Susan M. Edgerton

Association of Vascular Endothelial Growth Factor and Vascular Endothelial Growth Factor Receptor-2 Genetic Polymorphisms With Outcome in a Trial of Paclitaxel Compared With Paclitaxel Plus Bevacizumab in Advanced Breast Cancer: ECOG 2100

PURPOSE No biomarkers have been identified to predict outcome with the use of an antiangiogenesis agent for cancer. Vascular endothelial growth factor (VEGF) genetic variability has been associated with altered risk of breast cancer and variable promoter activity. Therefore, we evaluated the association of VEGF genotype with efficacy and toxicity in E2100, a phase III study comparing paclitaxel versus paclitaxel plus bevacizumab as initial chemotherapy for metastatic breast cancer. PATIENTS AND METHODS DNA was extracted from tumor blocks of patients from E2100. Three hundred sixty-three samples were available to evaluate associations between genotype and outcome. Genotyping was performed for selected polymorphisms in VEGF and VEGF receptor 2. Testing for associations between each polymorphism and efficacy and toxicity was performed. RESULTS The VEGF-2578 AA genotype was associated with a superior median overall survival (OS) in the combination arm when compared with the alternate genotypes combined (hazard ratio = 0.58; 95% CI, 0.36 to 0.93; P = .023). The VEGF-1154 A allele also demonstrated a superior median OS with an additive effect of each active allele in the combination arm but not the control arm (hazard ratio = 0.62; 95% CI, 0.46 to 0.83; P = .001). Two additional genotypes, VEGF-634 CC and VEGF-1498 TT, were associated with significantly less grade 3 or 4 hypertension in the combination arm when compared with the alternate genotypes combined (P = .005 and P = .022, respectively). CONCLUSION Our data support an association between VEGF genotype and median OS as well as grade 3 or 4 hypertension when using bevacizumab in metastatic breast cancer.

Metformin inhibits breast cancer cell growth, colony formation and induces cell cycle arrest in vitro

The anti-diabetic drug metformin reduces human cancer incidence and improves the survival of cancer patients, including those with breast cancer. We studied the activity of metformin against diverse molecular subtypes of breast cancer cell lines in vitro. Metformin showed biological activity against all estrogen receptor (ER) positive and negative, erbB2 normal and abnormal breast cancer cell lines tested. It inhibited cellular proliferation, reduced colony formation and caused partial cell cycle arrest at the G1 checkpoint. Metformin did not induce apoptosis (as measured by DNA fragmentation and PARP cleavage) in luminal A, B or erbB2 subtype breast cancer cell lines. At the molecular level, metformin treatment was associated with a reduction of cyclin D1 and E2F1 expression with no changes in p27kip1 or p21waf1. It inhibited mitogen activated protein kinase (MAPK) and Akt activity, as well as the mammalian target of rapamycin (mTOR) in both ER positive and negative, erbB2-overexpressing and erbB2-normal expressing breast cancer cells. In erbB2-overexpressing breast cancer cell lines, metformin reduced erbB2 expression at higher concentrations, and at lower concentrations within the therapeutic range, it inhibited erbB2 tyrosine kinase activity evidenced by a reduction of phosphorylated erbB2 (P-erbB2) at both auto- and Src- phosphorylation sites. These data suggest that metformin may have potential therapeutic utility against ER positive and negative, erbB2-overexpressing and erbB2-normal expressing breast cancer cells.

Metformin induces unique biological and molecular responses in triple negative breast cancer cells

Triple negative (TN) breast cancer is more frequent in women who are obese or have type II diabetes, as well as young Women of Color. These cancers do not express receptors for the steroid hormones estrogen or progesterone, or the type II receptor tyrosine kinase (RTK) Her-2 but do have upregulation of basal cytokeratins and the epidermal growth factor (EGFR). These data suggest that aberrations of glucose and fatty acid metabolism, signaling through EGFR and genetic factors may promote the development of TN cancers. The anti-type II diabetes drug metformin has been associated with a decreased incidence of breast cancer, although the specific molecular subtypes that may be reduced by metformin have not been reported. Our data indicates that metformin has unique anti-TN breast cancer effects both in vitro and in vivo. It inhibits cell proliferation (with partial S phase arrest), colony formation and induces apoptosis via activation of the intrinsic and extrinsic signaling pathways only in TN breast cancer cell lines. At the molecular level, metformin increases P-AMPK, reduces P-EGFR, EGFR, P-MAPK, P-Src, cyclin D1 and cyclin E (but not cyclin A or B, p27 or p21), and induces PARP cleavage in a dose- and time-dependent manner. These data are in stark contrast to our previously published biological and molecular effects of metformin on luminal A and B, or Her-2 type breast cancer cells. Nude mice bearing tumor xenografts of the TN line MDA-MB-231, treated with metformin, show significant reductions in tumor growth (p=0.0066) and cell proliferation (p=0.0021) as compared to untreated controls. Metformin pre-treatment, before injection of MDA-MB-231 cells, results in a significant decrease in tumor outgrowth and latency. Given the unique anti-cancer activity of metformin against TN disease, both in vitro and in vivo, it should be explored as a therapeutic agent against this aggressive form of breast cancer.

Relationship of Epidermal Growth Factor Receptor Expression to ErbB-2 Signaling Activity and Prognosis in Breast Cancer Patients

PURPOSE To examine the relationship of epidermal growth factor receptor (EGFR) expression to ErbB-2 signaling activity in breast cancer and the impact that this interaction has on the prognosis of patients with early-stage breast cancer. PATIENTS AND METHODS Paraffin tumor sections were collected retrospectively from 807 breast cancer patients diagnosed between 1976 and 1983. Immunohistochemical assays for ErbB-2, phosphorylated (activated) ErbB-2, and EGFR were performed, and the results were correlated with clinicopathologic variables and outcome. RESULTS EGFR expression was detectable in 15% of 807 invasive breast cancers, including 35% of the 306 ErbB-2-positive patients. Conversely, the majority (87%) of EGFR-positive tumors co-overexpressed ErbB-2. Ninety-seven percent of tumors with phosphorylated ErbB-2 co-overexpressed EGFR. Patients whose cancers demonstrated ErbB-2 phosphorylation or co-overexpression of ErbB-2 and EGFR had the shortest survival. In contrast, patients whose tumors were negative for all three markers and those tumors that expressed only EGFR or only nonphosphorylated ErbB-2 had a relatively favorable outcome. CONCLUSION These data provide the first clinical evidence that EGFR expression is linked to activation of ErbB-2 in human breast cancers. We have further shown that the adverse prognostic value of ErbB-2 overexpression is observed only when ErbB-2 is in the phosphorylated (activated) state or coexpressed with EGFR. These data suggest that ligand-dependent mechanisms of ErbB-2 activation are important in human breast cancer. These results also suggest that agents targeting EGFR may be useful in the treatment of tumors with activated ErbB-2.

Activation (Tyrosine Phosphorylation) of ErbB-2 (HER-2/neu): A Study of Incidence and Correlation With Outcome in Breast Cancer

PURPOSE We hypothesize that phosphorylated ErbB-2 (P-ErbB-2, identified by a novel antibody PN2A) may provide either more significant or additional prognostic marker data for breast cancer patients. This study was designed to compare the incidence and prognostic value of ErbB-2 (HER-2/neu) and P-ErbB-2 immunoexpression in archival breast cancer samples. MATERIALS AND METHODS Eight hundred sixteen invasive breast cancers with a median of 16.3 years of follow-up were immunostained for ErbB-2 (using antibody CB11) and P-ErbB-2 (using antibody PN2A). ErbB-2 and P-ErbB-2 data were compared with clinical, histologic, immunohistochemical, and outcome variables. RESULTS Of 816 primary breast cancers, 307 (38%) were positive for ErbB-2 and 37 (12% of ErbB-2 positive and 5% of the study population) expressed P-ErbB-2. P-ErbB-2 was not detected in ErbB-2-negative cases (n = 509). ErbB-2 immunohistochemical data were bimodal; patients with > or = 80% cellular expression had the shortest disease-free and disease-specific survival. P-ErbB-2 was associated with a higher percentage of ErbB-2-positive cells, a higher number of positive lymph nodes, and cellular proliferation. ErbB-2 and P-ErbB-2 were indicators of poor prognosis in node-positive patients in both univariate and multivariate analyses. We found that either P-ErbB-2 expression or high (> or = 80%) ErbB-2 expression provided the most significant prognostic value in node-positive cases by multivariate analyses. There were too few P-ErbB-2-positive cases and events in the node-negative patient group to allow statistical analysis of P-ErbB-2 in that subgroup. CONCLUSION PN2A immunostaining identified a subset (approximately 12% of ErbB-2-positive breast cancers) with activation (phosphorylation) of the receptor ErbB-2. P-ErbB-2 expression was strongly associated with higher levels of ErbB-2 expression (> or = 80%), although it was not a surrogate. Identification of cases with a high percentage of invasive breast cancer cells expressing ErbB-2 or determination of receptor activation via P-ErbB-2 may provide additional prognostic value in node-positive breast cancers.

HER2/Neu and the Ets transcription activator PEA3 are coordinately upregulated in human breast cancer.

HER2/Neu is overexpressed in 25 – 30% of all human breast cancers as a result of both gene amplification and enhanced transcription. Transcriptional upregulation of HER2/neu leads to a 6 – 8-fold increased abundance of its mRNA per gene copy and likely results from the elevated activity of transcription factors acting on the HER2/neu promoter. Here we report that transcripts of PEA3, an ETS transcription factor implicated in oncogenesis, were increased in 93% of HER2/Neu-overexpressing human breast tumor samples. Analyses to uncover the molecular basis for elevated PEA3 transcripts in HER2/Neu-positive breast tumors revealed that the HER2/Neu receptor tyrosine kinase initiated an intracellular signaling cascade resulting in increased PEA3 transcriptional activity; transcriptionally-activated PEA3 stimulated HER2/neu and PEA3 gene transcription by binding to sites in the promoters of these genes. PEA3 also activates transcription of genes encoding matrix-degrading proteinases, enzymes required for tumor cell migration and invasion. These findings implicate PEA3 in the initiation and progression of HER2/Neu positive breast cancer, and suggest that PEA3 and signaling proteins affecting its regulation are appropriate therapeutic targets.

Role of the androgen receptor in breast cancer and preclinical analysis of enzalutamide

IntroductionThe androgen receptor (AR) is widely expressed in breast cancers and has been proposed as a therapeutic target in estrogen receptor alpha (ER) negative breast cancers that retain AR. However, controversy exists regarding the role of AR, particularly in ER + tumors. Enzalutamide, an AR inhibitor that impairs nuclear localization of AR, was used to elucidate the role of AR in preclinical models of ER positive and negative breast cancer.MethodsWe examined nuclear AR to ER protein ratios in primary breast cancers in relation to response to endocrine therapy. The effects of AR inhibition with enzalutamide were examined in vitro and in preclinical models of ER positive and negative breast cancer that express AR.ResultsIn a cohort of 192 women with ER + breast cancers, a high ratio of AR:ER (≥2.0) indicated an over four fold increased risk for failure while on tamoxifen (HR = 4.43). The AR:ER ratio had an independent effect on risk for failure above ER % staining alone. AR:ER ratio is also an independent predictor of disease-free survival (HR = 4.04, 95% CI: 1.68, 9.69; p = 0.002) and disease specific survival (HR = 2.75, 95% CI: 1.11, 6.86; p = 0.03). Both enzalutamide and bicalutamide inhibited 5-alpha-dihydrotestosterone (DHT)-mediated proliferation of breast cancer lines in vitro; however, enzalutamide uniquely inhibited estradiol (E2)-mediated proliferation of ER+/AR + breast cancer cells. In MCF7 xenografts (ER+/AR+) enzalutamide inhibited E2-driven tumor growth as effectively as tamoxifen by decreasing proliferation. Enzalutamide also inhibited DHT- driven tumor growth in both ER positive (MCF7) and negative (MDA-MB-453) xenografts, but did so by increasing apoptosis.ConclusionsAR to ER ratio may influence breast cancer response to traditional endocrine therapy. Enzalutamide elicits different effects on E2-mediated breast cancer cell proliferation than bicalutamide. This preclinical study supports the initiation of clinical studies evaluating enzalutamide for treatment of AR+ tumors regardless of ER status, since it blocks both androgen- and estrogen- mediated tumor growth.

Invasive micropapillary carcinoma of the breast: A prognostic study☆

Invasive micropapillary carcinoma (IMC) of the breast is a rare variant of infiltrating ductal carcinoma that has been associated with an extremely high incidence of lymph node metastases. Follow-up studies on patients with pure IMC breast cancer histology have been limited by low patient numbers, short duration of follow-up, and a lack of multivariate analyses. Using invasive breast cancers from 1,287 patients (median follow-up, 13.8 years), histological review showed 21 cases (1.7%) with pure IMC histology. Pure IMC histology was associated with high-grade histology (P = .04), metastases to regional lymph nodes (P < .001), a high mitotic index (P = .02), and erbB-2 immunopositivity (P = .007). Univariate analyses showed a strong association between IMC histology and shortened survival (disease-free survival [DFS], P = .0052; median, 44 months for IMC and 63 months for non-IMC; disease-specific survival [DSS], P = .014; medians, 71 and 78 for IMC and non-IMC, respectively) only in an analysis of all patients. Because only 1 case of node-negative IMC histology was available, univariate analysis of IMC histology was performed only on node-positive patients without significance. Multivariate analyses comparing IMC histology with either node-positive or all other breast cancers failed to show independent prognostic significance. In summary, breast cancer patients with pure IMC histology showed survival rates similar to those of other patients with equivalent numbers of lymph node metastases.

Comparison of Mitotic Index, In Vitro Bromodeoxyuridine Labeling, and MIB-1 Assays to Quantitate Proliferation in Breast Cancer

PURPOSE To investigate the hypothesis that in vitro bromodeoxyuridine (BrDu) labeling might be superior to MIB-1 immunostaining for prognostic value, because it more selectively labels cells during the S phase. METHODS Four hundred eighty-six patients with breast cancers (59% lymph node-negative, 41% lymph node-positive) surgically excised between 1988 and 1993 (median follow-up, 62 months) were evaluated for cellular proliferation using prospective in vitro BrDu uptake assays, retrospective mitotic indices, and MIB-1 labeling. RESULTS MIB-1, BrDu labeling, and mitotic index-derived proliferation data were highly correlated. Each was similarly associated with most other markers of prognosis, although these relationships were not identical. By univariate analysis, nodal status was the most significant prognostic variable for all patients. Higher BrDu labeling index, MIB-1 immunolabeling, and mitotic index were also associated with shortened disease-free survival (DFS) and disease-specific survival for the entire patient group, as well as for node-negative patients. The association between cellular proliferation and survival was much weaker for node-positive patients. Multivariate models confirmed that nodal status, tumor size, and proliferation data predicted survival in all patients as well as those with node-negative disease, although MIB-1 was somewhat more closely associated with outcome than mitotic index or in vitro BrDu data. For patients with T1NOMO disease (n = 172), the only significant predictors of DFS were proliferation rate (mitotic index or MIB-1) and tumor grade. CONCLUSIONS Proliferation rate predicts recurrence and survival in breast cancer. This effect is more pronounced in node-negative patients. In vitro BrDu data are not superior to MIB-1 and mitotic counting.

Downregulation of erbB3 abrogates erbB2-mediated tamoxifen resistance in breast cancer cells.

Receptor tyrosine kinase activity is essential for erbB2 (HER2/neu) promotion of breast carcinogenesis, metastasis and therapeutic resistance. erbB2 kinase can be activated by dimerization with another erbB receptor, most of which bind ligands. Of these, the erbB2/erbB3 heterodimer is the most potent oncogenic complex. erbB2 reportedly requires erbB3 to promote cellular proliferation, although this may occur without changes in erbB2 tyrosine kinase activity in some model systems. Our investigations focus on the role(s) of erbB3 in erbB2‐associated kinase activity and tamoxifen resistance. Using tumor‐derived cell lines from wild type rat c‐neu transgenic mice and human breast cancers, we demonstrate that erbB3 plays a critical role in the activation of erbB2 tyrosine kinase activity and erbB2‐associated tumorigenesis. Mechanistically, downregulation of erbB3 by specific siRNA reduces erbB2 tyrosine phosphorylation, decreases the PI‐3K/Akt signaling, and inhibits mammary/breast cancer cell proliferation and colony formation. Specific erbB3 siRNA sensitizes erbB2 transfected MCF‐7 cells (MCF‐7/erbB2) to tamoxifen‐associated inhibition of both cell growth and colony formation and enhances tamoxifen‐induced apoptosis, in contrast to control siRNA transfected MCF‐7/erbB2 cells which are tamoxifen‐resistant. Our data indicates that erbB2/erbB3 heterodimerization is a prerequisite for erbB2 tyrosine kinase activation in mammary/breast cancer cells and that downregulation of erbB3 inhibits erbB2‐associated procarcinogenic activity via inactivation of the PI‐3K/Akt pathway. Furthermore, erbB3 also contributes to erbB2‐mediated tamoxifen resistance and therefore may be a clinically relevant therapeutic target in addition to erbB2.