HER2-negative Breast Tumors Research Articles

Prognostic relevance at 5 years of the early monitoring of neoadjuvant chemotherapy using FDG PET in luminal HER2-negative breast cancer.

11007 Background: To evaluate, in the luminal breast cancer subtype, the prognostic value of tumor glucose metabolism at baseline and of its changes after one cycle of neoadjuvant chemotherapy (NAC). Methods: This prospective study included 61 women with immunophenotypically defined luminal HER2-negative breast cancer treated with NAC. 18F-FDG PET was performed at baseline. Hepatic activity was used as a reference to distinguish between low-metabolic and hypermetabolic tumors. In hypermetabolic tumors, a PET exam was repeated after the first course of NAC. The relative change in the maximal Standardized Uptake Value of the tumor (ΔSUV), corresponding to the metabolic response, was calculated. Results: Forty-two women had hypermetabolic tumors at baseline, corresponding to more proliferative breast cancers with higher Ki-67 expression (p=0.017) and higher grade (p=0.04). Nineteen women had low-metabolic tumors with lower proliferation indexes. Worse overall survival was associated with larger tumor size (>5cm, HR=6.52, P=0.009) and with hypermetabolic tumors achieving a low metabolic response after one cycle of NAC (ΔSUV<16%, HR=10.63, P=0.004). Five-year overall survival in these poor-response patients was 49.22% (95% CI=[14.76%-76.90%]). In contrast, overall survival in women with low-metabolic tumors or hypermetabolic/good-response tumors (ΔSUV≥16%) was good, 100% and 96.15%, respectively (95% CI=[75.69%-99.45%]). Conclusions: In luminal HER2-negative breast tumors, tumor metabolism at baseline and changes after the first course of NAC are surrogate markers of patients’ survival. A subgroup of women with hypermetabolic/bad-responding tumors correlated with poor prognosis can be identified. These results may create the ability to tailor the NAC regimen to the metabolic response at an early stage.

Low expression of Beclin 1 and elevated expression of HIF-1α refine distant metastasis risk and predict poor prognosis of ER-positive, HER2-negative breast cancer

Even though ER-positive, HER2-negative breast tumors represent a subset of breast cancers with a better clinical outcome, approximately 12.7 % of patients in this subgroup ultimately develop cancer-related mortality. Recent studies had confirmed that hypoxia-induced autophagy-related gene Beclin 1 expression might be important for disease progression and be correlated with patient outcome in several tumors. Here, we examined the autophagic Beclin 1 and hypoxic HIF-1α levels in 378 ER-positive, HER2-negative breast cancer patients by immunohistochemistry using tissue microarray. We found that Beclin 1 was highly expressed in normal mammary gland epithelia. In contrast, it was either not expressed or only moderately expressed in 78.0 % of breast adenocarcinoma tissue. Compared to the subset overexpressing Beclin 1, the subset in which Beclin 1 levels were reduced had a poor 5-year overall survival rate (OS, 85.1 % vs. 94.1 %, P = 0.005) and a poor distant metastasis-free survival (DMFS, 79.1 % vs. 89.3 %, P = 0.037). Cox multivariate analysis confirmed that Beclin 1 was indeed an independent prognostic factor for OS and DMFS. Additionally, Beclin 1 positively correlated with HIF-1α expression (r = 0.206, P < 0.001). Importantly, among patients with HIF-1α overexpression, low levels of Beclin 1 predicted a worse OS. Our study confirmed that deficiency of Beclin 1 was a negative prognostic factor for OS and DMFS in ER-positive, HER2-negative breast cancer. The combination of Beclin 1 and HIF-1α refined the risk definition of the patient subset and provided a novel way to identify those with a high risk of relapse.

Abstract P5-03-09: Receptor Activator of Nuclear Factor Kappa B (RANK) as a potential therapeutic target in triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) has a poorer prognosis in comparison to other breast cancer subtypes because of its high metastatic rate. There is evidence that cancer stem cells (CSCs), known to have an epithelial-to-mesenchymal (EMT) phenotype, may contribute to the metastasis of breast cancer. Denosumab (an anti-RANK ligand antibody) is currently being explored as a treatment for breast cancer in phase II and III clinical trials. RANK is thought to regulate mammary stem cells. Therefore, we hypothesized that RANK promotes tumorigenicity and metastasis of TNBC via the regulation of EMT and enrichment of CSCs. We sought to determine the effects of RANK expression on the tumorigenic and metastatic activities of TNBC cells and whether RANK is a potential therapeutic target in TNBC. Methods: Primary breast tumor specimens from the MD Anderson Cancer Center Tumor Bank were stratified by ER/HER2 status. We analyzed RANK mRNA expression levels, obtained by Affymetrix gene chip array, in ER+/HER2-negative breast tumors (n = 22) and ER−/HER2-negative breast tumors including TNBC tumors (n = 18). To study the effects of RANK suppression in TNBC cells, we generated two stable RANK shRNA MDA-MB-231 TNBC cell lines and a negative shRNA cell line as a control and examined whether knockdown of RANK expression correlated with a decrease in mesenchymal markers, vimentin and snail, and stem cell marker, ALDH1, invasion, migration, and tumorigenicity in TNBC cells. Results: An analysis of RANK mRNA expression levels among human primary breast cancer specimens revealed increased RANK expression in ER−/HER2-negative primary breast tumors including TNBC tumors, compared with ER+/HER2-negative primary breast tumors (p = 0.034). Knocking down RANK in MDA-MB-231 cells significantly inhibited migration (p = 0.0005) and invasion (p = 0.0006) and reduced the stem cell subpopulation, as shown by a decrease in mammospheres (p = 0.0188) and ALDH1 activity. Further, in a three-dimensional (3D) cell culture model, we observed significant inhibition of projections in MDA-MB-231 cells with knockdown of RANK (p &amp;lt; 0.05), which correlated with downregulation of the mesenchymal markers vimentin and snail shown by qRT PCR analysis. Conclusion: Our data demonstrate that RANK may promote the tumorigenesis of TNBC by enriching cancer stem cells. In future studies, we will investigate the effect of RANK inhibition on metastasis in vivo using a TNBC xenograft model. Our long-term goal is to develop RANK-targeted therapy for TNBC. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-03-09.

Methylation profile of ER- and HER2-negative (ER-/HER2-) breast tumors.

e21098 Background: Breast cancer is a group of clinically, histopathologically and molecularly heterogeneous diseases, with diverse outcomes and responses to treatment. ER-/HER2- is an immunohistochemical description of breast cancers with a double negative staining for ER and HER2. These tumors have an aggressive clinical behavior and poor prognosis. The hypermethylation of CpG islands (CpGI) is a common epigenetic alteration for suppressing gene expression in breast cancer. Understanding how these epigenetic changes are involved in tumor progression is important for a better comprehension of tumor biology. Here we evaluated the methylation status of ER-/HER2- compared to luminal breast tumors. Methods: For the methylation analysis, we used the methyl specific-multiplex ligation probe amplification assay. Seventy invasive ductal carcinomas (IDC) were included in the study. In 20 IDCs we analyzed 127 CpGI and in 50 IDCs we analyzed 49 CpGI. Results: Among the islands studied we found positive association between ER-/HER2- tumors and methylated MGMT (p<0.002) and CD44 (p<0.01) and non methylated GSTP1 (p<0.04), PAX6 (p<0.04), MSH2 (p<0.002), MSH3 (p<0.001) and CACNA1A (p<0.049) genes. Conclusions: ER-/HER2- cancers present a clearly different methylation profile, compared to luminal tumors. These results could add knowledge to the behavior of these tumors and improve future treatment options.

Quantification and clinical relevance of gene amplification at chromosome 17q12-q21 in human epidermal growth factor receptor 2-amplified breast cancers

IntroductionHuman epidermal growth factor receptor 2 (HER2)-amplified breast cancers represent a tumor subtype with chromosome 17q rearrangements that lead to frequent gene amplifications. The aim of this study was to quantify the amplification of genes located on chromosome 17q and to analyze the relations between the pattern of gene amplifications and the patients' characteristics and survival.MethodsPatients with HER2-positive breast tumors (HER2 score of 3+ by immunohistochemistry or positive for HER2 amplification by fluorescence in situ hybridization (FISH)) (n = 86) and with HER2-negative breast tumors (n = 40) (negative controls) were included in this study. Using a quantitative polymerase chain reaction method and DNA extracted from frozen tumor specimens, 11 genes (MED1, STARD3, HER2, GRB7, THRA, RARA, TOP2A, IGFBP4, CCR7, KRT20, KRT19 and GAS), which are localized within Chr17q12-q21 and have a putative role in breast cancer development, were quantified. Relapse-free and overall survival rates were estimated from the date of surgery to the date of the event of interest (recurrence or death) using the Kaplan-Meier method.ResultsGene amplification was observed only in HER2-positive tumors, and the frequency of amplification decreased with the distance of the gene from HER2. HER2 presented the highest level of amplification. TOP2A was not included in the smallest region of amplification involving HER2. Amplification of RARA, KRT20 and KRT19 was significantly associated with node-positive breast cancer (P = 0.030, P = 0.002 and P = 0.033, respectively). During a median follow-up period of 55 months (range, 6 to 81 months), the subgroup of patients with hormone receptor-negative cancer and without TOP2A amplification showed the worst survival (relapse-free survival: hazard ratio (HR) = 0.29, 95% confidence interval (95% CI), 0.13 to 0.65, P = 0.001; and overall survival: HR = 0.28, 95% CI, 0.10 to 0.76, P = 0.008).ConclusionsHER2 amplification seems to drive genomic instability along chromosome 17q, leading to different patterns of gene amplification. This study confirms the clinical importance of identifying, among patients with HER2-positive breast tumors, the subgroup of patients with hormone receptor-negative and nonamplified TOP2A cancers as they have the worst prognosis.

Abstract P4-06-03: TMEPAI a Novel Prognostic Marker for TGF-β Dependency of Metastatic Breast Cancers: Converts Tumor Suppressive TGF-β into a Tumor Promoter

Abstract Background: The dependency of many aggressive and treatment resistant breast cancers on transforming growth factor-beta (TGF-β) for their growth and invasiveness is paradoxical because TGF-β is a classical tumor suppressor that inhibits the growth of normal and early neoplastic breast epithelium. Therefore, we addressed the question how does TGF-β signaling transform from its expected role as a tumor suppressor in early cancer to that of a tumor promoter that facilitates growth and invasion in the late stages? Materials and methods: TMEPAI RNA was quantitated by for qPCR with SYBR green in an Applied Biosystems 7500 Real Time PCR System. pLKO. 1 based lentiviral vector was used to knockdown TMEPAI. Immunoblotting, invasion assays and tumor xenografts were performed using standard methods. Results: TMEPAI expression is observed in ER/PR-negative and HER2-negative breast tumors compared to their matched normal/benign tissues. TMEPAI protein was found to be highly expressed in aggressive and invasive breast cancer cells compared to non-invasive ER-positive and HER2-negative breast cancer cells. Importantly, TGF-β induced late growth spurt in MDA-MB-231 cells and inhibited cell growth in normal mammary epithelial cells (HME). While TGF-β stimulated TMEPAI RNA by ∼40 fold and protein by ∼8-10 fold in MDA-MB-231 cells, it had minimal effect on HME. Both basal expression and TGF-β induction of TMEPAI were completely abolished by SB431542, a pharmacological inhibitor of TGF-β signaling as well as by overexpression of Smad7 or dominant negative TGF-β RI (DN Alk-5). We confirmed TMEPAI as a direct target gene of TGF-β signaling when breast cancer cells MDA-MB-468 and MCF-7 that do not express TMEPAI and are defective in either Smad4 or TGF-β receptor I respectively, by exogenously expressing Smad4 in MDA-MB-468 and Alk-5 in MCF-7 to restore TMEPAI induction by TGF-β. Notably, knockdown of TMEPAI has profound corrective effects on cancer cell growth in vitro and in vivo. These important biological effects were accompanied by increase of cellular levels of the tumor suppressor PTEN and p27kip1, decreased activation of the PI3K signaling mediator Akt, and diminished expression of the oncogenic proteins Hif-1a, and VEGF. Discussion: Our results indicate that increase of TMEPAI protein — further exacerbated by TGF-β — may play a crucial role in TGF-β mediated tumor promotion. We believe that increased expression of TMEPAI by genomic amplification and/or epigenetic events contributes to the cancer phenotype and oncogenic progression through regulation of critical proteins involved in growth, epithelial mesenchymal transition (EMT), and angiogenesis, thereby increasing the aggressiveness of breast carcinomas. Thus, increase of TMEPAI and its induction by TGF-β to pathological levels may be the key lynchpin that explains the conversion of tumor suppressive TGF-β into a tumor promoter and TGF-β dependent cancer cell aggressiveness. Moreover, the data suggest that TMEPAI might serve as a novel prognostic marker for aggressiveness, invasiveness and TGF-β dependency of metastatic breast cancers. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-06-03.

Whole Genome Profiling of Formalin-Fixed, Paraffin-Embedded Tissue Distinguishes HER2-Positive and -Negative Breast Tumors.

Abstract Background: Gene expression profiling is an important tool in prediction, prognosis, and cancer modeling. The DASL® assay (cDNA –mediated Annealing, extension, Selection, and Ligation; Illumina; San Diego, CA) is a gene expression profiling system designed for degraded RNAs such as those derived from formalin-fixed paraffin-embedded (FFPE) tumor samples. FFPE tumor samples are invaluable resources for cancer research, being the most widely available materials for which patient outcomes are known. Purpose: The objective of this study was to evaluate the performance of Illumina's Whole Genome DASL platform (24,526; 24K) at distinguishing HER2-positive (HER2+) from HER2-negative (HER2-) FFPE breast tumors. Methods: We measured the relative gene expression of biological and technical replicates using nine HER2+ and 11 HER2- primary breast tumors from patients that were all node-positive and estrogen receptor-positive. The HER2 status of the tumors was determined using the Dako Hercept Test following the 2007 ASCO/CAP guidelines for HER2 positivity. The non-background corrected probe-expression data were exported from Illumina BeadStudio software and normalized using the software R and the function fastlo. Log2 transformed expression values were used to obtain the relative gene expression per probe. Spearman correlations were calculated between both types of replicates. Unequal-variance t-tests were conducted to test for differences in expression levels between HER2+ and HER2- tumors. An overall ERBB2 gene expression level was calculated by taking the average of the three ERBB2 probes for each tumor across all replicates. To simulate the possible outcome if only one replicate of each tumor was measured for gene expression, differential expression was tested for all possible combinations of single replicate probe expression values. Results: Spearman correlations for technical and biological replicates within tumor samples ranged from 0.9804 – 0.9946, and 0.9822 – 0.9945, respectively. The overall ERBB2 gene expression was significantly lower in HER2- tumors than in HER2+ tumors (p = 0.0000018). The top five most differentially expressed genes (p &amp;lt; 0.0005) included ERBB2, GRB7, C17ORF37, PERLD1, and ASF1B. For each of the three ERBB2 probes, there were 65,536 possible ways to combine the individual probe expression values. The p-values for testing differential expression in each of these 65,536 combinations of HER2+ tumors versus HER2- tumors ranged from 0.00029 – 0.0091 for ILMN_1717902, 0.0000078 – 0.00026 for ILMN_1728761, and 0.000000002 – 0.0000032 for ILMN-2352131. Conclusions: Illumina's Whole Genome Platform provided highly reproducible gene expression values among biological and technical replicates of breast tumors. This platform also demonstrated significant differential ERBB2 gene expression between HER2+ and HER- breast tumors, which correctly classified the breast tumors as HER2+ and HER2-. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6127.

Overexpression of CXCR4 in Primary Tumor of Patients with HER-2 Negative Breast Cancer was Predictive of a Poor Disease-Free Survival: A Validation Study

Although HER-2 negative tumors are thought to be less aggressive than their counterpart, there is a subset that behaves poorly. The molecular mechanism to account for this is unknown. The chemokine receptor CXCR4 is often upregulated in a wide array of cancers. Using a training dataset, we previously reported that high CXCR4 overexpression portends a poor outcome among patients with HER-2 negative breast tumors. This study aims to validate these findings, using our validation dataset. There were 115 patients with stages I-III, HER-2 negative breast cancers who were prospectively accrued and analyzed. CXCR4 levels from primary tumors were detected using Western blots, and results were quantified against 1 microg of HeLa cells. CXCR4 expression was defined as low (<6.6 fold) or high (> or =6.6 fold). Primary endpoint was cancer recurrence. Statistical analysis performed included Spearman correlation, Fisher exact test, Kaplan-Meier survival analysis, Cox proportional hazard ratio model, and log-rank test. There were 13 patients in the high (> or =6.6 fold) and 102 patients in the low CXCR4 group (<6.6 fold). Overall survival (OS) and disease-free survival (DFS) for the cohort was 84 and 71%, respectively. The 5-year OS for the high CXCR4 group was 52% and for the low CXCR4 group was 86% (P = 0.08). The 5-year DFS for the high CXCR4 and low CXCR4 group was 38 and 74%, respectively (P = 0.01). We validated that high CXCR4 overexpression in primary tumors of patients with HER-2 negative tumors portend a poor outcome. These findings should be confirmed with either a prospective clinical trial and/or an external validation study.

Quantifying Cell-Surface Biomarker Expression in Thick Tissues with Ratiometric Three-Dimensional Microscopy

The burgeoning fields of in vivo three-dimensional (3D) microscopy and endomicroscopy, as well as ex vivo tissue cytometry have introduced new challenges for tissue preparation and staining with exogenous molecular contrast agents. These challenges include effective delivery of the agents, and once delivered, distinguishing between bound verses unbound molecular probes. If applied topically, there are additional issues with rinsing off unbound probe, which can be nonuniform and inefficient in thick tissues, thus leading to ambiguous contrast and a large nonspecific background that may obscure molecule-specific staining. Therefore, we have developed a ratiometric 3D microscopy scheme that not only reduces the effects of nonspecific sources of contrast, but also enables quantification of the relative binding affinity of imaging probes to their biomarker targets. Here we demonstrate this ratiometric approach by simultaneously imaging a HER2/neu (erbB2)-targeted monoclonal antibody labeled with one fluorophore and an isotype-matched negative control antibody labeled with another fluorophore. By taking a pixel-by-pixel calibrated ratio between the signals from each fluorescent image channel, accurate quantification of specific versus nonspecific binding affinity is achieved with cultured cells, yielding data that are in agreement with analyses via flow cytometry. We also demonstrate quantitative 3D microscopic imaging of biomarker expression in tissue models and in thick human biopsy samples of normal, HER2-negative, and HER2-positive breast tumors. This strategy enables rapid, quantitative, and unambiguous volumetric microscopy of biomarker expression in thick tissues, including whole biopsies, and will enable real-time optical assessment of disease markers in the living body.