Outcome Of Triple-negative Breast Cancer Patients Research Articles

Pretreatment TG/HDL-C Ratio Is Superior to Triacylglycerol Level as an Independent Prognostic Factor for the Survival of Triple Negative Breast Cancer Patients.

Purpose: Previous studies have reported that the triacylglycerol (TG) level and high-density lipoprotein cholesterol (HDL-C) are connected with breast cancer. However, the prognostic utility of the TG level and the TG/HDL-C ratio (THR) as conventional biomarkers in patients with triple negative breast cancer (TNBC) has not been elucidated. In this research, we investigate and compare the predictive value of the pretreatment serum TG level and THR in TNBC patients.Methods: We evaluated 221 patients with TNBC who had pretreatment conventional blood biochemical examinations and calculated the THR. Univariate and multivariate logistic regression analyses were used to assess the effect of the TG level and the THR on overall survival (OS) and disease-free survival (DFS).Results: The optimal cutoff values of the TG level and the THR were determined to be 0.935 mmol/L and 0.600, respectively. As shown in a Kaplan-Meier analysis, TNBC patients with a high TG level and THR had shorter OS and DFS than patients in the low-level groups (p < 0.05). The multivariate analysis suggested that the pretreatment THR level is an independent prognostic factor of OS (HR: 1.935; 95%CI: 1.032-3.629; p = 0.040) in TNBC patients.Conclusions: In conclusion, our data indicate that a high THR is an independent predictor and is superior to the TG level for predicting poor clinical outcomes in TNBC patients.

Survival outcomes in Egyptian patients with triple-negative breast cancer: Single institute experience.

158 Background: Triple-negative breast cancer (TNBC) is a unique subtype and consider as an aggressive disease without established targeted treatment options. This study conducted to determine the incidence, characteristics, and survival outcomes of TNBC patients in an Egyptian cancer institute. Methods: Medical records of 520 patients treated between 2010-2011 in Clinical Oncology Department-Ain Shams University-Egypt were analyzed. Cox proportional hazards models were used to evaluate the association between TNBC and DFS and OS after adjusting for other covariates. Results: Among the 520 patients, 139 were TNBC .The median age was 50 years (SD±11.767, Range 20-80 ) versus 52 years (SD±12.134, Range 20-80), median tumor diameter was 5 cm (SD± 1.408, Range 1-7) versus was 5 cm (SD± 1.401, Range 1-7) , and median number of positive axillary LN was 3 (SD± 4.779, Range 0-37) versus 3 (SD± 4.832, Range 0-25) in non TNBC and TNBC respectively . Median disease-free survival was 24 months (SD± 14.128, Range 1-69 ) versus 15 months (SD± 8.811, Range 1-43 ) and median overall survival was 41 months (SD± 16.249, Range 3-60) versus 31 months (SD± 12.184, Range 7-60 ) in non TNBC and TNBC respectively. About 85.6 % of the TNBC tumors were IDC, 4.4 % were ILC and 5% were mixed. About 1.4 % of the TNBC tumors were grade I, 70.5 % were grade II and 28.1% were grade III. Median disease-free survival was 24 months (95%CI 21.679- 26.321) versus 15 months (95%CI 12.587-17.413) (p&lt; 0.001) and median overall survival was 44 months (95%CI 41.396-46.604) versus 31 months (95%CI 29.460-32.540) (p&lt; 0.001) in non TNBC and TNBC respectively. In TNBC cohort , DFS was 12 months (95%CI 11.464-12.536) in patients with grade III tumors versus 25 months (95%CI 22.359-27.641 )in patients with other grades (p&lt; 0.001), this was also reflected in OS as 29 months (95%CI 25.129-32.871 ) versus 44 months (95%CI 41.238-46.762 ) (p&lt; 0.001). Conclusions: Multivariate analyses supported a conclusion that TNBC subtype was an independent adverse prognostic factor for survival along with other known risk factors such as tumor grade.

Stratifying triple-negative breast cancer prognosis using 18F-FDG-PET/CT imaging.

This study aims to stratify prognosis of triple-negative breast cancer (TNBC) patients using pre-treatment 18F-FDG-PET/CT, alone and with correlation to immunohistochemistry biomarkers. 200 consecutive TNBC breast cancer patients treated between 2008 and 2012 were retrieved. Among the full cohort, 79 patients had pre-treatment 18F-FDG-PET/CT scans. Immunostaining status of basal biomarkers (EGFR, CK5/6) and other clinicopathological variables were obtained. Three PET image features were evaluated: maximum uptake values (SUVmax), mean uptake (SUVmean), and metabolic volume (SUVvol) defined by SUV > 2.5. All variables were analyzed versus disease-free survival (DFS) using univariate and multivariate Cox analysis, Kaplan-Meier curves, and log-rank tests. The optimal cutoff points of variables were estimated using time-dependent survival receiver operating characteristic (ROC) analysis. All PET features significantly correlated with proliferation marker Ki-67 (all p < 0.010). SUVmax stratified the prognosis of TNBC patients with optimal cutoff derived by ROC analysis (≤3.5 vs. >3.5, AUC = 0.654, p = 0.006). SUVmax and EGFR were significant prognostic factors in univariate and multivariate Cox analyses. To integrate prognosis of biological and imaging markers, patients were first stratified by EGFR into low (≤15 %) and high (>15 %) risk groups. Further, SUVmax was used as a variable to stratify the two EGFR groups. In the high EGFR group, patients with high FDG uptake (SUVmax > 3.5) had worse survival outcome (median DFS = 7.6 months) than those patients with low FDG uptake (SUVmax ≤ 3.5, median DFS = 11.6 months). In the low EGFR group, high SUVmax also indicated worse survival outcome (17.2 months) than low SUVmax (22.8 months). The risk stratification with integrative EGFR and PET was statistically significant with log-rank p ≪ 0.001. Pre-treatment 18F-FDG-PET/CT imaging has significant prognostic value for predicting survival outcome of TNBC patients. Integrated with basal-biomarker EGFR, PET imaging can further stratify patient risks in the pre-treatment stage and help select appropriate treatment strategies for individual patients.

Abstract 1774: The effects of diarylthiourea analogs on triple negative breast cancer cell growth

Abstract Breast cancer is the most common cancer occurring in women and the second leading cause of cancer death in women after lung cancer. Breast cancer is often classified at the molecular level in terms of whether the cancer expresses the estrogen receptor, progesterone receptor and human epidermal growth factor 2 receptor. Hormone receptor positive breast cancer accounts for 75-80% of all breast cancer and the remaining 20-25% accounts for the most aggressive types of cancer which are negative for all three receptors– known as triple negative breast cancer (TNBC). The current treatment used to manage TNBC is chemotherapy. Although chemotherapy has been effective in improving patients’ prognosis, a significant number of patients either relapse or develop resistance to these drugs. There is a significant interest in developing new therapeutic drugs aimed at improving outcomes in TNBC patients. Many studies have shown that flexible heteroarotinoids (Flex-Het) are a promising new class of drugs due to their ability to regulate growth, differentiation and apoptosis in a variety of cancer cells without activating the nuclear retinoic acid receptors. Previous studies have shown ShetA2 as the leading Flex-Het anti-cancer drug with the greatest efficacy in treating various types of cancer cells (Liu et al. 2004, Benbrook et al. 2005, and Liu et al. 2007). Some of the limitations of the ShetA2 are its high lipophilicity, toxic side effects and the low yields associated with the laborious synthesis. Therefore, efforts have been made to develop 2nd generation diarylthiourea compounds that retain anti-cancer activity, but with minimal side effects. The objective of this study was to examine the effects of two 2nd generation drugs SL1-18 and SL1-09 on various TNBC cell lines including MDA-MB-453, MDA-MB-231 and MDA-MB-468. Our preliminary data shows that exposure to micromolar levels of the diarylthiourea compounds effectively inhibited cancer cell growth in all three TNBC cell lines; however additional studies are necessary to understand the molecular mechanism of how SL1-18 and SL1-09 work to block cell growth and whether these anti-cancer drugs are promising therapeutic leads for the treatment of triple negative breast cancer. Citation Format: Deborah Nambi, Maggie C. Louie, Shengquan Liu. The effects of diarylthiourea analogs on triple negative breast cancer cell growth. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1774. doi:10.1158/1538-7445.AM2015-1774

Abstract 5340: Dual PI3K and Wnt pathway inhibition is a synergistic combination against triple-negative breast cancer

Abstract Introduction: Triple negative breast cancer (TNBC) accounts for 15% of all breast cancer cases in the United States, and despite its lower incidence, contributes to a disproportionately higher rate of morbidity and mortality compared to other breast cancer subtypes. Because these tumors lack expression of the estrogen, progesterone, or HER-2 receptors (“triple negative”), TNBC patients do not respond to targeted therapies that have been successfully used against tumors that over-express these proteins. Thus, there exists a critical need to improve the outcomes of TNBC patients through the implementation of novel targeted agents. Methods: RNA-seq data from 94 TNBCs (from Indiana University and TCGA) and 20 microdissected normal breast tissues (Komen Tissue Bank) were merged and imported into Partek Genomics Suite. The merged transcript RPKMs were transformed, batch effect corrected, and analyzed for differential expression. Statistically significant genes were imported into Ingenuity Pathway Analysis (IPA) to identify therapeutic targets. For cell based studies, we tested a panel of seven TNBC cell lines using BKM120 and LGK974, a WNT pathway inhibitor, individually. To further enhance antitumor efficacy, we then tested these TNBC cell lines using variable dilutions of both drugs in combination with one another. Cell viability was assessed via Celltiter-Fluor. Synergy between the two drugs was calculated using the Chou-Talalay method. Results: Using next-generation RNA sequencing data of TNBCs and microdissected normal breast tissue, Ingenuity Pathway Analysis identified over-expression and hyper-activation of the PI3K/AKT/mTOR and Wnt pathways. When anti-tumor efficacy against these pathways was assessed, a significant reduction in cell viability in combination was observed across the panel of cell lines. Using the Chou-Talalay method, we found for MDA-MB-231 and Hs578T, a ∼50% reduction in cell viability at 100nM concentration of each drug that was highly synergistic (Combination Index = 0.33, and 0.36 respectively). For HCC70, this cell line was more resistant to the combination and we observed ∼50% reduction at 1uM each that had an additive, non-synergistic effect. Conclusion: PI3K/mTOR/AKT and Wnt pathways are a vital target for treatment of TNBC. Using small molecule inhibitors that are in phase trials (BKM120 and LGK974) we have found that there is a strong synergy when given at low nanomolar doses. In vitro studies of inhibitors of these two pathways in a panel of TNBC cell lines demonstrated significant efficacy in reducing cell viability with substantial synergy when used in combination. Furthermore, initial mouse studies display a similar synergy and actual amplification of drug concentration and half-life in the blood of both drugs when given in combination when compared to these two drugs alone. Citation Format: Jeffrey P. Solzak, Rutuja Atale, Brad Hancock, Milan Radovich. Dual PI3K and Wnt pathway inhibition is a synergistic combination against triple-negative breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5340. doi:10.1158/1538-7445.AM2015-5340

S100A14 is a novel independent prognostic biomarker in the triple-negative breast cancer subtype.

Triple-negative breast cancer (TNBC) represents a heterogeneous subgroup with generally poor outcome and lack of an effective targeted therapy. Prognostic or predictive biomarkers to guide treatment decisions for this group of patients are needed. To evaluate the potential of S100A14 protein as a novel biomarker in TNBC, the protein expression of S100A14 was correlated with clinical outcomes in a Pilot Sample set and a Danish cohort of predominantly TNBC patients. Kaplan-Meier analysis identified a prognostic impact of S100A14 on disease-free survival and overall survival, showing that tumors with high S100A14 protein expression levels were significantly correlated with poor outcome in TNBC patients (p = 0.017; p = 0.038), particularly those in the basal-like subgroup (p = 0.006; p = 0.037). Importantly, TNBC patients with high S100A14 expression, but tumor-negative axillary lymph nodes (N-), had equally poor outcomes as those with tumor-positive axillary lymph nodes (N+), while TNBC/N- patients with low S100A14 expression had a significantly better disease free survival (p = 0.013). Multivariate analysis revealed that S100A14 is an independent prognostic factor for TNBC patients (p = 0.024; p = 0.05). At the cellular level, S100A14 was found to be expressed in epithelial-like, but not in mesenchymal-like, TNBC cells in vitro. S100A14 is an independent prognostic factor in TNBC and a novel potential therapeutic target in TNBC.

BCL2 is an independent predictor of outcome in basal-like triple-negative breast cancers treated with adjuvant anthracycline-based chemotherapy.

Neither targeted therapies nor predictors for chemotherapy sensitivity are available for triple-negative breast cancer (TNBC). Our study included 187 patients with TNBC, 164 of whom were treated with anthracycline-based adjuvant chemotherapy. Eleven molecular biomarkers were analyzed. BCL2, epidermal growth factor receptor (EGFR), MYC, TOP2A, and Ki-67 protein expression was evaluated by immunohistochemistry. The status of the EGFR, MYC, and TOP2A genes and chromosomes 7, 8, and 17 was assessed using fluorescence in situ hybridization. High BCL2 expression predicted poor relapse-free survival (RFS) in patients treated with anthracycline-based adjuvant chemotherapy (p = 0.035), poor breast cancer-specific survival (BCSS) (p = 0.048), and a trend to poor overall survival (OS) (p = 0.085). High levels of BCL2 expression predicted poor OS in basal-like (BL) TNBC patients treated with adjuvant anthracycline-based regimens (log-rank p = 0.033, hazard ratio (HR) 3.04, 95 % confidence interval (CI) 1.04-8.91) and a trend to poor RFS (log-rank p = 0.079) and poor BCSS (log-rank p = 0.056). Multivariate analysis showed that BCL2 status, tumor size, and nodal status all had independent predictive significance for RFS (p = 0.005, p = 0.091, p = 0.003, respectively; likelihood ratio test for the whole model, p = 0.003), BCSS (p = 0.012, p = 0.077, p = 0.01, respectively; likelihood ratio test for the whole model, p = 0.016), and OS (p = 0.008, p = 0.004, p = 0.004, respectively; likelihood ratio test for the whole model, p = 0.0006). Similarly, multivariate analysis for BL TNBC showed BCL2, tumor size, and nodal status all had independent predictive significance for RFS (likelihood ratio test for the whole model, p = 0.00125), BCSS (p = 0.00035), and OS (p = 0.00063). High EGFR expression was associated with poor BCSS (p = 0.039) in patients treated with anthracycline-based adjuvant chemotherapy. Patients who underwent anthracycline-based adjuvant chemotherapy and exhibited CMYC amplification had a trend to worse BCSS (p = 0.066). In conclusion, high BCL2 expression is a significant independent predictor of poor outcome in TNBC patients treated with anthracycline-based adjuvant chemotherapy, and this is the first study showing the BCL2 prediction in BL TNBC. BCL2 expression analysis could facilitate decision making on adjuvant treatment in TNBC patients.

Triple negative breast cancer - BCL2 in prognosis and prediction. Review.

Breast cancer (BC), the most frequent malignancy in women worldwide, is currently diagnosed in about 1.4 million female patients annually. Approximately 10-20% of BC is represented by triple negative breast cancer (TNBC) which is aggressive, the prognosis is poor and patients cannot benefit from targeted treatment based on hormonal or HER2 receptors. For this reason, search for markers that can predict the efficacy of chemotherapy in TNBC is a priority. This review focuses on BCL2 protein as a prognostic marker in TNBC and its potential as a predictor of sensitivity to chemotherapy. BCL2 protein expression is a positive prognostic factor in BC. Better survival of patients with BCL2 positivity (BCL2+) has been explained by the correlation with estrogen receptor positive (ER+) status. BCL2+ is however not simply a surrogate marker for ER+. Moreover, BCL2 protein expression is also a positive prognostic marker in the TNBC subgroup. We and others show, that low BCL2 expression was associated with good outcome of TNBC patients treated with both adjuvant and neoadjuvant anthracycline-based chemotherapy. On the other hand, recent studies have shown that a subset of TNBC patients may benefit from the classical adjuvant CMF (cyclophosphamide, methotrexate, 5-fluorouracil) regimen. Given the heterogeneity of TNBC there is an urgent need to find and validate the sensitivity predictors to these regimens making them usable in clinical practice. BCL2 enrichment has been described in the mesenchymal stem-like (MSL) TNBC subgroup.

Pre-operative prognostic nutritional index predicts the outcomes for triple-negative breast cancer

The purpose of this study was to investigate whether pre-operative prognostic nutritional index (PNI), an indicator of nutritional and immunological status, has an impact on the long-term outcomes in triple-negative breast cancer (TNBC) patients. This retrospective study reviewed the medical records of 382 TNBC patients who had suffered from mastectomy. Pre-operative PNI was calculated as 10 × serum albumin (g/dl) + 0.005 × total lymphocyte count (per mm(3)). The receiver operating characteristic (ROC) curve analysis was performed to determine the optimal cutoff value of PNI. The correlations of PNI value with clinicopathological features were analyzed and the univariate and multivariate analysis were applied to identify the prognostic factors. The results showed that pre-operative PNI value was significantly related to advanced tumor status such as N stage (p = 0.011), T stage (p = 0.015), and recurrence incidents (p = 0.001). Survival analysis identified PNI as an independent prognostic factor for TNBC. Patients with higher PNI value had better 5-year disease-free survival (DFS) and 5-year overall survival (OS) than those with lower PNI value (DFS, p = 0.007; OS, p = 0.011). Taken together, our results suggest that the pre-operative PNI can be used as a simple and useful marker for predicting the long-term outcomes of TNBC patients.

Outcomes of triple-negative breast cancer (TNBC) versus non-TNBC patients: Does the survival vary for all stages?

e12034 Background: Triple-negative breast cancer (TNBC) poses a therapeutic challenge. In a cohort treated at a tertiary care North Indian center, outcomes of TNBC patients were compared with those...

Abstract P3-05-15: TP53 mutation and nestin protein expression patterns affect the outcome of patients with triple negative breast cancer (TNBC)

Abstract Background-aim: Nestin has recently been proposed as an ideal positive biomarker for calling basal-like breast cancer (BL). BL and TNBC overlap in 70-80% of cases and, although very heterogeneous at the genomic and phenotypic levels, most of these carry TP53 mutations. Herein, we investigated the effect of TP53 mutations and Nestin expression on the outcome of early high-risk TNBC patients who had received adjuvant chemotherapy (anthracyclines and/or taxanes). Methods: TP53 mutations were assessed in 190 FFPE TNBC DNA samples with massive parallel sequencing (entire coding region) in Ion Torrent PI chips. Variant calling and annotation (Ion Reporter 1.6) was followed by stringent read quality filtering (p&amp;lt;0.001). Deleterious and hot-spot mutations (Ingenuity &amp; Oncomine databases) in 183 informative tumors were accepted for analysis. Nestin protein expression was examined by immunohistochemistry (IHC) in 283 centrally assessed TNBC on tissue microarrays; positivity was set at 1% of cells regardless of staining intensity. Further BL and TNBC related IHC markers were also examined. Results: Nestin was expressed in 134/283 tumors (47.3%), more frequently in BL-TNBC (CK5+ and/or EGFR+, Fisher's exact p&amp;lt;0.001) and in highly proliferative tumors (p&amp;lt;0.001), while it was positively associated with high tumor grade (p = 0.017) and with P-cadherin IHC (p = 0.001). TP53 mutations were observed in 141/183 tumors (77%), out of which 30 carried truncating TP53 mutations (21.3%) and 59 double damaging mutations (41.8%) usually at similar coverage; among these, truncating mutations occurred in 16 cases. TP53 mutations were more common in BL-TNBC (p = 0.029). Double mutations were positively associated with E- and P-cadherin protein expression (p = 0.016 and p = 0.020, respectively) Although TP53 mutations in general and the presence of double mutations were not associated with p53 IHC, tumors with truncating mutations were almost exclusively p53 IHC negative (p&amp;lt;0.001) in line with the detection capacity of the employed antibody. As single markers, neither Nestin nor TP53 mutations (any pattern) had significant effects on patient outcome. When combined, a DFS-related interaction between Nestin and double TP53 mutations was observed (Wald's p = 0.016). Nestin positive tumors with double TP53 mutations (n = 22) conferred median DFS of 45 mo. This interval was significantly shorter as compared to that of patients with Nestin positive tumors without double TP53 mutations (n = 62; median DFS 56 mo; HR 2.2; 95%CI 1-4.5; p = 0.045) and Nestin negative tumors with double TP53 mutations (n = 25) that were associated with the most favorable DFS (median: 66 mo; HR 4; 95%CI 1.3-12.4; p = 0.016). Nestin negative tumors without double TP53 mutations performed similarly to both positive tumors, although not statistically significant. Conclusions: The presence of double TP53 mutations in TNBC may reflect the genetic instability and heterogeneity characterizing these tumors. This feature in combination with the putative cancer stem cell and BL marker Nestin seems to aggravate the outcome of TNBC patients treated with standard adjuvant chemotherapy regimens. These novel findings might be worthy pursuing for validation in larger patient series. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-05-15.

Abstract P4-07-12: miR-206 inhibits cell migration through direct targeting of the actin-binding protein coronin 1C in triple-negative breast cancer

Abstract Patients with triple-negative breast cancer (TNBC) have an overall poor prognosis, which is primarily due to a high metastatic capacity of these tumors. Novel therapeutic approaches to target the signaling pathways that promote metastasis are desirable, in order to improve the outcome of TNBC patients. A loss of function of a microRNA, miR-206, is related to increased metastasis potential in breast cancers but the mechanism remains to be elucidated. In this study, we show that miR-206 was decreased in TNBC clinical tumor samples and cell lines whereas one of its predicted targets, actin-binding protein CORO1C, was increased. Expression of miR-206 significantly reduced proliferation by inducing a G1-S cell cycle arrest and migration and also repressed CORO1C mRNA and protein levels. We demonstrate that miR-206 interacts with the 3’-untranslated region (3’-UTR) of CORO1C and regulates this gene post-transcriptionally. Further, silencing of CORO1C reduced tumor cell migration and affected the actin skeleton and cell morphology, similar to miR-206 expression, but did not reduce proliferation. Our findings suggest that miR-206 targets CORO1C in TNBC cells, thereby affecting the actin filaments which results in a repressed tumor cell migration. This pathway is a novel mechanism offering a mechanistic basis whereby the metastatic potential of TNBC tumors could be targeted. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-07-12.

Abstract P6-04-09: Dual PI3K and Wnt pathway inhibition is a synergistic combination against triple negative breast cancer

Abstract Introduction: Triple negative breast cancer (TNBC) accounts for 15% of all breast cancer cases in the United States, and despite its lower incidence, contributes to a disproportionately higher rate of morbidity and mortality compared to other breast cancer subtypes. Because these tumors lack expression of the estrogen, progesterone, or HER-2 receptors (“triple negative”), TNBC patients do not respond to targeted therapies that have been successfully used against tumors that over-express these proteins. Thus, there exists a critical need to improve the outcomes of TNBC patients through the implementation of novel targeted agents. Methods: RNA-seq data from 94 TNBCs (from Indiana University and TCGA), 20 microdissected normal breast tissues (Komen Tissue Bank), and 10 adjacent normal tissues (TCGA), were merged and imported into Partek Genomics Suite. The merged transcript RPKMs were transformed, batch effect corrected, and analyzed for differential expression. Statistically significant genes were imported into Ingenuity Pathway Analysis (IPA) to identify therapeutic targets. For cell based studies, we tested a panel of eight PI3K/AKT/mTOR inhibitors that are either currently in clinical trials against a panel of seven TNBC cell lines. To further enhance anti-tumor efficacy, we then tested these TNBC cell lines using increasing concentrations of FH535, a WNT pathway inhibitor. Cell viability was assessed via Celltiter-Fluor. Results: Using next-generation RNA sequencing data of TNBCs and microdissected normal breast tissue, Ingenuity Pathway Analysis identified over-expression and hyper-activation of the PI3K/AKT/mTOR and Wnt pathways. To assess anti-tumor efficacy against these pathways, we tested eight PI3K/AKT/mTOR inhibitors, and observed significant reductions in cell viability with all inhibitors. Because of the reported clinical efficacy of BKM120, a pan-isoform PI3K inhibitor, we focused our cell viability experiments with BKM120 and observed an average IC50 of 0.7uM across TNBC cell lines. We then tested FH535, a Wnt pathway inhibitor, and observed an average IC50 of 4.86μM. All TNBC cell lines were then treated with 1, 10, and 100nM BKM120 in combination with increasing doses of FH535 starting at 1nM. At 10nm BKM120, we observed significant synergy with FH535 at low doses. The average IC50 of FH535 when combined with 10nm BKM120 was 4.5nM across cell lines. While variation in sensitivity was seen across cell lines, the highest FH535 IC50 for the combination was 10nM in MDA-MB-453 cells, still demonstrating significant synergy at low doses. RNA-seq analyses of TNBC cells after treatment with BKM120 to understand the nature of this synergy are currently underway. Conclusion: Next-generation RNA-sequencing has identified PI3K/mTOR/AKT and Wnt pathways as key targets for the treatment of TNBC and that inhibition of these pathways using small molecule inhibitors in combination is highly synergistic. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-04-09.

Spectrum of EGFR Gene Copy Number Changes and KRAS Gene Mutation Status in Korean Triple Negative Breast Cancer Patients

Anti-epidermal growth factor receptor (EGFR) therapy has been tried in triple negative breast cancer (TNBC) patients without evaluation of molecular and clinical predictors in several randomized clinical studies. Only fewer than 20% of metastatic TNBCs showed response to anti-EGFR therapy. In order to increase the overall response rate, first step would be to classify TNBC into good or poor responders according to oncogenic mutation profiles. This study provides the molecular characteristics of TNBCs including EGFR gene copy number changes and mutation status of EGFR and KRAS gene in Korean TNBC patients. Mutation analysis for EGFR, KRAS, BRAF and TP53 from a total of 105 TNBC tissue samples was performed by direct sequencing, peptide nucleic acid-mediated PCR clamping method and real-time PCR. Copy number changes of EGFR gene were evaluated using multiplex ligation-dependent probe amplification. Out of all 105 TNBCs, 15.2% (16/105) showed EGFR copy number changes. Among them, increased or decreased EGFR copy number was detected in 13 (5 single copy gain, 2 amplification and 4 high-copy number amplification) and 3 cases (3 hemizygous deletion), respectively. The mutation frequencies of KRAS, EGFR and TP53 gene were 1.9% (G12V and G12D), 1.0% (exon 19 del) and 31.4%, respectively. There was no BRAF V600E mutation found. Future studies are needed to evaluate the clinical outcomes of TNBC patients who undergo anti-EGFR therapy according to the genetic status of EGFR.

High expression of LC3B is associated with progression and poor outcome in triple-negative breast cancer

Microtubule-associated protein 1 light chain 3B (LC3B) was involved in autophagosome formation and had been as a marker of autophagy which played an important role in the development of breast cancer. The purpose of this study was to explore the level of LC3B expression in four stages of triple-negative breast cancer (TNBC) and to evaluate the prognostic significance of LC3B expression in TNBC. The ultimate aim was to identify the new factor that could be useful in predicting clinical behavior of TNBC. We evaluated the expression level of LC3B protein in four kinds of TNBC tissue samples, including intraductal carcinoma in situ (DCIS), invasive ductal carcinoma (IDC) without metastases, IDC with lymph node metastases (LNM), and IDC with distant metastases (DM). Hundred and four primary TNBC patients were involved in present study, and the expression level of LC3B protein was assessed by immunohistochemistry. Medical records of these patients were reviewed, and the clinicopathological analysis was performed. High expression of LC3B was observed in 7.7 % of DCIS (1 of 13 cases), 16.2 % of IDC (6 of 37 cases), 35.7 % of LNM (15 of 42 cases), and 58.3 % of DM (7 of 12 cases). LC3B high expression was significantly associated with tumor size (P = 0.028), lymph node metastasis (P = 0.002), and Ki-67 expression (P = 0.047). LC3B high expression patients showed poorer DFS and OS rates compared with LC3B low expression patients (P = 0.024, and P = 0.047, respectively). Multivariate analyses confirmed that high LC3B expression was an independent and significant factor for predicting the poor outcome of TNBC patients. These preliminary results demonstrated that high LC3B expression was associated with lymph node and distant metastasis. Furthermore, high LC3B protein expression was correlated with shorter survival in patients with triple-negative breast carcinoma. These findings provided preliminary evidence for the function of LC3B on the progression of TNBC and suggested LC3B was a useful marker in prognostic evaluation for patients with TNBC.

Radiotherapy Can Improve the Disease-Free Survival Rate in Triple-Negative Breast Cancer Patients with T1–T2 Disease and One to Three Positive Lymph Nodes After Mastectomy

Several studies have demonstrated poor locoregional control in patients with triple-negative breast cancer (TNBC), compared with other molecular subtypes of breast cancer. We sought to evaluate whether or not postmastectomy radiotherapy (PMRT) improves locoregional recurrence-free survival (LRFS) and disease-free survival (DFS) outcomes in TNBC patients. Between January 2000 and July 2007, 553 TNBC patients treated with modified radical mastectomy from a single institution were analyzed retrospectively. Patients were categorized into three groups: low risk (stage T1-T2N0), intermediate risk (stage T1-T2N1), and high risk (stage T3-T4 and/or N2-N3). Cox proportional hazards models were used to evaluate the association between PMRT and LRFS and DFS times after adjusting for other clinicopathologic covariates. With a median follow-up of 65 months (range, 1-140 months), 51 patients (9.2%) developed locoregional recurrence and 135 patients (24.4%) experienced disease recurrence. On multivariate analysis, PMRT was associated with significantly longer LRFS and DFS times in the entire cohort. In the intermediate-risk group, PMRT was associated with a longer DFS time but not with the LRFS interval. In the high-risk group, PMRT was associated with significantly longer LRFS and DFS times. PMRT is associated with longer LRFS and DFS times in high-risk TNBC patients and a longer DFS time in intermediate-risk TNBC patients. Prospective randomized studies are needed to investigate the best locoregional treatment approaches for patients with this molecular subtype of breast cancer.

Predictors of neoadjuvant chemotherapy for triple-negative breast cancer: a meta-analysis with 723 cases

Neoadjuvant chemotherapy (NAC) was increasingly used as a systemic therapy for triple-negative breast cancer (TNBC). The pathological complete response (PCR) rates of neoadjuvant chemotherapy in TNBC were higher than other types of breast cancer with fluctuate data. Predictors to identify which subgroup TNBC was more likely to achieve PCR in neoadjuvant chemotherapy would give us some hints on how to improve outcomes of TNBC patients. The meta-analysis was conducted to contrast the prognostic function of some clinicopathological parameters in the PCR rates of neoadjuvant chemotherapy for TNBC. Studies were selected from the PubMed database. The relevant parameters to PCR rates in TNBC group were recorded. Review Manager and MIX were used to estimate prognostic function of some biological markers and clinicopathological parameters in PCR rates of TNBC. The analysis included 6 studies with 723 patients, the aggregate PCR rate was 27.9% in TNBC group. The association of lymph nodes metastasis, Ki-67 expression, p53 expression and CK5/6 expression with PCR rate of TNBC was investigated in the analysis, and the odds ratios were 0.50, 9.87, 1.17 and 0.53 respectively. This meta-analysis demonstrated that Ki-67 expression and lymph nodes metastasis were predictors of PCR rate for TNBC in neoadjuvant chemotherapy, while p53 and CK5/6 expression could not be confirmed for the prognostic function.

Abstract 3449: Impact of BRCA2 mutation in primary human triple negative breast cancer xenografts on the responses to ionizing radiation and in vivo tumor growth after treatment

Abstract Introduction: Triple negative breast cancer (TNBC) is an aggressive disease associated with a high risk of distant recurrence and poor overall survival and, as for other BC subtypes, loco-regional treatment relies on surgery and radiotherapy. To prevent relapses, there is a need for both better systemic therapies and improvements in local treatments. Primary human TNBC xenografts are a useful tool for such preclinical studies. Using two TNBC models, one of which carrying a BRCA2 mutation (De Plater et al, BJC 2010;103:1192-200), we have investigated ionizing radiation sensitivity and the impact of the treatment on the growth of irradiated tumors. Experimental design: Two TNBC models, the BRCA2 HBCx-17 and the wild-type HBCx-12A xenografts were subcutaneously transplanted into the flanks of nude mice. After immobilization of the animals, the tumors were locally irradiated with low energy photons generated by an X-Ray tube (voltage: 200 kV, filter: 1 mm Al + 0.3 mm Cu, mean energy: 100 keV). Irradiation fields adapted to the targeted tissue were obtained using Cerrobend shields. With a daily fraction of 3.25 Gy, the cumulative dose ranged between 0 Gy to 32.25 Gy. In addition for both models, three tumor fragments from non irradiated tumors, 9.25 Gy-irradiated tumors with initial partial local control and subsequent progressive disease, and 19.5 Gy-irradiated tumors with initial complete remission and subsequent relapse were regrafted 3 months after treatment into nude mice. In all experiments, tumor take and growth were assessed by measuring tumor volume with a caliper twice a week for 5 months. Results: In both TNBC models, a strong sensitivity to ionizing radiation was observed with the occurrence of complete remission seen at higher doses. However, after 32.25 Gy, this was 100% in the BRCA2-mutated xenograft and only 50% in the HBCx-12A model (p &amp;lt; 0.05). Similarly, relapses occurred more frequently in the wild-type xenograft (57.1% vs 14.9%; p &amp;lt; 0.001). With respect to the treated or untreated tumors that were regrafted into mice, no differences of tumor take, in vivo tumor growth nor kinetics were observed for the BRCA2-mutated model for the 3 tumor types, in contrast to the HBCx-12A xenograft where 9.25 Gy-irradiated tumors grew faster than the 2 other groups. Conclusion: Our preclinical results confirm the sensitivity of TNBC to ionizing radiation and the impact of BRCA2 mutations on this sensitivity. In addition the presence of BRCA2 mutations would appear to modulate the post-radiotherapy growth of the tumors making them less aggressive. Such a characterization of highly relevant preclinical models supports their use for pharmacological assessments that will combine both radiotherapy and new therapeutic approaches to improve the outcome of TNBC patients. 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 3449. doi:1538-7445.AM2012-3449

P3-18-05: Triple-Negative Breast Cancer: Stem Cells, Cancer and New Treatment Strategy.

Abstract Background: Women with triple negative breast cancer (TNBC) have a worse prognosis compared with other breast cancer subtypes. Hormonal or Herceptin-based therapies were found to be ineffective because of the loss of target receptors such as ER, PR and HER-2 amplification. TNBC has increased recurrence and poor survival, despite initial response to neoadjuvant chemotherapy. The aim of this study was to determine whether current drug treatment(s) eliminates bulk of tumor cells, but it has a minimal effect on cancer stem cells (CSCs) leading to tumor recurrence. Materials and Methods: Cancer stem cells were identified by ALDH-positive population by fluorescence-activated cell sorting. CSCs ability to form mammospheres, are generated in nonadherent and nondifferentiated conditions. Apoptosis was assessed using the Cell Death Detection ELISAPLUS kit. Xenograft tumor formation was evaluated using Nod/Scid mice. Cancer stem cell levels in human TNBC and ER+, PR+ and HER2− tumors were obtained by CD44+/ CD24low/− immunohistochemistry. Results: TNBC tumors express relatively higher levels of cancer stem cells compared to ER-positive, PR-positive and HER2−negative breast tumors. We studied the effects of PARP inhibitors (AZD2281 and BSI-201), paclitaxel, docetaxel, cisplatin and cisplatin plus TRAIL on CSCs derived from CRL-2335 and MDA-MB-468 TNBC cells in vitro. The in vitro data indicates that cisplatin plus TRIAL treatment was most effective in eliminating CSCs compared to PARP inhibitors, cisplatin, paclitaxel and docetaxel. Treatment with cisplatin plus TRAIL also inhibits Wnt-1 signaling and its downstream target, β-catenin, phospho β-catenin, cyclin D1, increased apoptosis, reduced proliferation and mammosphere formation. Inhibition of Wnt-1 by siRNA significantly reduced the ability of CSCs to form mammospheres compared to control. However, maximum effect was seen in cisplatin plus TRAIL treated cells. The combination of cisplatin/TRAIL also inhibited the growth of CRL-2335 Xenografts. Discussion: We demonstrate for the first time that cisplatin plus TRAIL is highly effective in reducing CSCs compared to PARP inhibitors, docetaxel or paclitaxel in TNBC cells in vitro. Inhibition of Wnt-1 signaling pathways by siRNA or cisplatin plus TRAIL reduces cell proliferation and increases apoptosis in CSCs. Taken together the data suggests that cisplatin plus TRAIL treatment has the potential of providing a new strategy for improving the therapeutic outcome in TNBC patients. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-18-05.

P2-11-09: EGFR Overexpression in Triple Negative Breast Cancer (TNBC) and Its Association with the Prognosis.

Abstract Objective: The aim of this study is to investigate EGFR expression in Triple Negative Breast Cancer (TNBC), and to find the relationship between EGFR overexpression and prognosis of TNBC, further to clarify the significance of EGFR in TNBC and provide valuable information for TNBC therapy. Methods: 42 triple ***negandection ***ssitive breast cancer patients(studying group) and 40 HER2(3+) breast cancer patients(controling group) who underwent surgery from January 2000 to December 2005 were analyzed. 82 cases of paraffin-embedded specimens were detected by Immunohistochemistry(IHC), fluorescence in situ hybridization(FISH) and polymerase chain reaction(PCR) to investigate the overexpression, amplification and mutation of EGFR gene. The distant-free survival(DFS) and overall survival(OS) of these patients were used to investigate the relationship between EGFR overexpression and the prognosis of TNBC. Results: 34(43.9%) EGFR overexpression was observed in all cases, while gene amplification was only 7(9.1%) cases. No EGFR gene mutation was found in all cases. Overexpression of EGFR occurring in 57.1% patients in TNBC group and 25.0% patients in HER2 group, and we didn't found any correlation between EGFR overexpression and clinicopathology. 50(61.0%) patients relapsed (TNBC 28,HER2 22) and 27(32.9%) patients died(TNBC 18,HER2 9) were observed during the more than 5 years follow-up. The 5-year DFS was 57.1% and 77.5% respectively, the 5-year OS was 71.4% and 87.5% in TNBC and HER2 groups. In TNBC group, the survival of the EGFR-overexpressing group patients was significantly lower than that of the non-EGFR-overexpressing group patients (p=0.018 for DFS, p=0.026 for OS); In HER2 group, no statistical difference was found (p=0.079 for DFS, p=0.055 for OS). Conclusions: This study showed that EGFR overexpression increased significantly in TNBC patients, which was no correlation with their clinico-pathological data. EGFR gene amplification was much less frequent than its overexpression. It suggested that EGFR gene amplification may not be the unique mechanism of EGFR overexpression in TNBC. There may be other possible mechanisms and pathways that cause EGFR overexpression. In addition, it may suggest that gefitinib therapy is useless in TNBC patients because we did not find any mutations in the tested exons of TNBC. EGFR overexpression may associate with a poor outcome of TNBC patients which suggest it could be a significant prognostic factor for TNBC patients. EGFR may play important role for molecular-targeting therapy of TNBC. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-11-09.