Neu Oncoprotein Expression Research Articles

Detection of HER-2/neu gene amplification in breast carcinomas using quantitative real-time PCR — A comparison with immunohistochemical and FISH results

The aim of our study was to evaluate the value of quantitative real-time-PCR (qPCR) in the determination of HER-2/neu amplification status of human breast carcinomas by comparing qPCR, FISH and immunohistochemistry results from the same samples. A total of 210 breast carcinomas were examined. Ready-to-use CB11 antibody was applied to detect HER-2/neu oncoprotein expression. In 76 out of 210 cases FISH was performed, and 162 cases were investigated with qPCR. Seventy-five tumors were 2+ or 3+ positive with immunohistochemistry, while 135 samples were either completely negative or 1+. In 45 cases results from all three methods were available. Out of these, in twenty negative and sixteen positive cases both FISH and qPCR led to similar results. The mean qPCR amplification ratio in the concordant positive cases was 5.424 while in the qPCR+/FISH- group the mean ratio was 2.765. Out of 121 samples with scores of 0 or 1+ immunohistochemical result, analyzed also with qPCR, 26 showed HER-2/neu gene amplification. In these cases the mean amplification ratio was 2.53. Comparison of FISH and qPCR together with immunohistochemistry shows that qPCR is more sensitive to detect HER-2/neu gene amplification in tumors scored as 2+ with immunohistochemistry, but the diagnostic cut-off ratio should be defined above 2.7 to avoid high number of false positive cases. Amongst the immunohistochemistry score 2+ cases, 10 of 18 showed gene amplification by qPCR while 10 of 26 by FISH. In conclusion, a well calibrated HER-2/neu qPCR assay may serve as useful alternative to FISH in breast cancer patients.

Evaluation of HER-2/neu expression in clinically localized prostate cancer

4768 Background: The overexpression of HER-2/neu oncoprotein is found in 20–30% of patients with breast carcinoma and is an adverse prognostic factor. HER-2/neu overexpression has been reported to occur in up to 60% of patients with prostate carcinoma and may serve a possible new target for anti-tumour therapy. However, no conclusive studies have related oncogene activation with the development of prostatic neoplasia. Reported rates of HER-2/neu overexpression in patients with prostate carcinoma vary greatly. Methods: In order to determine the immunohistochemical expression of HER-2/neu protein in clinically localized prostate cancers and to compare immunohistochemical results and clinical prognostic factors, we have studied archival formalin-fixed, paraffin-embedded radical prostatectomy specimens. The HercepTest (DakoCytomation) was used to assess HER-2/neu expression. The level of HER-2/neu expression was evaluated on a scale from 0 (no staining) to 3+ according to the published guidelines. Fluorescent in situ hybridization for gene amplification was performed on all specimens. Results: Out of 114 cases 77 specimens (67,5%) had no staining, 15 (13,2%) specimens had 1+ staining, 14 (12,3%) specimens had 2+ staining and 8 (7%) specimens has 3+ staining. There was an association between the level of HER-2/neu expression and tumor stage (P = 0.03) as well as Gleason grade (P = 0.02), but none of the specimens had HER-2/neu gene amplification. This study demonstrates that more than a third of all clinically localized prostate cancers express HER-2/neu oncoprotein via immunohistochemistry using HercepTest on archival material of clinically localized prostate cancer. Conclusions: Although HER-2/neu oncoprotein expression was detected, no positive case demonstrated gene amplification. HER-2/neu oncoprotein expression does not appear to be a prognostic marker for prostate cancer on archival specimens. No significant financial relationships to disclose.

Does endogenous fatty acid metabolism allow cancer cells to sense hypoxia and mediate hypoxic vasodilatation? Characterization of a novel molecular connection between fatty acid synthase (FAS) and hypoxia‐inducible factor‐1α (HIF‐1α)‐related expression of vascular endothelial growth factor (VEGF) in cancer cells overexpressing her‐2/neu oncogene

Her-2/neu (erbB-2) oncogene overexpression is associated with increased tumor progression and metastasis. Fatty acid synthase (FAS), the key lipogenic enzyme responsible for the endogenous synthesis of fatty acids, has been shown to be one of the genes regulated by Her-2/neu at the level of transcription, translation, and biosynthetic activity. Interestingly, we recently established that both pharmacological inhibition of FAS activity and silencing of FAS gene expression specifically suppress Her-2/neu oncoprotein expression and tyrosine-kinase activity in breast and ovarian Her-2/neu overexpressors. Unraveling the functional organization of this novel bi-directional molecular connection between Her-2/neu and FAS-dependent neoplastic lipogenesis is a major challenge that the field is only beginning to take on. Considering that Her-2/neu overexpression correlates with increased expression of the hypoxia inducible factor-1alpha (HIF-1alpha), which, in a mitogen-activated protein kinase (MAPK)-dependent manner, plays a key role in the expression of several genes including cytokines such as vascular endothelial growth factor (VEGF), we hypothesized that FAS blockade should result in a concomitant down-regulation of VEGF. Unexpectedly, the specific inhibition of the de novo fatty acid synthesis with the small-molecule inhibitor of FAS activity C75 resulted in a dramatic dose-dependent enhancement (up to 500% increase) of VEGF secretion in Her-2/neu-overexpressing SK-Br3, BT-474, and SKOV3 cancer cells. Concurrently, FAS blockade drastically activated MAPK and promoted further a prominent accumulation of HIF-1alpha in Her-2/neu overexpressors. Moreover, U0126-induced inhibition of MAPK activity completely abolished C75-induced up-regulation of HIF-1alpha expression and VEGF secretion, whereas it did not modulate C75-induced down-regulation of Her-2/neu oncogene. Importantly, RNA interference (RNAi)-mediated silencing of the FAS gene recapitulated C75's effects by up-regulating VEGF secretion, MAPK activation and HIF-1alpha expression. Therefore, it appears that perturbation of cancer-associated endogenous fatty metabolism triggers a "hypoxia-like" (oxygen-independent) condition that actively rescues Her-2/neu-dependent MAPK --> HIP-1alpha --> VEGF cascade. It is tempting to suggest that an intact FAS-catalyzed endogenous fatty acid metabolism is a necessary metabolic adaptation to support the enhanced ability of Her-2/neu-overexpressing cancer cells to survive cellular hypoxia in a HIF-alpha-dependent manner.

P-638 Does HER-2/neu oncoprotein expression predict resistance to neoadjuvant therapy in locally advanced non-small cell lung cancer?

P-638 Does HER-2/neu oncoprotein expression predict resistance to neoadjuvant therapy in locally advanced non-small cell lung cancer?

Evaluation of HER-2 neu oncoprotein expression as a prognostic indicator of local recurrence in conservatively treated breast cancer: A case-control study

The purpose of this study is to determine the prognostic significance of overexpression of the HER-2 neu oncoprotein with respect to local relapse following conservative surgery and radiation therapy (CS + RT). Twenty consecutive patients who sustained a local recurrence as the first and only site of failure following CS + RT comprised the case population base for this study. Only patients who received no adjuvant systemic chemotherapy or tamoxifen were selected for analysis. Following the identification of 20 consecutive local-relapse patients, the patient database was searched for 20 matching control patients who did not sustain a local relapse. Each control patient was matched to the index case with respect to age, menopausal status, follow-up, primary histology, axillary nodal status, and primary tumor size. Both index cases and the matched control group received full course radiation therapy, to a total dose of 64 Gy to the tumor bed. The paraffin-embedded blocks of the original primary tumor of the local-relapse cases and the primary tumor of 20 matched controls were processed for immunohistochemical staining of the HER-2 neu oncoprotein. Each slide was rated on the 3-point scale, 0 representing no stain, 1+ indicating light staining, and 2+ denoted heavy staining (overexpression). Of the 20 index cases, with each of the matched controls, 16 were evaluable for analysis. The 4 cases that were eliminated had inadequate paraffin-embedded material in either the case or the match control for adequate staining. By design of the study, the index case group and control group had similar ages (52 years index vs. 51.4 control), follow-up (10.8 years index vs. 10.5 years control), and histologies. For the immunostaining, a value of 2+ was considered to denote high activity and overexpression, and 0 and 1+ were considered negative values. Using these criteria, a total of 9 of the 16 index cases (56%) exhibited overexpression of HER-2 neu oncoprotein, and only 3 of the 16 control cases (18%) demonstrated high immunoreactivity. The difference in immunostaining between the index and control cases was statistically significant by a Pierson chi-square analysis at the p = 0.03 level. In this matched case-control study, overexpression of the HER-2 neu oncoprotein appears to have prognostic significance with respect to local relapse in the conservatively treated breast. The correlation of overexpression of HER-2 neu by multivariable analysis with other prognostic factors for local recurrence warrants further investigation. The clinical implications of the study are discussed.

Prognostic value of immunohistochemically detected HER-2/neu oncoprotein in endometrial cancer

HER-2/neu (c-erbB-2) oncoprotein is a transmembrane glycoprotein and may function as a growth factor receptor being involved in the regulation of cell growth and cell transformation. We performed an analysis of 100 patients with endometrial cancer stage FIGO I to IV using an immunoperoxidase technique on formalin-fixed, paraffin-embedded tissue samples in order to determine HER-2/neu oncoprotein expression. HER-2/neu oncoprotein was expressed in the tumors of 21 patients (21%). Clinical stage, histologic stage, histologic grade and depth of invasion did not correlate with HER-2/neu oncoprotein expression. We found HER-2/neu oncoprotein in all clinical stages and therefore it does not seem to be a late event in the natural history of endometrial cancer. HER-2/neu oncoprotein expression was associated with poor overall survival (log-rank P-value 0.04).

Prognostic value of immunohistochemically detected HER-2/neu oncoprotein in endometrial cancer

HER-2/neu (c-erbB-2) oncoprotein is a transmembrane glycoprotein and may function as a growth factor receptor being involved in the regulation of cell growth and cell transformation. We performed an analysis of 100 patients with endometrial cancer stage FIGO I to IV using an immunoperoxidase technique on formalin-fixed, paraffin-embedded tissue samples in order to determine HER-2/neu oncoprotein expression. HER-2/neu oncoprotein was expressed in the tumors of 21 patients (21%). Clinical stage, histologic stage, histologic grade and death of invasion did not correlate with HER-2/neu oncoprotein expression. We found HER-2/neu oncoprotein in all clinical stages and therefore it does not seem to be a late event in the natural history of endometrial cancer. HER-2/neu oncoprotein expression was associated with poor overall survival (log-rank P-value 0.04).

140 Evaluation of HER-2 neu oncoprotein expression as a prognostic indicator of local recurrence in conservatively treated breast cancer: A case-control study

140 Evaluation of HER-2 neu oncoprotein expression as a prognostic indicator of local recurrence in conservatively treated breast cancer: A case-control study

Prognostic significance of HER-2/neu oncoprotein expression in node-positive breast cancer. The influence of the pattern of immunostaining and adjuvant therapy

The influence of HER-2/neu on prognosis of breast cancer is controversial. The authors investigated by immunohistochemistry in node-positive disease the influence of the pattern of immunostaining (membranous or cytoplasmic) on outcome and the prognostic significance of this marker in patients receiving or not receiving adjuvant therapy. The immunostaining for HER-2/neu oncoprotein was performed on formaldehyde-solution-fixed, paraffin-embedded sections of 888 node-positive breast cancers resected between 1980 and 1986 and for which a follow-up of 2.5-10.5 years was available. The staining was performed using a polyclonal antibody (dilution, 1/15). One hundred forty-three cases (16.1%) revealed a positive membrane staining with or without additional cytoplasmic contribution, whereas cytoplasmic staining alone was noted in 118 cases (13.3%). Positive membrane staining was correlated with more involved lymph nodes (P = 0.005), aneuploidy (P = 0.002), poor nuclear (P < 0.0001) and histologic (P = 0.007) grades, absence of estrogen (P < 0.0001) and progesterone (P < 0.0001) receptor content, and cathepsin D expression (P = 0.009). No relation was found (P > 0.05) with either age, tumor size, or HSP27 expression. Membrane staining was strongly associated with poor distant metastasis-free or overall survival rate (P < 0.0001), whereas cytoplasmic staining had no prognostic significance. Two hundred thirty-two patients (26.1%) received no additional treatment after surgery. The difference in survival rates between cases with positive and negative staining was only significant among patients submitted to adjuvant chemotherapy or hormone therapy. This study strongly supports the association of HER-2/neu oncoprotein expression with poor prognosis in node-positive breast cancer and demonstrates that membranous but not cytoplasmic staining is prognostically relevant. It also shows that HER-2/neu oncoprotein expression is useful in predicting survival time only in patients receiving adjuvant therapy, thus suggesting that it may be a marker of drug resistance.

Prognostic significance of cathepsin-D expression in node-positive breast carcinoma: an immunohistochemical study.

The prognostic significance of cathepsin-D expression was evaluated by immunohistochemistry in 638 node-positive breast carcinomas diagnosed between 1980 and 1986. A minimum of 2.5 years of follow-up was available for each patient (maximum: 9.5 years). Cathepsin-D expression was assessed separately both in cancer and in stromal cells using a commercially available polyclonal antibody. While cancer-cell immunostaining was not associated with prognosis, positive staining of stromal elements was related to shorter metastasis-free survival. The difference in distant metastasis-free survival between positive and negative expressors was greatest in the sub-group of patients submitted to adjuvant chemotherapy, with a hazard ratio for occurrence of distant metastasis of 1.76 by multivariate analysis, but was lowest for those receiving hormone therapy. Cathepsin-D expression by stromal cells was related to HER-2/neu oncoprotein expression, HSP-27 expression, poor nuclear grade, aneuploidy, and absence of estrogen and progesterone receptors. No association was found with the number of involved lymph nodes, tumor size, age, histologic grade, S-phase fraction, or vascular invasion. Our study suggests that cathepsin-D expression by stromal cells (and not by cancer cells) affects the prognosis of breast cancer, that stromal cells probably play a key role in local invasion and metastatic dissemination of the tumor, and that the prognostic significance of cathepsin-D expression may vary according to the type of adjuvant therapy.

Prediction of Response to Adjuvant Chemotherapy in Premenopausal Lymph Node Positive Breast Cancer Patients with Morphometry, DNA Flow Cytometry and Her-2/Neu Oncoprotein Expression: Preliminary Results

The value of morphometry, DNA flow cytometry and HER-2/neu oncoprotein expression for prediction of response to adjuvant chemotherapy in premenopausal lymph node positive breast cancer patients was evaluated in a group of CMF treated patients and controls with long-term follow-up. In the treated group, the Morphometric Prognostic Index (cutpoint 1.1) was the best prognosticator (p less than 0.0001, MC = 16.9), followed by the Mitotic Activity Index, the volume percentage epithelium and the number of positive nodes. For the controls, only the % HER-2/neu oncoprotein expression revealed significant differences (p less than 0.0001, MC = 16.3). When directly comparing treated patients and controls stratified for a certain parameter, no significant differences were obtained, although a trend towards improved survival in the treated group was present for some of the subgroups for several parameters. These preliminary results indicate that morphometric features and quantitative HER-2/neu oncoprotein expression may be important factors for identifying cases that will or will not respond to adjuvant chemotherapy.

Study of the biological and prognostic significance of the antigen CaMBr8 on breast carcinoma.

We previously reported that the expression on the primary tumour of the antigen CaMBr8 was related to a short survival, attributable either to higher tumour aggressiveness or a poor response to oophorectomy. To further verify the CaMBr8 prognostic value, we analysed retrospectively 862 breast cancer patients with a 19 year follow-up. In this series, CaMBr8 expression was found to be associated to some negative prognostic factors (premenopausal status, lymphnode invasion, a high number of mitosis and HER-2/neu oncoprotein expression), but had no influence on the patients' survival. Direct association with a poor prognosis was only evident in patients with lobular or mixed breast carcinoma, which however represent only a small fraction of the total breast cancers. Another possibility was that CaMBr8 could identify a subgroup of patients which did not respond to hormone therapy. To verify this hypothesis we evaluated on a second series of 116 patients the relationship between CaMBr8 expression and hormone-receptor levels. A negative association emerged which was also observed in vitro in the human breast cancer line MCF-7 treated with Sodium Butyrate, a differentiation inducer, which reduced hormone-receptor levels and increased CaMBr8 expression. In conclusion, the longer survival of CaMBr8 negative tumour patients observed in the initial study, was probably related to a better response to oophorectomy, due to the hormone-receptor level of their tumours.