Malgorzata Beryt

Inhibitory effects of combinations of HER-2/ neu antibody and chemotherapeutic agents used for treatment of human breast cancers

Previous studies have demonstrated a synergistic interaction between rhuMAb HER2 and the cytotoxic drug cisplatin in human breast and ovarian cancer cells. To define the nature of the interaction between rhuMAb HER2 and other classes of cytotoxic drugs, we applied multiple drug effect/combination index (CI) isobologram analysis to a variety of chemotherapeutic drug/rhuMAb HER2 combinations in vitro. Synergistic interactions at clinically relevant drug concentrations were observed for rhuMAb HER2 in combination with cisplatin (CI=0.48, P=0.003), thiotepa (CI=0.67, P=0.0008), and etoposide (CI=0.54, P=0.0003). Additive cytotoxic effects were observed with rhuMAb HER2 plus doxorubicin (CI=1.16, P=0.13), paclitaxel (CI=0.91, P=0.21), methotrexate (CI=1.15, P=0.28), and vinblastine (CI=1.09, P=0.26). One drug, 5-fluorouracil, was found to be antagonistic with rhuMAb HER2 in vitro (CI=2.87, P=0.0001). In vivo drug/rhuMAb HER2 studies were conducted with HER-2/neu-transfected, MCF7 human breast cancer xenografts in athymic mice. Combinations of rhuMAb HER2 plus cyclophosphamide, doxorubicin, paclitaxel, methotrexate, etoposide, and vinblastine in vivo resulted in a significant reduction in xenograft volume compared to chemotherapy alone (P<0.05). Xenografts treated with rhuMAb HER2 plus 5-fluorouracil were not significantly different from 5-fluorouracil alone controls consistent with the subadditive effects observed with this combination in vitro. The synergistic interaction of rhuMAb HER2 with alkylating agents, platinum analogs and topoisomerase II inhibitors, as well as the additive interaction with taxanes, anthracyclines and some antimetabolites in HER-2/neu-overexpressing breast cancer cells demonstrates that these are rational combinations to test in human clinical trials.

The effect of HER-2/neu overexpression on chemotherapeutic drug sensitivity in human breast and ovarian cancer cells

Recent studies indicate that oncogenes may be involved in determining the sensitivity of human cancers to chemotherapeutic agents. To define the effect of HER-2/neu oncogene overexpression on sensitivity to chemotherapeutic drugs, a full-length, human HER-2/neu cDNA was introduced into human breast and ovarian cancer cells. In vitro dose-response curves following exposure to 7 different classes of chemotherapeutic agents were compared for HER-2- and control-transfected cells. Chemosensitivity was also tested in vivo for HER-2- and control-transfected human breast and ovarian cancer xenografts in athymic mice. These studies indicate that HER-2/neu overexpression was not sufficient to induce intrinsic, pleomorphic drug resistance. Furthermore, changes in chemosensitivity profiles resulting from HER-2/neu transfection observed in vitro were cell line specific. In vivo, HER-2/neu-overexpressing breast and ovarian cancer xenografts were responsive to different classes of chemotherapeutic drugs compared to control-treated xenografts with no statistically significant differences between HER-2/neu-overexpressing and non-overexpressing xenografts. We found no instance in which HER-2/neu-overexpressing xenografts were rendered more sensitive to chemotherapeutic drugs in vivo. HER-2/neu-overexpressing xenografts consistently exhibited more rapid regrowth than control xenografts following initial response to chemotherapy suggesting that a high rate of tumor cell proliferation rather than intrinsic drug resistance may be responsible for the adverse prognosis associated with HER-2/neu overexpression in human cancers.

Association between HER-2/neu and Vascular Endothelial Growth Factor Expression Predicts Clinical Outcome in Primary Breast Cancer Patients

Purpose: Activation or overexpression of HER-2/neu is associated with up-regulation of vascular endothelial growth factor (VEGF) in human breast cancer cells in vitro. Preclinical experiments indicate that increased expression of VEGF may in part mediate the biologically aggressive phenotype of HER-2/neu-overexpressing human breast cancer. It was the purpose of this study to: (a) evaluate the association between HER-2/neu and VEGF expression in a large clinical cohort of primary breast cancer patients; (b) compare the prognostic significance of VEGF isoforms; and (c) analyze the combined effects of HER-2/neu and VEGF on clinical outcome. Experimental Design: HER-2/neu and VEGF were measured by ELISA in primary breast tumor tissue lysates from 611 unselected patients with a median clinical follow-up of 50 months. At least six VEGF isoforms consisting of 121, 145, 165, 183, 189, or 206 amino acids are generated as a result of alternative splicing. The VEGF121–206 ELISA uses antibodies that bind to VEGF121 and, therefore, detects all of the VEGF isoforms with 121 and more amino acids. The VEGF165–206 ELISA uses antibodies that bind to VEGF165 and, therefore, detects all of the VEGF isoforms with 165 and more amino acids. VEGF121–206 and VEGF165–206 were analyzed both as continuous and categorical variables, using detectable expression as a cutoff for positivity. Cell lines with defined HER-2/neu expression levels were used to establish a cutoff point for HER-2/neu overexpression in breast tumor samples. Results: Our findings indicate a significant positive association between HER-2/neu and VEGF expression. VEGF121–206 and VEGF165–206 expression was detectable in 88 (77.2%) and 100 (87.7%), respectively, of the 114 patients with HER-2/neu-overexpressing tumors, in contrast to 271 (54.5%) and 353 (71.0%), respectively, of the 497 patients with nonoverexpressing tumors (χ2 test: P < 0.001 for both VEGF121–206 and VEGF165–206). VEGF121–206 and VEGF165–206 demonstrate a comparable prognostic significance for survival in unselected primary breast cancer patients (univariate analysis: VEGF121–206, P = 0.0068; VEGF165–206, P = 0.0046; multivariate analysis: VEGF121–206, P = 0.1475; VEGF165–206, P = 0.1483). When the analyses were performed separately for node-negative and node-positive patients, VEGF121–206 and VEGF165–206 were of prognostic significance for survival only in node-positive patients (univariate analysis: VEGF121–206, P = 0.0003; VEGF165–206, P = 0.0038; multivariate analysis: VEGF121–206, P = 0.0103; VEGF165–206, P = 0.0150). A biological concentration-effect relationship between VEGF expression and survival (VEGF121–206, P = 0.0280; VEGF165–206, P = 0.0097) suggests that VEGF levels, as determined by ELISA, could be of importance as a predictive marker for therapeutic strategies that target VEGF. Combining HER-2/neu and VEGF121–206/VEGF165–206 results in additional prognostic information for survival (VEGF121–206, P = 0.0133; VEGF165–206, P = 0.0092). Conclusion: The positive association between HER-2/neu and VEGF expression implicates VEGF in the aggressive phenotype exhibited by HER-2/neu overexpression, and supports the use of combination therapies directed against both HER-2/neu and VEGF for treatment of breast cancers that overexpress HER-2/neu.

Human epidermal growth factor receptor (HER-1:HER-3) Fc-mediated heterodimer has broad antiproliferative activity in vitro and in human tumor xenografts

All four members of the human epidermal growth factor (EGF) receptor (HER) family are implicated in human cancers. Although efficacious in a subset of patients, resistance to single-targeted anti-HER therapy [i.e., cetuximab (Erbitux) and trastuzumab (Herceptin)] is often associated with coexpression of other HER family members. This may be overcome by a HER ligand binding molecule that sequesters multiple EGF-like ligands, preventing ligand-dependent receptor activation. Toward this end, we have combined the HER-1/EGFR and HER-3 ligand binding domains, dimerized with fusion of an Fc fragment of human IgG1. This resulted in a mixture of HER-1/Fc homodimer (HFD100), HER-3/Fc homodimer (HFD300), and HER-1/Fc:HER-3/Fc heterodimer (RB200), also termed Hermodulins. The purified first-generation RB200 bound EGF and neuregulin 1 (NRG1)-β1 ligands, determined by cross-linking and direct binding studies. The binding affinity for both was ∼10 nmol/L by dissociation-enhanced lanthanide fluorescence immunoassay using europium (Eu)-labeled ligands. Competition studies with RB200 using Eu-EGF or Eu-NRG1-β1 revealed that RB200 bound HER-1 ligands, including transforming growth factor-α and heparin-binding EGF, and HER-3 ligands NRG1-α and NRG1-β3. RB200 inhibited EGF- and NRG1-β1–stimulated tyrosine phosphorylation of HER family proteins, proliferation of a diverse range of tumor cells in monolayer cell growth assays, tumor cell proliferation as a single agent and in synergy with tyrosine kinase inhibitors, lysophosphatidic acid–stimulated cell proliferation, and tumor growth in two human tumor xenograft nude mouse models. Taken together, the data reveal that RB200 has the potential to sequester multiple HER ligands and interfere with signaling by HER-1, HER-2, and HER-3. [Mol Cancer Ther 2008;7(10):3223–36]

Drug interactions and cytotoxic effects of paclitaxel in combination with carboplatin, epirubicin, gemcitabine or vinorelbine in breast cancer cell lines and tumor samples.

The purpose of this study was to analyze the drug interactions of paclitaxel (PTX) with epirubicin (EPI), carboplatin (CBDCA), gemcitabine (GEM) and vinorelbine (VIN) in human breast cancer cells and compare the cytotoxic activity of each drug combination in primary breast cancer samples. These experiments were intended to identify the most active agents in combination with PTX, and to provide a preclinical rational for future clinical investigations in breast cancer. Multiple drug effect/combination index (CI) isobologram analysis was applied to combinations of PTX with either CBDCA, EPI, GEM or VIN in MCF-7, MDA-MB-231 and SK-BR-3 human breast cancer cell lines. Drug concentrations were limited to the ranges achievable in humans in vivo, and the drugs were applied simultaneously at fixed molar ratios for each drug combination. Interactions were assessed at multiple effect levels (IC10–IC90). Additionally, the cytotoxic activity of these combinations was assessed in tumor samples of 50 primary breast cancer patients, utilizing the ATP-tumorchemosensitivity assay (ATP-TCA). Drug interactions were shown to be strongly dose-related in the human breast cancer cell lines investigated. At clinically relevant concentrations, CBDCA/PTX demonstrated synergistic (MCF-7) or additive (MDA-MB-231, SK-BR-3) interactions, and EPI/PTX showed additive (SK-BR-3, MCF-7) and antagonistic (MDA-MB-231) interactions. GEM/PTX and VIN/PTX, however, demonstrated antagonism over multiple dose effect levels at clinically relevant drug concentrations in all three cell lines tested. At plasma peak concentrations, EPI/PTX, CBDCA/PTX, GEM/PTX and VIN/PTX achieved ≥90% tumor growth inhibition in 93, 86, 63 and 50%, respectively, of primary breast cancer samples investigated with the ATP-TCA. Cumulative dose-response plots of primary breast cancer tumor cells responding in vitro with ≥90% growth inhibition showed a strong dose dependence for both EPI/PTX and CBDCA/PTX. In conclusion, the current data indicate favorable drug interactions for CBDCA/PTX at clinically relevant drug concentrations in breast cancer cells, and demonstrate superior in vitro cytotoxicity of EPI/PTX and CBDCA/PTX compared to GEM/PTX and VIN/PTX in primary breast cancer cultures.

HER-2/neu overexpression and in vitro chemosensitivity to CMF and FEC in primary breast cancer.

Available clinical and experimental data on the effect of HER-2/neu overexpression on chemosensitivity are controversial. It was the purpose of this in vitro study to define the association between HER-2/neu overexpression and the sensitivity to the chemotherapeutic drug combinations of cyclophosphamide, methotrexate and 5-fluorouracil (CMF) and 5-fluorouracil, epirubicin and cyclophosphamide (FEC) of breast cancer cells derived from 140 chemotherapy-naïve patients at the time of primary surgery. Both drug combinations were tested at six different concentrations ranging from 6.25–200% peak plasma concentration (PPC). Immunohistochemical detection of HER-2/neu overexpression was performed with the HER-2/neu antibodies, CB11, TAB250 and AO485, in the same tumor specimens. Immunoreactions were determined as negative (0/1+), weakly positive (2+) and strongly positive (3+). However, the antibodies varied in their degrees of sensitivity. Breast cancer samples with strong (3+) HER-2/neu overexpression demonstrated 90% growth inhibition (IC90) at significantly lower PPC values, using the CB11 (p=0.048), TAB250 (p=0.007) and AO485 (p≤0.01) antibodies, and showed 50% growth inhibition (IC50) at significantly lower PPC values, using the CB11 antibody (p=0.01) compared to their counterparts with lower levels of HER-2/neu expression. When analyzing the group of patients with intermediate and strong HER-2/neu overexpression (2+ and 3+), an association between HER-2/neu overexpression and increased chemosensitivity was seen with the TAB250 (p=0.044) and AO485 (p=0.032) antibodies, but not with the CB11 antibody (p=0.8) at the IC90 level. Differences in chemosensitivity between samples with strong HER-2/neu overexpression and those with lower levels were then analyzed separately for CMF and FEC. Both regimens achieved 90% tumor growth inhibition at lower PPC values in samples with strong HER-2/neu overexpression (3+) compared to their counterparts with lower expression levels (AO485 p=0.011 for CMF, and p=0.09 for FEC). Cumulative concentration-response plots of tumors responding in vitro with 90% tumor cell inhibition showed a stronger dose dependence for both CMF and FEC among tumor samples with strong HER-2/neu overexpression compared to those with lower levels of expression. In conclusion, the data show that HER-2/neu overexpression was not associated with in vitro drug resistance to CMF or FEC. In contrast, tumors with strong HER-2/neu overexpression demonstrated increased dose-dependent in vitro sensitivity to both the FEC and CMF regimens.