HER2 testing in breast cancer: Opportunities and challenges

Human epidermal growth factor receptor 2 (HER2) is overexpressed in 15-25% of breast cancers, usually as a result of HER2 gene amplification. Positive HER2 status is considered to be an adverse prognostic factor. Recognition of the role of HER2 in breast cancer growth has led to the development of anti-HER2 directed therapy, with the humanized monoclonal antibody trastuzumab (Herceptin (R)) having been approved for the therapy of HER2-positive metastatic breast cancer. Clinical studies have further suggested that HER2 status can provide important information regarding success or failure of certain hormonal therapies or chemotherapies. As a result of these developments, there has been increasing demand to perform HER2 testing on current and archived breast cancer specimens. This article reviews the molecular background of HER2 function, activation and inhibition as well as current opinions concerning its role in chemosensitivity and interaction with estrogen receptor biology. The different tissue-based assays used to detect HER2 amplification and overexpression are discussed with respect to their advantages and disadvantages, when to test (at initial diagnosis or pre-treatment), where to test (locally or centralized) and the need for quality assurance to ensure accurate and valid testing results

The role of Herceptin in early breast cancer

Herceptin is widely regarded as the most important development in the treatment of breast cancer since Tamoxifen and the development of the multidisciplinary team (MDT). It is particularly exciting from an oncological polint of view as it represents success in the emerging field of specific targeted therapies to specific molecular abnormalities in tumour cells. This review will focus on the nature of the Her2 overexpression and the role of herceptin in the treatment of early breast cancer

Dose escalation and pharmacokinetic study of a humanized anti-HER2 monoclonal antibody in patients with HER2/neu-overexpressing metastatic breast cancer

We conducted a phase I pharmacokinetic dose escalation study of a recombinant humanized anti-p185HER2 monoclonal antibody (MKC-454) in 18 patients with metastatic breast cancer refractory to chemotherapy. Three or six patients at each dose level received 1, 2, 4 and 8 mg kg–1 of MKC-454 as 90-min intravenous infusions. The first dose was followed in 3 weeks by nine weekly doses. Target trough serum concentration has been set at 10 μg ml–1 based on in vitro observations. The mean value of minimum trough serum concentrations at each dose level were 3.58 ± 0.63, 6.53 ± 5.26, 40.2 ± 7.12 and 87.9 ± 23.5 μg ml–1 respectively. At 2 mg kg–1, although minimum trough serum concentrations were lower than the target trough concentration with a wide range of variation, trough concentrations increased and exceeded the target concentration, as administrations were repeated weekly. Finally 2 mg kg–1 was considered to be sufficient to achieve the target trough concentration by the weekly dosing regimen. One patient receiving 1 mg kg–1 had grade 3 fever, one at the 1 mg kg–1 level had severe fatigue defined as grade 3, and one at 8 mg kg–1 had severe bone pain of grade 3. No antibodies against MKC-454 were detected in any patients. Objective tumour responses were observed in two patients; one receiving 4 mg kg–1 had a partial response in lung metastases and the other receiving 8 mg kg–1 had a complete response in soft tissue metastases. These results indicate that MKC-454 is well tolerated and effective in patients with refractory metastatic breast cancers overexpressing the HER2 proto-oncogene. Further evaluation of this agent with 2–4 mg kg–1 weekly intravenous infusion is warranted. © 1999 Cancer Research Campaig

Suppression of the c-erbB-2 gene product decreases transformation abilities but not the proliferation and secretion of proteases of SK-OV-3 ovarian cancer cells

The overexpression of the c-erbB-2 oncogene product has been reported in approximately 20–30% of human ovarian cancers and has been correlated with a poor prognosis in ovarian cancer patients. To investigate the function of p185c-erbB-2 in human ovarian cancer cells, a c-erbB-2-specific single-chain antibody (scFv-5R) was expressed in the c-erbB-2-overexpressing SK-OV-3 cell line using a retroviral expression vector. Eight individual clones expressing the single-chain antibody were isolated. These clones have a prominent retention of the cell surface p185c-erbB-2. In this study we compared the proliferation rate, the anchorage-independent growth, the secretion of matrix metalloproteases and of the urokinase-type plasminogen activator. The clones expressing the c-erbB-2 single-chain antibody, the control cells harbouring the empty vector and the parental SK-OV-3 cells they all had similar proliferation rates in the presence of 10% serum and secreted similar amounts of matrix metalloproteases and of the urokinase-type plasminogen activator. However, the expression of the c-erbB-2 oncogene product offers a strong growth advantage under serum-reduced conditions with 1% serum. In contrast to the parental SK-OV-3 and empty vector control cells, the scFv-5R-expressing clones were not able to grow anchorage-independently. These findings suggest that c-erbB-2 enhances transformation abilities of SK-OV-3 ovarian cancer cells without affecting the secretion of proteases and the proliferation of SK-OV-3 ovarian cancer cells in the presence of high concentrations of serum. © 1999 Cancer Research Campaig

Upregulated HSP27 in human breast cancer cells reduces Herceptin susceptibility by increasing Her2 protein stability

<p>Abstract</p> <p>Background</p> <p>Elucidating the molecular mechanisms by which tumors become resistant to Herceptin is critical for the treatment of Her2-overexpressed metastatic breast cancer.</p> <p>Methods</p> <p>To further understand Herceptin resistance mechanisms at the molecular level, we used comparative proteome approaches to analyze two human breast cancer cell lines; Her2-positive SK-BR-3 cells and its Herceptin-resistant SK-BR-3 (SK-BR-3 HR) cells.</p> <p>Results</p> <p>Heat-shock protein 27 (HSP27) expression was shown to be upregulated in SK-BR-3 HR cells. Suppression of HSP27 by specific siRNA transfection increased the susceptibility of SK-BR-3 HR cells to Herceptin. In the presence of Herceptin, Her2 was downregulated in both cell lines. However, Her2 expression was reduced by a greater amount in SK-BR-3 parent cells than in SK-BR-3 HR cells. Interestingly, co-immunoprecipitation analysis showed that HSP27 can bind to Her2. In the absence of Herceptin, HSP27 expression is suppressed and Her2 expression is reduced, indicating that downregulation of Her2 by Herceptin can be obstructed by the formation of a Her2-HSP27 complex.</p> <p>Conclusion</p> <p>Our present study demonstrates that upregulated HSP27 in human breast cancer cells can reduce Herceptin susceptibility by increasing Her2 protein stability.</p

Combination treatment with Grb7 peptide and Doxorubicin or Trastuzumab (Herceptin) results in cooperative cell growth inhibition in breast cancer cells

Grb7 has potential importance in the progression of cancer. We have previously identified a novel peptide that binds to the SH2 domain of Grb7 and inhibits its association with several different receptor tyrosine kinases. We have synthesised the Grb7 peptide, G7-18NATE, with two different cell penetrating peptides, Penetratin and Tat. In this study, we have shown that both Penetratin- and Tat-conjugated G7-18NATE peptides are able to inhibit the proliferation of SK-BR-3, ZR-75-30, MDA-MB-361 and MDA-MB-231 breast cancer cells. There was no significant effects on breast cancer MCF-7cells, non-malignant MCF 10A or 3T3 cells. In addition, there was no significant inhibition of proliferation by Penetratin or Tat alone or by their conjugates with arbitrary peptide sequence in any of the cell lines tested. We determined the EC50 of G7-18NATE-P peptide for SK-BR-3 cell proliferation to be 7.663 × 10−6 M. Co-treatment of G7-18NATE-P peptide plus Doxorubicin in SK-BR-3 breast cancer cells resulted in an additional inhibition of proliferation, resulting in 56 and 84% decreases in the Doxorubicin EC50 value in the presence of 5 × 10−6 and 1.0 × 10−5 M G7-18NATE-P peptide, respectively. Importantly, the co-treatment with Doxorubicin and the delivery peptide did not change the Doxorubicin EC50. Since Grb7 associates with ErbB2, we assessed whether the peptide inhibitor would have a combined effect with a molecule that targets ErbB2, Herceptin. Co-treatment of Herceptin plus 1.0 × 10−5 M G7-18NATE-P peptide in SK-BR-3 cells resulted in a 46% decrease in the Herceptin EC50 value and no decrease following the co-treatment with Herceptin and penetratin alone. This Grb7 peptide has potential to be developed as a therapeutic agent alone, in combination with traditional chemotherapy, or in combination with other targeting molecules

The role of the ubiquitination–proteasome pathway in breast cancer: Ubiquitin mediated degradation of growth factor receptors in the pathogenesis and treatment of cancer

Aberrant activity of growth factor receptors has been implicated in the pathogenesis of a wide variety of malignancies. The negative regulation of signaling by growth factor receptors is mediated in large part by the ubiquitination, internalization, and degradation of the activated receptor. Over the past few years, considerable insight into the mechanisms that control receptor downregulation has been gained. There are also data suggesting that mutations that lead to inhibition of downregulation of growth factor receptors could play a role in the pathogenesis of cancer. Therapies directed at enhancing the degradation of growth factor receptors offer a promising approach to the treatment of malignancies

Modulation of MicroRNA-194 and cell migration by HER2-targeting trastuzumab in breast cancer

Conceived and designed the experiments: XFL GAC RCB. Performed the experiments: XFL MIA WM RS MSN SZ. Analyzed the data: XFL SR. Contributed reagents/materials/analysis tools: YW GAC. Wrote the paper: XFL RCB.Trastuzumab, a humanized monoclonal antibody directed against the extracellular domain of the HER2 oncoprotein, can effectively target HER2-positive breast cancer through several mechanisms. Although the effects of trastuzumab on cancer cell proliferation, angiogenesis and apoptosis have been investigated in depth, the effect of trastuzumab on microRNA (miRNA) has not been extensively studied. We have performed miRNA microarray profiling before and after trastuzumab treatment in SKBr3 and BT474 human breast cancer cells that overexpress HER2. We found that trastuzumab treatment of SKBr3 cells significantly decreased five miRNAs and increased three others, whereas treatment of BT474 cells significantly decreased two miRNAs and increased nine. The only change in miRNA expression observed in both cell lines following trastuzumab treatment was upregulation of miRNA-194 (miR-194) that was further validated in vitro and in vivo. Forced expression of miR-194 in breast cancer cells that overexpress HER2 produced no effect on apoptosis, modest inhibition of proliferation, significant inhibition of cell migration/invasion in vitro and significant inhibition of xenograft growth in vivo. Conversely, knockdown of miR-194 promoted cell migration. Increased miR-194 expression markedly reduced levels of the cytoskeletal protein talin2 and specifically inhibited luciferase reporter activity of a talin2 wild-type 39-untranslated region, but not that of a mutant reporter, indicating that talin2 is a direct downstream target of miR-194. Trastuzumab treatment inhibited breast cancer cell migration and reduced talin2 expression in vitro and in vivo. Knockdown of talin2 inhibited cell migration/invasion. Knockdown of trastuzumab-induced miR-194 expression with a miR-194 inhibitor compromised trastuzumab-inhibited cell migration in HER2-overexpressing breast cancer cells. Consequently, trastuzumab treatment upregulates miR-194 expression and may exert its cell migration-inhibitory effect through miR-194-mediated downregulation of cytoskeleton protein talin2 in HER2-overexpressing human breast cancer cells.This work was supported by the Anne and Henry Zarrow Foundation, kind gifts from Stuart and Gaye Lynn Zarrow and from Mrs. Delores Wilkenfeld, the Laura and John Arnold Foundation, the RGK Foundation, and the MD Anderson NCI CCSG P30 CA16672. G.A.C. is supported as a Fellow at the University of Texas MD Anderson Research Trust, as a University of Texas System Regents Research Scholar and by the CLL Global Research Foundation

Prognostic impact of epidermal growth factor receptor (EGFR) expression on loco-regional recurrence after preoperative radiotherapy in rectal cancer

BACKGROUND: Epidermal growth factor receptor (EGFR) represents a major target for current radiosensitizing strategies. We wished to ascertain whether a correlation exists between the expression of EGFR and treatment outcome in a group of patients with rectal adenocarcinoma who had undergone preoperative radiotherapy (RT). METHODS: Within a six-year period, 138 patients underwent preoperative radiotherapy and curative surgery for rectal cancer (UICC stages II-III) at our institute. Among them, 77 pretherapeutic tumor biopsies were available for semi-quantitative immunohistochemical investigation evaluating the intensity and the number (extent) of tumor stained cells. Statistical analyses included Cox regression for calculating risk ratios of survival endpoints and logistic regression for determining odds ratios for the development of loco-regional recurrences. RESULTS: Median age was 64 years (range: 30–88). Initial staging showed 75% and 25% stage II and III tumors, respectively. RT consisted of 44-Gy pelvic irradiation in 2-Gy fractions using 18-MV photons. In 25 very low-rectal-cancer patients the primary tumor received a boost dose of up to 16 Gy for a sphincter-preservation approach. Concomitant chemotherapy was used in 17% of the cases. All patients underwent complete total mesorectal resection. Positive staining (EGFR+) was observed in 43 patients (56%). Median follow-up was 36 months (range: 6–86). Locoregional recurrence rates were 7 and 20% for EGFR extent inferior and superior to 25%, respectively. The corresponding locoregional recurrence-free survival rate at two years was 94% (95% confidence interval, CI, 92–98%) and 84% (CI 95%, 58–95%), respectively (P = 0.06). Multivariate analyses showed a significant correlation between the rate of loco-regional recurrence and three parameters: EGFR extent superior to 25% (hazard ratio = 7.18, CI 95%, 1.17–46, P = 0.037), rectal resection with microscopic residue (hazard ratio = 6.92, CI 95%, 1.18–40.41, P = 0.032), and a total dose of 44 Gy (hazard ratio = 5.78, CI 95%, 1.04–32.05, P = 0.045). CONCLUSION: EGFR expression impacts on loco-regional recurrence. Knowledge of expression of EGFR in rectal cancer could contribute to the identification of patients with an increased risk of recurrences, and to the prediction of prognosis