Platelet-derived growth factor expression in phyllodes tumors and fibroadenomas of the breast

The development of normal breast tissue and the pathogenesis of various tumors are influenced by growth factor-mediated epithelial-stromal interactions. Similar interactions may occur in fibroepithelial breast tumors. We have studied the expression of platelet-derived growth factor (PDGF) and PDGF beta receptor (PDGFRbeta) in 46 phyllodes tumors (18 benign, 15 borderline, 13 malignant), 11 fibroadenomas, and 6 samples of normal breast. There was neoplastic stromal cell positivity for PDGFRbeta in almost 50% of phyllodes tumors and for PDGF in 24%. Both were associated with prominent nuclear pleomorphism (P<.01), PDGF with high grade (P<.01), and a higher mean Ki-67 labeling index (P = .013), and PDGFRbeta with conspicuous stromal overgrowth (P<.01). Co-positivity for stromal PDGF and PDGFRbeta was found in 15% of phyllodes tumors, and for epithelial PDGF and stromal PDGFRbeta in 43%. Both types of co-positivity were associated with prominent nuclear pleomorphism and the latter type with conspicuous stromal overgrowth (all P<.01). Follow-up of 41 phyllodes tumors showed that disease-related death was associated with established histologic features of malignancy including mitotic count, stromal overgrowth, an infiltrative tumor margin, and nuclear pleomorphism. In addition, stromal PDGFRbeta positivity (P =.013) and epithelial PDGF/stromal PDGFRbeta co-positivity (P =.0075) were associated with disease-related death. Stromal PDGF and PDGFRbeta expression in fibroadenomas was less common and less extensive (P<.05) than in phyllodes tumors. The results suggest that PDGF influences the pathogenesis of fibroepithelial breast tumors and that PDGF-dependent paracrine and autocrine mechanisms may operate. Also, it is possible that assessment of PDGF and PDGFRbeta expression could contribute to the management of these tumors in the future.     

Determination of EGF receptors (epidermal growth factor) in the placenta and breast tumors

Receptors for Epidermal Growth Factor (EGF) have been identified in different types of tumors and they are often associated with other characteristics which are related to a bad prognosis. A technic has been established to measure membrane receptors in human placenta, which is appropriate for the study of tumors. Preparations of placental membranes were incubated either with increasing amounts (0.1 to 3.0, 10(-9) M) of EGF labeled with 125I (method by saturation) or with 0.2, 10(-9) M labeled EGF and increasing concentrations (0 to 1.6, 10(-9) M) of cold EGF (method by competition). The KD obtained with both technics are not significantly different: 0.85 and 0.77, 10(-9) M respectively, as well as the maximum binding capacity: 5.9 and 4.4 pmol/mg protein respectively. A preliminary study in breast tumors showed that 2 out of 9 contained significant amounts of receptors (58 and 114 fmol/mg protein). One of these two tumors held both estradiol and progesterone receptors. In the other 7 tumors without EGF receptors only one did not contained any estradiol or progesterone receptors. Further studies are needed to evaluate the relationships between EGF/sex hormone receptor status and the degree of breast tumor malignancy.     

Beta-catenin abnormalities and associated insulin-like growth factor overexpression are important in phyllodes tumours and fibroadenomas of the breast

The aim of this study was to assess the expression of IGF-I and IGF-II in phyllodes tumours and fibroadenomas and to see if there is any correlation between nuclear beta-catenin expression and IGF-I and IGF-II expression in these tumours. In a previous study, it has been shown that Wnt signalling is important in the pathogenesis of phyllodes tumours of the breast. Epithelial Wnt5a overexpression and stromal Wnt2 overexpression were associated with abnormal, nuclear localization of beta-catenin in the stromal cells of these tumours. However, not all tumours with beta-catenin accumulation showed Wnt overexpression. One other possible cause of beta-catenin accumulation is overexpression of insulin-like growth factors (IGFs), as both IGF-I and IGF-II have been shown to stabilize beta-catenin. In this study, 30 fibroadenomas of the breast were assessed for beta-catenin expression using immunohistochemistry and the results were compared with previous data from 119 phyllodes tumours. In situ hybridization was used to assess IGF-I and IGF-II expression in 23 phyllodes tumours and 16 fibroadenomas. Nineteen phyllodes tumours (83%) showed widespread overexpression of IGF-II and 5/23 (22%) showed widespread overexpression of IGF-I. IGF-I expression correlated with nuclear beta-catenin staining in phyllodes tumours. Malignant phyllodes tumours showed no or little IGF-I expression. There was a degree of nuclear beta-catenin expression in the stroma (weak in 33%, moderate in 27%, and strong in 40%) in all fibroadenomas and nuclear beta-catenin staining correlated with IGF-I overexpression. Extensive IGF-II overexpression was also found in the majority of fibroadenomas (12/16). These results support the hypothesis that IGF-I and IGF-II overexpression may be important in the pathogenesis of fibroepithelial neoplasms of the breast and that IGF-I overexpression is likely to be contributing to the nuclear beta-catenin localization observed in the tumours.     

Expression of Cbl linking with the epidermal growth factor receptor system is associated with tumor progression and poor prognosis of human gastric carcinoma

Cbl proteins play important roles in downregulation of growth factor receptors by acting as ubiquitin ligases and multi-adapter proteins. Ligand-induced desensitization of the epidermal growth factor receptor (EGFR) has been shown to be controlled by Cbl. In the present study, we examined the expression of Cbl in gastric carcinomas and studied the correlation of Cbl expression with clinicopathological characteristics as well as EGFR expression. Cbl protein was expressed in 67% (82/122) of gastric carcinomas, and diffuse expression of Cbl was detected in 29% (35/122) of the cases. The incidence of cases with diffuse expression of Cbl was significantly higher in advanced cases (28/70, 40%) than in early cases (7/52, 14%) (P=0.0010). Diffuse expression of Cbl was significantly associated with metastasis of tumor cells in lymph nodes (P=0.0318). Diffuse expression of EGFR was significantly associated with depth of invasion (P=0.0057), lymph-node metastasis (P=0.0371) and tumor stages (P=0.0278). As the grades of Cbl expression became stronger, the cases with diffuse EGFR expression increased, the positive correlation being significant (P=0.049). All the cases with diffuse expression of Cbl and EGFR were found to show nodal metastasis and to be at an advanced stage. Moreover, the prognosis of the patients with synchronous diffuse expression of Cbl and EGFR was significantly poorer than that of the patients negative for Cbl and focal or negative for EGFR (P=0.0086). The expression of Cbl protein was clearly induced in gastric carcinoma cell lines by transforming growth factor- treatment. These results suggest that Cbl in connection with the EGFR system may be associated with stomach carcinogenesis, invasion and metastasis. Cbl may serve as a novel molecular marker for aggressive gastric carcinoma.     

Radioimmunohistochemistry of epidermal growth factor receptor in breast cancer

CONTEXT: Conflicting reports of epidermal growth factor receptor (EGFR) expression in breast cancer and inconstant relationships with established prognostic indicators and outcomes suggest difficulties with EGFR measurement. OBJECTIVE: To compare EGFR measurement in a panel of cell lines and in breast carcinomas by radioimmunohistochemistry (R-IHC), conventional immunohistochemistry (IHC), and a ligand binding (LB) assay. DESIGN: Eight EGFR-expressing cell lines and 50 primary breast carcinoma specimens were analyzed for EGFR by IHC, R-IHC, and LB assays. A further 153 primary breast cancer specimens were analyzed by R-IHC alone. RESULTS: All 3 assays were in good agreement for the cell lines. In the subset of the 50 carcinoma specimens, EGFR was detected by LB assays in 19 (38%) and by IHC in 24 (48%). However, R-IHC detected EGFR in 46 (92%) of 50 and in 186 (92%) of all 203 carcinoma specimens. The LB assay agreed poorly with R-IHC of carcinomas, possibly because the LB assay is sensitive to EGFR-expressing nontumor breast parenchyma in the tissue analyzed. Both IHC and R-IHC on carcinoma specimens agreed better, but 26 carcinoma specimens (52%) in which EGFR was not detectable by IHC had a 10-fold range in receptor level detectable by R-IHC. CONCLUSION: To elucidate the role of EGFR or other growth factor receptors in breast cancer requires accurate, sensitive receptor assays. With its dynamic range, R-IHC returned meaningful results over the entire range of expression actually present in breast cancer, which LB assays and IHC failed to do.     

Exon 8 amplification of epidermal growth factor receptor (EGFR) in invasive breast carcinomas

Souka E, Alexiadis E, Theohari I, Giannopoulou I, Papadimitriou C & Nakopoulou L
(2012) Histopathology  61, 644–651 Exon 8 amplification of epidermal growth factor receptor (EGFR) in invasive breast carcinomas Aims: The rate of EGFR amplification in breast cancer ranges between 0% and 15%. Recent studies have focused on the amplification status of cytosine–adenine (CA) repeats in intron 1 of the gene and correlated it with increased EGFR protein. The aim of this study was to investigate, for the first time, the significance of coding exon 8 amplification of EGFR in invasive breast cancer. Methods and results: We investigated, by means of real‐time polymerase chain reaction (PCR), the amplification status of exon 8 of the EGFR gene in 148 paraffin‐embedded tissue sections, 115 with invasive breast carcinoma and 33 controls. Immunohistochemistry was utilized to detect EGFR and human epidermal growth factor receptor 2 (HER2) expression. Univariate and multivariate statistical analyses were performed for the evaluation of our results. EGFR amplification was observed in 7.8% of the patients, and EGFR was immunodetected in 9.6%. EGFR amplification was correlated positively with EGFR expression (P < 0.0001), HER2 expression (P = 0.023), coexpression of EGFR/HER2 (P < 0.0001) and nuclear grade (P = 0.047), and inversely with ER protein expression (P = 0.047). Conclusions: It appears that amplification of the coding sequence exon 8 exhibits similar biological behaviour to amplification of the regulatory sequence in intron 1, leading to elevated levels of EGFR protein.     

Expression of epidermal growth factor receptor in breast carcinoma.

A series of 90 patients with primary operable breast cancer and with up to 36 months of follow up were investigated for expression of epidermal growth factor receptor (EGFR) in their tumours by immunocytochemical staining with the monoclonal antibody EGFR 1. Tumour samples were snap frozen in liquid nitrogen immediately after resection and subsequently stained using a standard indirect immunocytochemical method. Tumour staining was assessed by two observers and scored on a four point scale (0-3). Thirteen (14%) tumours showed positive immunoreactivity. A strong correlation between distinct EGFR expression and short disease free interval was observed. Significant correlations were also shown with oestrogen and progesterone receptor expression and tumour nuclear size. No significant association was found with tumour size, lymph node stage, and histological grade. The association with disease free interval remained significant in multivariate analysis.     

neu oncogene protein and epidermal growth factor receptor are independently expressed in benign and malignant breast tissues

The neu oncogene protein, p185, and epidermal growth factor receptor (EGFR) were localized immunohistochemically in benign and malignant human breast tissues using monoclonal antibodies. Both benign and malignant epithelial cells were positive for these oncogene proteins in acetone-postfixed frozen sections. Stromal cells were negative for p185, but occasionally positive for EGFR. Myoepithelial cells were consistently positive for EGFR, and p185 was localized predominantly in duct-lining cells, where the basolateral plasma membrane was the normal expression site of both substances. Paraformaldehyde-prefixed frozen sections were less sensitive for antigen demonstration. Based on the intensity of immunoreactivity, 11 of 37 acetone-postfixed breast carcinomas (30%) were judged neu overexpressors, while none of 24 benign tissues overexpressed neu. Epidermal growth factor receptor was demonstrated in 18 of 36 acetone-postfixed cancer tissues (50%) and was overexpressed in three (8%). At the cellular level, heterogenous expression of p185 and EGFR was occasionally observed in both benign and malignant tissues, and a single case of cancer overexpressing both neu and EGFR showed reciprocal patterns of staining, indicating their independent expression. In some carcinomas, EGFR was localized only in stromal cells. Our findings confirmed mutually independent expression of the two closely related protooncogenes in benign and malignant breast tissues.     

Oestrogen receptor and epidermal growth factor receptor expression in male breast carcinoma.

Male breast carcinomas are probably hormone-dependent, but receptor studies are few because this is a relatively rare tumour. We have studied 21 cases of male breast carcinoma immunohistochemically for oestrogen receptor (ER) and epidermal growth factor receptor (EGFR) expression employing the antibodies ER-ICA and 12E on formalin-fixed, paraffin-embedded material. In our series, 86 per cent of male breast cancers were ER-positive and 76 per cent were EGFR-positive. Male breast carcinomas do not exhibit the inverse correlation between ER and EGFR expression that characterizes female breast carcinomas. Owing to the limitations of a small series, we were unable to comment on the relationship between ER and EGFR expression and patient survival. However, the relatively high incidence of ER expression may provide a growth advantage for this tumour in a male environment characterized by low levels of oestrogen. In addition, high EGFR expression may also contribute to a poor prognosis independent of ER status.     

Deletion of the inhibitor of growth 4 (ING4) tumor suppressor gene is prevalent in human epidermal growth factor 2 (HER2)-positive breast cancer

Inhibitor of growth 4 (ING4) is a candidate tumor suppressor gene that was shown to be deleted in 10% to 20% of breast cancers by array comparative genome hybridization analysis. We developed fluorescent in situ hybridization to detect the ING4 gene directly in the tissue samples on tumor tissue microarrays. We evaluated the ING4 gene status in 1033 breast cancer tissue samples and observed that ING4 was deleted in 16.5% (170/1033) of all breast cancers. ING4 deletion was significantly associated with Her2 overexpression: of the tumors with ING4 deletion, 23.8% (39/164) were human epidermal growth factor 2 (HER2) positive, as compared with 14.1% (115/814) of the tumors without ING4 deletion (P = .002). In addition, the tumors with ING4 deletion were more likely to belong to the HER2 molecular subtype (estrogen receptor negative/progesterone receptor negative/human epidermal growth factor positive) of breast cancer, compared with the other subtypes (28.4% HER2 versus 15.7% all, P = .002). ING4 deletion did not affect survival outcome of all patients with breast cancer (P = .797) or of the patients with HER2-positive tumors (P = .792). We conclude that ING4 deletion in breast cancer is relatively common, as 1 in 6 breast cancer harbors ING4 deletion. Furthermore, ING4 deletion is more prevalent in HER2-positive tumors, suggesting a functional antagonistic relationship between the ING4 tumor suppressor and the HER2 oncogene. These results sustain the view that ING4 is a tumor suppressor in breast cancer and suggest that ING4 deletion may contribute to the pathogenesis of HER2-positive breast cancer.     

R497K polymorphism in epidermal growth factor receptor gene is associated with the risk of acute coronary syndrome

Background  

Previous studies suggested that genetic polymorphisms in the epidermal growth factor receptor (EGFR) gene had been implicated in the susceptibility to some tumors and inflammatory diseases. EGFR has been recently implicated in vascular pathophysiological processes associated with excessive remodeling and atherosclerosis. Acute coronary syndrome (ACS) is a clinical manifestation of preceding atherosclerosis. Our purpose was to investigate the association of the EGFR polymorphism with the risk of ACS. In this context, we analyzed the HER-1 R497K and EGFR intron 1 (CA)_n repeat polymorphisms in 191 patients with ACS and 210 age- and sex-matched controls in a Chinese population, using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) strategy and direct sequencing.     

Immunohistochemical expression of transforming growth factor alpha and epidermal growth factor receptor in pancreatic endocrine tumors.

Coexpression of transforming growth factor alpha (TGF-alpha) and its receptor epidermal growth factor receptor (EGFR) is known to be associated with aggressive biologic behavior and adverse clinical outcome in a variety of tumors, including pancreatic adenocarcinomas. However, very little information is currently available as to whether this is true of pancreatic endocrine tumors (PETs) as well. Thirty-five PETs were retrospectively studied for immunohistochemical expression of TGF-alpha, the intracellular and extracellular domains of EGFR, and various hormonal secretory products. Proliferative activity was additionally studied (in 20 cases only) using the MIB-1 antibody. Thirty-one (89%) of 35 tumors were reactive for 1 or more of the peptide hormones tested; 22 (63%) tumors were positive for TGF-alpha; and 23 (65%) were positive for the intracellular and/or extracellular domain of EGFR. Based on their TGF-alpha and EGFR expression, these tumors could be classified into 4 groups. Of the 10 tumors in group I (positive for TGF-alpha and the complete EGFR molecule), 3 were malignant, 6 were >2 cm in diameter, 5 were functional, and 1 had a proliferative index of >40%. The 12 tumors in group II (positive for TGF-alpha but negative for the intracellular and/or extracellular domain of EGFR) included 4 malignant tumors, 4 PETs >2 cm in diameter, 8 functional, and 1 with a proliferative index of >40%. The 7 PETs in group III (positive for the intracellular/extracellular domain of EGFR alone) included 3 malignant tumors, 3 PETs >2 cm in diameter, and 3 functional tumors. The 6 tumors in group IV (completely negative for both TGF-alpha and EGFR) included 4 malignant tumors, 3 PETs >2 cm in diameter, and 4 functional lesions. Therefore, immunohistochemical expression of TGF-alpha and EGFR, either alone or in concert, shows no correlation with size, functional status, secretory profile, or biologic behavior and hence cannot be used as a marker of malignancy in this group of tumors.     

Expression of Yes-associated protein (YAP) in breast phyllodes tumor

This study aimed to identify expression profiles of Yes-associated protein (YAP) and its phosphorylated form (pYAP) in phyllodes tumor (PT) of human breast and verify the clinical implications. We selected PTs from the pathologic archive and reviewed the histologic features (141 benign, 27 borderline, and 15 malignant). We made tissue microarray (TMA) block from the formalin-fixed paraffin-embedded (FFPE) tissue corresponding to the representative section. Using TMA block, we performed immunohistochemical staining of YAP and pYAP. In the stromal component, expressions of YAP and pYAP were increased in borderline/malignant PT with comparison of benign PT (P = 0.002, and P < 0.001, respectively). In the epithelial component, cytoplasmic expression of YAP was highest in borderline PT (P = 0.001). Stromal YAP expression (P < 0.001) and stromal pYAP expression (P = 0.042) were associated with shorter disease-free survival (DFS) and stromal pYAP expression (P = 0.001) was associated with shorter overall survival (OS) in univariate Cox analysis. In multivariate Cox analysis, stromal YAP expression was an independent prognostic factor associated with shorter DFS (Hazard ration: 3.206, 95% CI: 1.000-10.27, P = 0.050). In conclusion, expression level of YAP in stromal component was increased along with histologic grade of PT and YAP expression in PT was related to tumor progression and poor prognosis.     

Enzyme immunoassay of human epidermal growth factor receptor (hEGFR)

Overexpession of EGFR has been reported in a variety of human cancers and serves as a target for diagnosis and therapy. In the case of breast cancer, about 48% EGFR and have poor clinical prognosis. Besides the prognostic factors like tumor size, nodal status, histological grade etc., which are significant in the management of breast cancer, EGFR level might also serve as an additional parameter. Immunocytochemical assay has been extensively used to study the expression of EGFR in various cancers. We have generated a panel of monoclonal antibodies against human EGFR with a view to evaluate their application for the diagnosis and therapy of these cancers. In the present study, an EIA has been developed using 2 monoclonal antibodies against hEGFR designated as CIBCNSH3 as the capture antibody and CIBCRGC1 as the detector antibody. EGFR isolated from MDA MB 468, a human breast carcinoma cell line, with high expression of EGFR and purified by conA affinity chromatography and HPLC has been used to develop the EIA procedure. Sera samples of 150 healthy women donors, of 300 breast cancer patients with different histological types of malignancies and of various other types of cancers have been analyzed. The control women had a range for serum EGFR level of 7-162 fmol/ml, whereas the 300 breast cancer patients studied had a range of 126-1587 fmol/ml with a cut off value of 180 fmol/ml. It is interesting to note that 67.5% of breast cancer patients had elevated levels of circulating EGFR. These results might suggest that serum EGFR level can be used as prognostic marker for breast cancer. The serum EGFR level will be compared with disease free interval and patient survival.     

Correlation of epidermal growth factor receptor expression with tumor microdensity vessels and with vascular endothelial growth factor expression in ovarian carcinoma

We analyzed in advanced ovarian serous G3 carcinoma the correlation between epidermal growth factor receptor (EGFR) overexpression and tumor angiogenesis and their relation with clinical outcome. Microvessel density (MVD) and vascular endothelial growth factor (VEGF) were statistically correlated with disease-free interval and death from disease both in univariate and multivariate analyses while EGFR expression was not correlated with clinical outcome. MVD was significantly associated with progression of disease during chemotherapy while VEGF and EGFR expression were not correlated with responsiveness to chemotherapy (Fisher's exact test). VEGF expression was correlated with MVD (Fisher's exact test). EGFR showed a trend to correlation with MVD. Further studies focusing on the use of angiogenesis inhibitors in addition to EGFR inhibitors on ovarian carcinoma cells may produce therapeutic strategies in the selection of tailored therapies in ovarian cancer patients.     

p53 protein expression and gene mutation in phyllodes tumors of the breast

The malignant potential of mammary phyllodes tumors is difficult to assess on initial pathologic examination. Studies on the p53 tumor suppressor gene have shown that it has an important role in the development of a variety of malignancies, yet the specific contribution to the pathogenesis and development of the malignant potential of phyllodes tumor is largely unknown. We studied p53 protein expression in 25 cases of phyllodes tumors histologically classified as either malignant (12 cases) or benign (13 cases). Using microdissection approach, we also analyzed the p53 gene sequence in a case that demonstrated progression from benign to malignant phenotype. Nuclear p53 staining was detected in various proportions (1-90%) of neoplastic stromal cells of malignant tumors. No staining was found in benign tumors. Progression from benign to malignant phenotype was associated with a significant increase in the accumulation of p53 (more than 20 times). This was caused by an underlying missense mutation in exon 7, resulting in a change from Arg248 to Trp248 in the malignant component of the tumor. Stromal p53 over-expression was observed only in neoplasms histologically classified as malignant and was associated with an increased proliferation index (MIB-1 staining). These two markers may be used as useful adjuncts in the diagnosis of malignancy in difficult cases or when only a limited sample size is available. Somatic mutation in exon 7 of p53 gene in malignant phyllodes tumor points toward the importance of p53 in the malignant transformation of phyllodes tumors.     

Clinical value of epidermal growth factor receptor expression in primary breast cancer

EGFR expression in primary breast cancer has been extensively investigated for its prognostic and predictive value. However overall there is no consensus on its potential to guide such prognostication. This is largely because of the great heterogeneity in study designs and methods used to assay the EGFR protein. The impetus to standardize such studies is much needed as there are now several tyrosine kinase inhibitors directed against the EGF receptor and phase II trials are showing significant promise.