying the gene expression-based model of Lehman (2011): basal-like 1 (BL1), basal-like 2 (BL2), immunomodulatory (IM), mesenchymal (MES), mesenchymal stem-like (MSL), luminal androgen receptor (LAR), and unstable (UNS). Among the 116 identified TNBC specimens, 61 had level-three data available on both methylation and RNAseq expression. For methylation, beta values were extracted based on Illumina's Infinium Human DNA Methylation 450K platform; for expression, RNAseqV2 Expectation/Maximization normalized counts (RSEM: Li and Dewey, 2011) were extracted on 20,531 genes and a logarithmic transformation applied. Data were filtered to identify methylation changes specific to each TNBC subtype based on a novel, generalized statistic (Switchenko and Kowalski, 2013) and tested for median difference between subtypes using p &amp;lt; 0.10 to define significance. Results: Among the 61 TNBC samples, the following molecular subtypes were represented: BL1 (n=6), BL2 (n=6), IM (n=12), LAR (n=5), MES (n=15), MSL (n=4), and UNS (n=13). A number of genes were determined to be hypermethylated with corresponding decreased expression by RNAseq in several subtypes. Methylated genes specific to the LAR TNBC subtype included IGF1R (61% LAR vs. 2% other) and N4BP2L1 (44% LAR vs. 3% other). Methylated genes in the MES subtype included ACSL5 (80% vs. 50%), RHOH (82% vs. 6%) and OAS2 (26% vs. 7%), and specific to the BL2 subtype were EN1 (35% vs. 8%) and TRAFIP3 (60% vs. 35%). Two hypomethylated genes, PIK3CD and CD69, associated with respective subtypes, MSL and IM, showed corresponding increased expression levels. Conclusion: Our results provide further insight into the molecular heterogeneity surrounding TNBC patients by examining methylation changes specific to molecular subtypes, which may be further examined for their effect on clinical outcome. Citation Format: Jeanne Kowalski, Jeffery M. Switchenko, Bhakti Dwivedi, Dana Nickleach, Carlos S. Moreno. Methylation signatures specific to triple negative breast cancer subtypes. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr B40.

Heat Shock Factor 1 (HSF1) Controls Chemoresistance and Autophagy through Transcriptional Regulation of Autophagy-related Protein 7 (ATG7)

Heat shock factor 1 (HSF1), a master regulator of heat shock responses, plays an important role in tumorigenesis. In this study we demonstrated that HSF1 is required for chemotherapeutic agent-induced cytoprotective autophagy through transcriptional up-regulation of autophagy-related gene ATG7. Interestingly, this is independent of the HSF1 heat shock response function. Treatment of cancer cells with the FDA-approved chemotherapeutic agent carboplatin induced autophagy and growth inhibition, which were significantly increased upon knockdown of HSF1. Mechanistic studies revealed that HSF1 regulates autophagy by directly binding to ATG7 promoter and transcriptionally up-regulating its expression. Significantly, breast cancer patient sample study revealed that a higher ATG7 expression level is associated with poor patient survival. This novel finding was further confirmed by analysis of two independent patient databases, demonstrating a prognostic value of ATG7. Furthermore, a strong positive correlation was observed between levels of HSF1 and ATG7 in triple-negative breast cancer patient samples, thus validating our in vitro findings. This is the first study identifying a critical role for HSF1 in controlling cytoprotective autophagy through regulation of ATG7, which is distinct from the HSF1 function in the heat shock response. This is also the first study demonstrating a prognostic value of ATG7 in breast cancer patients. These findings strongly argue that combining chemotherapeutic agents with autophagy inhibition by repressing HSF1/ATG7 axis represents a promising strategy for future cancer treatment.

Reduced E-Cadherin Expression Is a Predictor of Lower Overall Survival and Metastatic Disease in Invasive Ductal Breast Cancer

Background: E-cadherin, a transmembrane adhesion molecule, is implicated in the development of many solid tumors as well as in the acquisition of metastatic potential of epithelial tumors. Its clinical use has yet to be established. Patients and Methods: The prognostic value of E-cadherin expression in 134 invasive ductal breast carcinoma patients over a 10-year follow-up period was investigated. Additionally, the correlation between E-cadherin expression and other traditional prognostic factors was investigated. Results: A statistically significant influence on overall survival was found for estrogen receptor, tumor size, histological and nuclear grade, HER2, lymph node involvement, vascular invasion, proliferative index, and E-cadherin. E-cadherin expression had a significant impact on overall survival and development of metastases in the group of patients not receiving chemotherapy, while it had no such effect in the group of patients who received chemotherapy. Conclusion: We conclude that determination of E-cadherin expression can be used as an adjunct in selecting patients who may benefit from adjuvant chemotherapy in the presence of otherwise favorable prognostic factors

Abstract 688: Lamin A/C a novel potential prognostic marker in early operable primary breast cancer

Abstract Background Lamins A/C, a major component of the nuclear lamina of highly differentiated mammalian cells is encoded by LMNA gene. Its involvement in tumorignesis is of interest due to its role in maintaining the nuclear integrity, regulation of gene expression, cell proliferation and apoptosis. Reduced lamin A/C expression in cancer has been reported to be a sign of poor prognosis. However there is few data on its clinical significance in breast cancer. This study aimed to evaluate expression of lamin A/C in a large series of patients with early operable invasive breast cancer; we analyzed the association of lamin A/C expression with clinicopathological parameters and long term clinical outcome. Methods Using immunohistochemistal staining of tissue microarrays, expression of Lamin A/C was evaluated in a large well characterized series of early operable invasive breast carcinoma patients (n=938) obtained from Nottingham Tenovus Primary Breast Carcinoma Series between the period of 1988 and 1998 who underwent primary surgery. All patients were managed following a standard management guidelines by a single team of clinicians and the decision of adjuvant systemic therapy was made on the basis of Nottingham prognostic Index (NPI) score and hormonal status. Using X-tile bioinformatics software a positivity cut-off of H-score &amp;gt;150 was found to be most informative. The expression was then correlated with clinicopathological parameters (age, tumour grade, tumour stage, tumour size, nodal status, vascular invasion, and NPI) using Chi-squared analyses. Association of lamin A/C expression with prognosis [loco-regional recurrence, distant metastasis and breast cancer specific survival (BCSS)] was evaluated by Kaplan Meier survival analysis and Cox proportional hazards model was used to assess the statistical independence and significance of the marker on BCSS. Results The median follow-up of patients was 146 months. Positive expression rate of Lamin A/C was 42.2%. (n=398). Lamin A/C expression was significantly associated with low histological grade (p&amp;lt;0.001), tumour size &amp;lt;1.5 cm (p=0.004), good NPI score (p&amp;lt;0.001), absence of vascular invasion (p=0.014) and absence of distant metastasis (p = 0.027). No association was found between cells expressing lamin A/C and lymph node stage, distant metastases, loco-regional recurrence and patient age. Survival analysis showed that lamin A/C positivity was significantly associated with a longer breast cancer specific survival (p = 0.008) but it was not independent of tumour stage, grade or size for predicting BCSS (p=0.304). Conclusion This study suggests lamin A/C expression as a potential prognostic biomarker for early operable invasive breast cancer. Our findings indicate that lamin A/C expressing tumours are associated with better breast cancer specific survival. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 688. doi:1538-7445.AM2012-688

Genetic alterations in benign, preneoplastic and malignant breast lesions

The c-erbB-2 proto-oncogene is a member of the epidermal growth factor receptor family and has been associated with a more aggressive breast tumor biology and resistance to some types of treatments. The aim is to investigate the correlation among bcl-2 and c-erbB-2 and the micronucleus (MN) formation in patients with early breast cancer (BC). This study was conducted between May 2010 and December 2011. We analyzed the MN frequencies in 15 patients with invasive breast carcinoma (IBC), 13 patients with intraductal proliferative lesion (IDPL) and 12 benign breast lesion (BBL). The sample consisted of 40 formalin-fixed, paraffin-embedded blocks of benign and malignant breast tissue. The specimens were evaluated for bcl-2 or c-erbB-2 immunoreactivity was semi-quantitatively evaluated in at least 1000 cells examined under the microscope at 40Χ magnification and recorded as the percentage of c-erbB-2 and bcl-2 positive tumor cells over the total number of cells examined in the same area. The percentage scores were subsequently categorized using the 5% cut-off point for positive staining. The MN was significantly increased in IBC and in IDPL patients compared to BBL patients (3.82 ± 0.17 and 2.37 ± 0.52, respectively, vs. 1.61 ± 0.40, P < 0.001). On other hand, the MN frequencies in IBC patients were higher than those in IDPL patients (3.82 ± 0.17 vs. 2.37 ± 0.52, P < 0.01). c-erbB-2, had the highest record in IBC (60%), and the score was not observed in both IDPL and BBL: bcl-2 immunostaining was also assessed, the lowest recorded score was in IBC (46.66%) and the highest in both BBL and IDPL (100%). Furthermore, there was a significantly difference in the mean MN frequency between c-erbB-2 positive IBC patients (4.06 ± 0.48) and c-erbB-2 negative IBC patients (3.44 ± 0.39) (P < 0.05). Our results suggest that increased chromosome / DNA instabilities may be associated with the pathogenesis of early BC.

P1-03-05: Fibrocystic Changes Have Different Age-Dependent Patterns in Benign, In Situ, and Invasive Breast Cancer Patients.

Abstract Background: Fibrocystic changes (FCCs) have been known to develop in the aging process. Breast cancers in younger and elderly women are known to have different characteristics. Less is known about alterations in the microenvironment for disease development in middle-aged women who undergo menopausal changes. The Walter Reed Army Medical Center, through the Clinical Breast Care Project, has enrolled over 2000 subjects undergoing a biopsy; all the pathology was reviewed by a single pathologist. These subjects provide an opportunity to study the age-dependent pattern of FCC across benign, in situ (IS) and invasive breast carcinoma (IBC) patient populations. Methods: Subjects were enrolled following IRB-approved protocols. A total of 2060 subjects were identified for this study, including 1226 benign/atypical, 193 IS, and 641 IBC patients. Patients were divided into three age groups: &amp;lt;=45 years, 46–65 years, and &amp;gt;=66 years. Chi-Square test in the SAS was used for statistical analysis across the age groups for the occurrence of FCCs. For IS and IBC patients, additional analysis was performed on high grade (grade 3) and non-high grade (grades 1 and 2 combined) cancers. For IS, grade analysis was performed on ductal carcinoma (DCIS). Results: As shown in the Table, FCC occurrence rate is significantly lower in younger patients in the benign population. In the IBC population, this rate is significantly higher in middle-aged patients. This higher rate is significant in the non-high grade patients but disappears in the high grade patients. In the IS population, the same trend exists that the FCC rate is higher in middle-aged patients. This trend is retained in non-high grade DCIS but again lost in high grade DCIS. Discussion: The observation that FCC increases with age in the benign population, but is bell-shaped in IBC and IS populations, supports a hypothesis that physiological changes in the middle-aged women may alter the microenvironment for disease development. The observation that the bell-shaped pattern is due to non-high grade DCIS but not due to high-grade DCIS suggests that changes in the microenvironment contribute to breast disease development. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-03-05.

Serum TIMP-1 Predicts Survival Outcomes of Invasive Breast Carcinoma Patients: A Meta-analysis

Tissue inhibitor of metalloproteinase-1 (TIMP-1) is a small secretory glycoprotein with multifunctional activity including anti-apoptosis and the inhibition of matrix metalloproteinase in invasive breast carcinomas. There have been contradictory results as to whether TIMP-1 is a poor or good prognostic factor in breast cancer patients. To address this controversy, we conducted a meta-analysis for the relationship between TIMP-1 levels and prognostic parameters in the breast cancer. The relevant published studies were pooled according to the defined selection criteria. The effect sizes of overall survival and prognostic parameters were calculated by a hazard ratio (HR) or an odds ratio (OR). HRs or ORs were combined using a random-effects model. Survival outcomes between high or elevated and low or normal serum TIMP-1 levels were compared by uni- and multivariate analyses involving 886 and 844 breast cancer patients, respectively. Patients with high or elevated serum TIMP-1 levels had unfavorable survival outcomes compared to patients with low or normal serum TIMP-1 levels in the uni- and multivariate analyses (HR, 1.7 and 2.4; p <0.001 and p= 0.033, respectively). However, no survival difference was evident in the data from tissue TIMP-1 levels by enzyme-linked immunosorbent assay and the expression of tissue TIMP-1 mRNA. The high or positive immunohistochemical expression of tissue TIMP-1 protein was not related to adjusted and unadjusted HRs, lymph node metastasis, and clinical stages. This meta-analysis indicates that serum TIMP-1 levels may be useful for predicting survival outcomes of invasive breast cancer patients.