Purpose: To critically assess the accuracy and reproducibility of human epidermal growth factor receptor type 2 (HER-2) testing in
outside/local community-based hospitals versus two centralized ...reference laboratories and its effect on selection of women
for trastuzumab (Herceptin)–based clinical trials.
Experimental Design: Breast cancer specimens from 2,600 women were prospectively evaluated by fluorescence in situ hybridization (FISH) for entry into Breast Cancer International Research Group (BCIRG) clinical trials for HER-2-directed
therapies.
Results: HER-2 gene amplification by FISH was observed in 657 of the 2,502 (26%) breast cancers successfully analyzed. Among 2,243 breast
cancers with central laboratory immunohistochemistry (10H8-IHC) analysis, 504 (22.54%) showed overexpression (2+ or 3+). Outside/local
laboratories assessed HER-2 status by immunohistochemistry in 1,536 of these cases and by FISH in 131 cases. Overall, the
HER-2 alteration status determined by outside/local immunohistochemistry showed a 79% agreement rate κ statistic, 0.56; 95% confidence interval (95% CI), 0.52-0.60, with FISH done by the central laboratories. The agreement
rate comparing BCIRG central laboratory 10H8-IHC and outside/local laboratory immunohistochemistry was 77.5% ( κ statistic, 0.51; 95% CI, 0.46-0.55). Finally, HER-2 status, determined by unspecified FISH assay methods at outside/local
laboratories, showed a 92% agreement rate ( κ statistic, 0.83; 95% CI, 0.73-0.93), with FISH done at the BCIRG central laboratories.
Conclusions: Compared with the HER-2 status determined at centralized BCIRG reference laboratories, these results indicate superiority
of FISH to accurately and reproducibly assess tumors for the HER-2 alteration at outside/local laboratories for entry to clinical
trials.
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 VEGF 121–206 ELISA uses antibodies that bind to VEGF 121 and, therefore, detects all of the VEGF isoforms with 121 and more amino acids. The VEGF 165–206 ELISA uses antibodies that bind to VEGF 165 and, therefore, detects all of the VEGF isoforms with 165 and more amino acids. VEGF 121–206 and VEGF 165–206 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. VEGF 121–206 and VEGF 165–206 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 (χ 2 test: P < 0.001 for both VEGF 121–206 and VEGF 165–206 ). VEGF 121–206 and VEGF 165–206 demonstrate a comparable prognostic significance for survival in unselected primary breast cancer patients (univariate analysis:
VEGF 121–206 , P = 0.0068; VEGF 165–206 , P = 0.0046; multivariate analysis: VEGF 121–206 , P = 0.1475; VEGF 165–206 , P = 0.1483). When the analyses were performed separately for node-negative and node-positive patients, VEGF 121–206 and VEGF 165–206 were of prognostic significance for survival only in node-positive patients (univariate analysis: VEGF 121–206 , P = 0.0003; VEGF 165–206 , P = 0.0038; multivariate analysis: VEGF 121–206 , P = 0.0103; VEGF 165–206 , P = 0.0150). A biological concentration-effect relationship between VEGF expression and survival (VEGF 121–206 , P = 0.0280; VEGF 165–206, 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 VEGF 121–206 /VEGF 165–206 results in additional prognostic information for survival (VEGF 121–206 , P = 0.0133; VEGF 165–206 , 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 .
Amplification and overexpression of the HER-2/neu gene occurs in 25-30% of human breast cancers. This genetic alteration is associated with a poor clinical prognosis in women with either node ...negative or node positive breast cancers. The initial studies testing this association were somewhat controversial and this controversy was due in large part to significant heterogeneity in both the methods and/or reagents used in testing archival material for the presence of the alteration. These methods included a number of solid matrix blotting techniques for DNA, RNA and protein as well as immunohistochemistry. Fluorescence in situ hybridization (FISH) represents the newest methodologic approach for testing for this genetic alteration. In this study, FISH is compared to Southern, Northern and Western blot analyses as well as immunohistochemistry in a large cohort of archival human breast cancer specimens. FISH was found to be superior to all other methodologies tested in assessing formalin fixed, paraffin embedded material for HER-2/neu amplification. The results from this study also confirm that overexpression of HER-2/neu rarely occurs in the absence of gene amplification in breast cancer (approximately 3% of cases). This method of analysis is rapid, reproducible and extremely reliable in detecting presence of HER-2/neu gene amplification and should have clinical utility.
The heregulins are a family of ligands with ability to induce phosphorylation of the p185HER-2/neu receptor. Various investigators have reported a variety of responses of mouse and human breast and ...ovarian cells to this family of ligands including growth stimulation, growth inhibition, apoptosis and induction of differentiation in cells expressing the HER-2/neu receptor. Some of the disparity in the literature has been attributed to variations in the cell lines studied, ligand dose applied, methodologies utilized or model system evaluated (i.e. in vitro or in vivo). To evaluate the effects of heregulin on normal and malignant human breast and ovarian epithelial cells expressing known levels of the HER-2/neu receptor, this report presents the use of several different assays, performed both in vitro and in vivo, in vitro proliferation assays, direct cell counts, clonogenicity under anchorage-dependent and anchorage-independent conditions, as well as the in vivo effects of heregulin on human cells growing in nude mice to address heregulin activity. Using a total of five different biologic assays in nine different cell lines, across two different epithelia and over a one log heregulin dose range, we obtained results that clearly indicate a growth-stimulatory role for this ligand in human breast and ovarian epithelial cells. We find no evidence that heregulin has any growth-inhibitory effects in human epithelial cells. We also quantitated the amount of each member of the type I receptor tyrosine kinase family (RTK I, i.e. HER-1, HER-2, HER-3 and HER-4) in the cell lines employed and correlated this to their respective heregulin responses. These data demonstrate that HER-2/neu overexpression itself affects the expression of other RTK I members and that cells expressing the highest levels of HER-2/neu have the greatest response to HRG.
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DOBA, EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, IZUM, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UILJ, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
In a new study, compelling new evidence is presented regarding the association between alterations in the human epidermal growth factor receptor type 2 (HER2) amplicon and incremental sensitivity to ...anthracycline-based adjuvant therapy for breast cancers. This report is the latest in a series of publications that question the generalized assumption that there is an incremental benefit to all breast cancer patients who receive anthracycline-based adjuvant therapy as opposed to non-anthracycline-containing adjuvant therapy. This premise has dominated the design of studies and clinical practice utilization for the vast majority of adjuvant regimens worldwide over the past 30 years. The widespread use of adjuvant anthracyclines is almost entirely based on the well-known meta-analysis published by the Early Breast Cancer Trialists' Collaborative Group, frequently referred to as the Oxford "Overview", rather than results from any single study.
The management of human breast cancer frequently includes radiation therapy as an important intervention, and improvement in the clinical efficacy of radiation is desirable. Overexpression of the ...HER-2 growth factor receptor occurs in 25-30% of human breast cancers and correlates with poor clinical outcome, including earlier local relapse following conservative surgery accompanied by radiation therapy. In breast cancer cells with overexpression of HER-2 receptor, recombinant humanized monoclonal antibodies (rhuMAbs) to HER-2 receptors (rhuMAb HER-2) decrease cell proliferation in vitro and reduce tumor formation in nude mice. Therapy with rhuMAb HER-2 enhances tumor sensitivity to radiation at doses of 1-5 Gy, exceeding remission rates obtained with radiation alone. This benefit is specific to cells with HER-2 overexpression and does not occur in cells without overexpression. Treatment of cells with radiation (2-4 Gy) alone provokes a marked increase in unscheduled DNA synthesis, a measure of DNA repair, but HER-2-overexpressing cells treated with a combination of rhuMAb HER-2 and radiation demonstrate a decrease of unscheduled DNA synthesis to 25-44% of controls. Using an alternate test of DNA repair, i.e., radiation-damaged or undamaged reporter DNA, we introduced a cytomegalovirus-driven beta -galactosidase into HER-2-overexpressing breast cancer cells that had been treated with rhuMAb HER-2 or control. At 24 h posttransfection, the extent of repair assayed by measuring reporter DNA expression was high after exposure to radiation alone but significantly lower in cells treated with combined radiation and rhuMAb HER-2 therapy. To further characterize effects of rhuMAb HER-2 and the combination of antibody and radiation on cell growth, analyses of cell cycle phase distribution were performed. Antibody reduces the fraction of HER-2-overexpressing breast cancer cells in S phase at 24 and 48 h. Radiation treatment is also known to promote cell cycle arrest, predominantly at G sub(1), with low S-phase fraction at 24 and 48 h. In the presence of rhuMAb HER-2, radiation elicits a similar reduction in S phase at 24 h, but a significant reversal of this arrest appears to begin 48 h postradiation exposure. The level of S-phase fraction at 48 h is significantly greater than that found at 24 h with the combined antibody-radiation therapy, suggesting that early escape from cell cycle arrest in the presence of antireceptor antibody may not allow sufficient time for completion of DNA repair in HER-2-overexpressing cells. Because it is well known that failure of adequate p21WAF1 induction after DNA damage is associated with failure of cell cycle arrest, we also assessed the activity of this critical mediator of the cellular response to DNA damage. The results show induction of p21WAF1 transcripts and protein product at 6, 12, and 24 h after radiation treatment; however, increased levels of p21WAF1 transcript and protein are not sustained in HER-2-overexpressing cells exposed to radiation in the presence of rhuMAb HER-2. Although transcript and protein levels increase at 6-12 h, they are both diminished by 24 h. Levels of p21WAF1 transcript and protein at 24 h are significantly lower than in cells treated by radiation without antibody. A reduction in the basal level of p21WAF1 transcript also occurred after 12-24 h exposure to antibody alone. The effect of HER-2 antibody may be related to tyrosine phosphorylation of p21WAF1 protein. Tyrosine phosphorylation of p21WAF1 is increased after treatment with radiation alone, but phosphorylation is blocked by combined treatment with antireceptor antibody and radiation. This dysregulation of p21WAF1 in HER-2-overexpressing breast cells after treatment with rhuMAb HER-2 and radiation appears to be independent of p53 expression levels but does correlate with reduced levels of mdm2 protein. These data indicate that human breast cancer cells damaged by radiation may be especially vulnerable to injury if they are also deprived of essential signal transduction pathways provided by the HER-2 growth factor receptor pathway.
Amplification of the HER-2/neu (c-erbB-2) gene resulting in overexpression of the p185HER-2 growth factor receptor occurs in approximately 25% of early stage breast cancers. HER-2/neu has been ...established as an important independent prognostic factor in early stage breast cancer in large cohorts of patients and in cohorts with very long (30 year) follow-up duration. New data are emerging to suggest that HER-2/neu may be useful not only as a prognostic factor but also as a predictive marker for projecting response to chemotherapeutics, antiestrogens, and therapeutic anti-HER-2/neu monoclonal antibodies. In this review we highlight recent data on HER-2/neu as a predictive marker of response to breast cancer therapy and discuss the clinical implications of this information. The difficulty in comparing results from different data sets due to the wide variety of reagents and technologies used to detect HER-2/neu amplification/overexpression in clinical specimens is also discussed. Finally, we report results from experimental models of HER-2/neu overexpression which have been used in an effort to understand the relationship between HER-2/neu and response to chemotherapeutics and antiestrogens in breast cancer.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Approximately 30% of human breast and ovarian cancers have amplification and/or overexpression of HER-2/neu gene which encodes a cell surface growth-factor receptor. Overexpression of this receptor, ...p185HER-2/neu, is associated with poor outcome and may predict clinical response to chemotherapy. Antibodies to HER-2/neu receptor have a cytostatic effect in suppressing growth of cells with overexpression of p185HER-2/neu. To elicit a cytocidal effect, therapy with antireceptor antibody was used in combination with the DNA-damaging drug, cisplatin, and this combined treatment produced a synergistic decrease in cell growth. In addition, antibody mediated an increased sensitivity to cisplatin in drug-resistant ovarian carcinoma cells containing multiple copies of HER-2/neu gene. To evaluate the mechanism for this synergy, unscheduled DNA synthesis was measured in cancer cells using incorporation of 3Hthymidine and autoradiography, and formation and repair of cisplatin-induced DNA adducts was also measured. Treatment with cisplatin led to a marked, dose-dependent increase in unscheduled DNA synthesis which was significantly reduced by combined treatment with antireceptor antibody in HER-2/neu-overexpressing cells. Therapy with antibody to HER-2/neu receptor also led to a 35-40% reduction in repair of cisplatin-DNA adducts after cisplatin exposure and, as a result, promoted drug-induced killing in target cells. This phenomenon which we term receptor-enhanced chemosensitivity may provide a rationale for more selective targeting and exploitation of overexpressed growth factor receptors in cancer cells, thus leading to new strategies for clinical intervention.
The pivotal phase II and III Herceptin trials proved the efficacy and safety of second- or third-line single-agent Herceptin and first-line Herceptin in combination with chemotherapy, respectively. ...In the current trial, 114 patients were randomized to one of two dose groups of first-line Herceptin monotherapy: standard dose of 4 mg/ kg initial dose followed by 2 mg/kg intravenous (i.v.) weekly; or high dose of 8 mg/kg initial dose followed by 4 mg/kg i.v. weekly. The regimen was generally well tolerated. A similar incidence of adverse events was demonstrated in the two dose groups with the possible exception of acute infusion-related events such as fever and chills as well as rash and dyspnea, which appear to be more prevalent in the higher dose group. The overall response rate was 26% and response rates were similar between the two dose groups (24% for the standard Herceptin dose group and 28% for the high Herceptin dose group). Subgroup analysis determined a higher response rate in IHC 3+ patients (35%) and FISH-positive patients (41%). When women with stable disease for > or =6 months were included with responders, the clinical benefit rate in IHC 3+ patients was 47%. Median survival was 24.4 months, which is comparable with the survival rate seen in the pivotal phase III combination trial (25 months). Therefore, single-agent Herceptin is an important new option for the first-line treatment of HER2-positive metastatic breast cancer patients.
The HER‐2/neu proto‐oncogene encodes a 185 kDa transmembrane receptor tyrosine kinase with significant sequence homology to other members of the class I receptor tyrosine kinase family. The HER‐2/neu ...gene is amplified and/or overexpressed in 25%‐30% of human breast and ovarian cancers, and overexpression of the receptor is associated with poor prognosis. Tyrosine phosphorylation and activation of the HER‐2 receptor lead to activation of specific signal transduction pathways in breast and ovarian cancer cells, including the ras/MAP kinase cascade, phosphatidylinositol 3‐kinase, and phospholipase C‐γ. HER‐2/neu signal transduction pathways ultimately converge on the cell nucleus, where the expression of diverse genes is induced after activation of the receptor. A more complete understanding of HER‐2/neu signal transduction pathways may allow the development of specific therapeutics for the treatment of those human breast and ovarian cancers containing this alteration.
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