HER2 is unlikely to be involved in directly regulating angiogenesis in human breast cancer.

Angiogenesis is a fundamental component of oncogenesis. Angiogenic factors such as vascular endothelial growth factor (VEGF) and platelet derived-endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP) are generated from tumor cells to provide tumor growth and are thought to be regulated via the HER2 oncogene, whose amplification is the most common genetic alteration in breast cancer. The present study aimed to evaluate the immunoreactivity of angiogenic factors (VEGF, PD-ECGF/TP) and microvessel density (MVD) via epidermal growth factor receptor (EGFR) and HER2, and to correlate their expression with clinicopathologic features. Two hundred one invasive human breast cancer specimens were tested immunohistochemically for the expression of these proteins. In addition, MVD was examined using computerized image analysis. VEGF could be an additional interesting prognostic variable, as it was significantly associated with tumor grade (P=0.002), stage (P=0.018), and negative estrogen receptor status (P=0.011). EGFR was significantly related to invasive ductal carcinoma (P=0.030), tumor grade (P=0.009), VEGF expression (P=0.013), PD-ECGF/TP expression (P=0.024), and MVD (P=0.050). The finding that VEGF is not correlated to MVD does not rule out a crucial role of VEGF as a key factor in angiogenesis. HER2 could not be correlated to MVD, VEGF expression, or PD-ECGF/TP expression, indicating that this factor is unlikely to be involved in directly regulating angiogenesis, whereas the significant correlations between EGFR and histologic tumor type, tumor grade, the angiogenic factors VEGF and PD-ECGF/TP, and MVD point out that EGF is the major modulating growth factor for angiogenesis in breast cancer.     

Expression of growth factors,growth inhibiting factors,and their receptors in invasive breast cancer. I: An inventory in search of autocrine and paracrine loops

The aim of the present study was to investigate which growth factors, receptors, and growth inhibiting factors are expressed in invasive breast cancer. Five (angiogenic) growth factors and their receptors: platelet-derived growth factor A chain (PDGF-AA) and PDGF receptor alpha (PDGFαR), PDGF-BB and PDGF beta receptor, transforming growth factor alpha (TGFα) and its receptor epidermal growth factor receptor (EGFR), and vascular endothelial growth factor (VEGF) and its receptors vascular endothelial growth factor receptor I (Flt-1) and vascular endothelial growth factor receptor II (Flk-1/KDR); two growth inhibiting factors: transforming growth factor beta-1 (TGFβ1) and TGFβ2) and their receptor couple transforming growth factor beta receptor I (TGFβR-I) and TGFβR-II; and basic fibroblast growth factor (bFGF) were stained by standard immunohistochemistry on frozen sections in 45 cases of invasive carcinoma of the breast. Staining was scored as negative or positive in tumour epithelium, stroma, and blood vessels. TGFβ1 and TGFβ2 were expressed in the tumour cells in 67 per cent and 76 per cent of cases, respectively, whereas PDGFβR and TGFβR-II were expressed in 0 per cent and 2 per cent, respectively. The other factors showed variable expression in tumour cells. All factors were expressed in the stroma in most cases, except Flt-1, Flk-1/KDR, TGFβ2, and TGFβR-II, which showed variable expression, and EGFR, which showed no expression. The endothelium was in most cases positive for bFGF, PDGF-AA, PDGF-BB, VEGF, PDGFαR, PDGFβR, and TGFβ1 but TGFβ2 was negative in most cases and TGFα, EGFR, Flt-1, Flk-1/KDR, TGFβR-I, and TGFβR-II were variably expressed. The most interesting possible auto/paracrine loops, as demonstrated on serial sections and by fluorescence double staining, were the TGFα/EGFR, TGFβs/TGFβR, VEGF/Flt-1, and the VEGF/Flk-1 combinations. In conclusion, growth factors, growth inhibiting factors, and their receptors are frequently expressed in invasive breast cancer. Indications for some possible auto-and paracrine loops have been found, which should encourage further study on the role of these factors in breast cancer proliferation and angiogenesis. © 1998 John Wiley & Sons, Ltd.     

Ductal carcinoma in situ with basal-like phenotype: a possible precursor to invasive basal-like breast cancer.

Basal-like carcinomas have recently been identified in gene expression profiling studies as a subtype of invasive breast cancer. These lesions are estrogen receptor (ER)-negative, progesterone receptor (PR)-negative, and HER2-negative (triple negative), and typically express basal cytokeratins, epidermal growth factor receptor (EGFR), and/or c-kit. As poorly differentiated invasive ductal carcinomas, they presumably have a ductal carcinoma in situ (DCIS) precursor with similar cytologic and immunophenotypic features. However, the frequency and even the existence of a DCIS lesion with an immunophenotype analogous to that of invasive basal-like carcinomas have not been previously evaluated. We studied 66 cases of high nuclear grade DCIS using antibodies to ER, PR, HER2, three basal cytokeratins, EGFR, and c-kit to determine the frequency of the triple negative phenotype, and to determine the relationship between the triple negative phenotype and expression of basal cytokeratins and other biomarkers characteristically expressed by invasive basal-like carcinomas. Four cases (6%) exhibited the triple negative phenotype; the remaining cases showed other combinations of ER, PR, and HER2 expression (nontriple negative). Basal cytokeratins, EGFR, or both were expressed by all four triple negative lesions, but by only 21 of 51 (42%) nontriple negative cases (P = 0.04). We conclude that a small proportion of high-grade ductal carcinomas in situ exhibit an ER-negative/PR-negative/HER2-negative (triple negative) phenotype, and these lesions more commonly show expression of basal cytokeratins and/or EGFR than nontriple negative high-grade DCIS. Given that invasive breast cancers typically share immunophenotypic features with the ductal carcinoma in situ from which they arise, our findings raise the possibility that the triple-negative, basal cytokeratin and/or EGFR-positive DCIS lesions we identified represent a precursor lesion to invasive basal-like carcinomas.     

Inactivation of the PTEN gene protein product is associated with the invasiveness and metastasis, but not angiogenesis, of breast cancer

PTEN is a novel tumor-suppressor gene located on chromosomal band 10q23. Loss of PTEN function has been implicated in the progression of several types of cancer, but the correlation between loss of PTEN expression and advanced carcinomas is not well established. The capacity for angiogenesis of a tumor is known to play a very important role in growth and metastasis, and there have been reports that PTEN relates to angiogenesis. In the present study, formalin-fixed and paraffin embedded tissues from 101 patients with breast carcinomas, including 88 cases of invasive ductal carcinomas and 13 cases of ductal carcinoma in situ (DCIS), were evaluated by immunohistochemical methods for the expression of PTEN and vascular endothelial growth factor (VEGF), as well as microvessel density (MVD). The results were compared with the clinicopathologic parameters. There was no loss of PTEN expression in any of the cases of DCIS, but 28 (32%) of the 88 invasive cases did not express PTEN. Loss of PTEN expression was associated with lymph node metastasis ( P  = 0.03), but did not correlate with tumor size, tumor grade, MVD or recurrence. VEGF expression significantly correlated with lymph node metastasis in invasive ductal carcinoma ( P  = 0.01). There was no correlation between the expression of PTEN and that of VEGF ( P  = 0.63). The present study suggests that loss of PTEN expression is common and correlates with tumor progression and lymph node metastasis in breast carcinoma. The relationship between loss of PTEN and progression of breast cancer may not be explained by modulation of angiogenesis.     

P-cadherin and cytokeratin 5: useful adjunct markers to distinguish basal-like ductal carcinomas in situ

Gene expression profiles of invasive breast carcinomas have identified a subgroup of tumours with worse prognosis, which have been called "basal-like". These are characterized by a specific pattern of expression, being estrogen receptor (ER) and HER2 negative, and frequently expressing at least one basal marker such as basal cytokeratins or epidermal growth factor receptor (EGFR). Previously, our group characterized basal-like tumours in a series of invasive breast carcinomas using P-cadherin (P-CD), p63 and cytokeratin 5 (CK5). Based on that study, we hypothesized that those high-grade basal-like invasive carcinomas might have a pre-invasive counterpart, which could be identified using the same approach. A series of 79 ductal carcinomas in situ (DCIS) were classified into distinct subgroups according to their ER, HER2 and basal markers expression. Luminal DCIS expressed ER and constituted 64.6% of the series; the HER2 overexpressing tumours did not express ER and represented 25.3% of the cases, whereas 10.1% lack the expression of ER and HER2 and expressed at least one basal marker (P-CD, CK5, CK14, p63, vimentin and/or EGFR). These basal-like DCIS were mostly high-grade, with comedo-type necrosis, and consistently showed expression of P-CD and CK5. In conclusion, DCIS with a basal-like phenotype represent a small percentage in our series, being P-CD and CK5, the most useful adjunct markers to distinguish this subset of carcinomas in situ of the breast.     

Expression of MT-1 MMP, MMP2, MMP9 and TIMP2 mRNAs in ductal carcinoma in situ and invasive ductal carcinoma of the breast

We investigated the expression of membrane type-1 (MT1)-MMP, MMP2, MMP9 and TIMP2 mRNAs and their roles in ductal carcinoma in situ (DCIS) and T1 and T2 invasive ductal carcinoma of the breast. We further compared these two types of carcinomas for differences in microvessel density, and expression of angiogenic factors and CD44std. MT1-MMP, MMP2, MMP9 and TIMP2 mRNA were expressed in both DCIS and invasive ductal carcinomas. Expression rates of MT1-MMP, MMP2, MMP9 and TIMP2 mRNAs were not statistically different between DCIS and invasive ductal carcinomas, nor did they differ statistically when grouped by tumor size, histologic grade or nuclear grade of invasive ductal carcinoma. Microvessel density and expression of VEGF and TGF-beta1 were not statistically different between DCIS and invasive ductal carcinoma. CD44std expression was significantly increased in DCIS compared to invasive ductal carcinoma (p < 0.05) and it was also significantly increased in lower clinical stage, histologic grade and nuclear grade of invasive ductal carcinoma (p < 0.05). Axillary node metastasis was significantly correlated with MT1-MMP mRNA, VEGF and TGF-beta1 expression (p < 0.05) and MT1-MMP mRNA was positively correlated with VEGF expression and TIMP2 mRNA (p < 0.05). In summary, patterns of MMP mRNA expression in DCIS and invasive ductal carcinoma suggest that the invasive potential of breast carcinoma is already achieved before morphologically overt invasive growth is observed. As MT1-MMP mRNA expression is significantly correlated with axillary nodal metastasis, it may be useful as a prognostic indicator of invasive ductal carcinoma. Considering the positive correlation of MT1-MMP mRNA and TIMP2mRNA expression, our finding supports a role for TIMP2 in tumor growth, as well as the utility of CD44std as a prognostic indicator of breast cancer.     

Angiopoietins 1 and 2 and Tie-2 receptor expression in human ductal breast disease

Staton C A, Hoh L, Baldwin A, Shaw L, Globe J, Cross S S, Reed M W & Brown N J
(2011) Histopathology 59 , 256–263 Angiopoietins 1 and 2 and Tie‐2 receptor expression in human ductal breast disease Aims: This study aimed to identify the involvement of the angiopoietin/Tie‐2 receptor system in breast cancer development, progression, metastasis and angiogenesis. Methods and results: We quantified and correlated angiopoietin‐1 (Ang‐1), Ang‐2 and Tie‐2 expression in sections of normal human breast, benign and premalignant hyperplastic tissue, pre‐invasive and invasive cancer, and compared these findings with our previously published data on vascular endothelial growth factor (VEGF) and microvessel density (MVD) in the same samples. A breast cancer tissue microarray was used to evaluate the prognostic value of these factors. Histological analysis revealed a significant decrease in Ang‐1 expression (P = 0.001) and an inverse correlation with MVD (r = ?0.442, P = 0.008) and VEGF (r = ?0.510, P = 0.002) in the non‐invasive lesions. In contrast Ang‐2 expression increased significantly (P = 0.0004) with increasing severity of lesion and correlated with MVD (r = 0.570; P = 0.0002), while Tie‐2 expression remained relatively unchanged. Expression of all three factors was reduced in invasive breast cancer and did not correlate with oestrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), lymph node status or tumour grade. Conclusions: These data suggest that a change in the angiopoietin balance in favour of Ang‐2 is associated with the angiogenic switch at the onset of hyperplasia in the breast. However, angiopoietins and the Tie‐2 receptor are not related to known prognostic indicators in invasive breast cancer.     

Angiogenesis and expression of thymidine phosphorylase by inflammatory and carcinoma cells in ductal carcinoma in situ of the breast

Angiogenesis is essential for tumour growth and important in metastasis and for prognosis in invasive carcinoma of the breast. Two patterns of increased vascularity have been shown in mammary ductal carcinoma in situ (DCIS): a cuff of vessels close to the involved ducts, and vessels in the interductal stroma. Inflammation may potentially promote angiogenesis by release of angiogenic factors and digestive enzymes. A correlation has previously been found between the intensity of perivascular inflammation and stromal vascularity in DCIS, but no strong relationship has been observed between inflammation and angiogenesis in invasive carcinoma. Tumour angiogenesis is regulated by a number of angiogenic factors, including thymidine phosphorylase (platelet-derived endothelial cell growth factor), which is expressed at high levels in macrophages. Using immunohistochemical methods, thymidine phosphorylase expression and vascularity have been studied in DCIS (n = 34) and invasive carcinoma (n = 32). Stromal vascularity in DCIS was associated with thymidine phosphorylase expression in the perivascular inflammatory cells and in the cytoplasm of carcinoma cells. In invasive carcinoma, no relationship was found between vascularity and thymidine phosphorylase expression in either the carcinoma or the inflammatory cells. This study suggests that thymidine phosphorylase expression in both inflammatory and carcinoma cells may contribute to one of the patterns of vascularity in DCIS, but not in invasive disease. Copyright © 1999 John Wiley & Sons, Ltd.     

Caveolin-1 is down-regulated and inversely correlated with HER2 and EGFR expression status in invasive ductal carcinoma of the breast

AIMS: To evaluate the caveolin-1 status of invasive ductal carcinoma and its correlation with other important parameters of breast carcinogenesis. Caveolin-1, the main structural protein of caveolae, is involved in the regulation of several intracellular signalling pathways and also functions as a tumour suppressor in breast carcinogenesis. METHODS AND RESULTS: One hundred and thirty cases of invasive ductal carcinomas with matched normal breast tissue were evaluated immunohistochemically for caveolin-1 expression. Using a tissue microarray, caveolin-1 expression was also correlated with the expression of other antigens such as eostrogen receptor, progesterone receptor, epidermal growth factor receptor (EGFR), HER2, beta-catenin, E-cadherin, p53, Ki67 and with clinicopathological parameters. Immunohistochemical results showed strong expression of caveolin-1 in all normal breast epithelial cells, but a reduction of caveolin-1 expression in 56 cases (43.1%) of invasive ductal carcinoma. Furthermore, a statistically significant inverse correlation between caveolin-1 and EGFR and HER2 was noted (P < 0.001). CONCLUSIONS: Our results indicate a reduction in caveolin-1 expression in invasive ductal carcinoma of the breast, which supports in vitro studies of its role as a tumour suppressor. Caveolin-1 also shows an inverse correlation with EGFR and HER2, which fits with its function as a negative regulator of signal transduction.     

Expression of the vascular endothelial growth factor receptor-2/Flk-1 in breast carcinomas: correlation with proliferation

Vascular endothelial growth factor (VEGF) and its receptor Flk-1/KDR play an important role in vascular permeability and tumor angiogenesis. Prompted by the hypothesis that VEGF/Flk-1 system may have regulatory roles in breast carcinogenesis, we investigated the expression of Flk-1 in 141 invasive breast carcinomas in correlation with clinical and immunohistochemical prognostic parameters, including proliferation indices like Ki-67 and Topoisomerase IIalpha (Topo-IIalpha). The immunohistochemical avidin-biotin-peroxidase method was performed on paraffin sections for the detection of Flk-1, p53, Bcl-2, c-erbB-2, Ki-67, Topo-IIalpha, ER, and PR. Flk-1 was detected in 91 of 141 (64.5%) of invasive breast carcinomas showing a widespread cytoplasmic expression in most of the neoplastic cells. Flk-1 expression was correlated with the menopausal status (P = 0.051) of the patient and the nuclear grade of the invasive breast carcinoma (P = 0.003), but demonstrated no correlation with histologic grade, stage, and patient survival. It is interesting that Flk-1 expression demonstrated a significant correlation with 2 well-established proliferation indices, Ki-67 (P = 0.037) and topo-IIalpha (P = 0.009), whereas there was no correlation with the expression of ER, PR, p53, Bcl-2, and c-erbB-2. Moreover, Flk-1 expression showed an inverse correlation with TIMP-1 mRNA localization in intratumoral stromal cells (P = 0.013). In conclusion, the significant correlation of Flk-1 expression in invasive breast carcinomas with proliferation indices like Ki-67 and topo-IIalpha suggests that VEGF may exert a growth factor activity on mammary cancer cells through its receptor Flk-1. On the other hand, the inverse correlation of Flk-1 with TIMP-1 mRNA in intratumoral stromal cells supports the notion that TIMP-1 may have an inhibitory role on angiogenesis.     

HER2 protein overexpression in estrogen receptor-positive ductal carcinoma in situ of the breast: frequency and implications for tamoxifen therapy.

Recent clinical data have suggested that the efficacy of tamoxifen in reducing the risk of local recurrence following lumpectomy and radiation therapy in patients with ductal carcinoma in situ (DCIS) is limited to patients with estrogen receptor (ER)-positive lesions. However, it is currently not known if HER2 protein overexpression might be associated with reduced tamoxifen benefit in patients with ER-positive DCIS, as has been suggested in patients with ER-positive invasive breast cancer and in preclinical models. Moreover, the frequency of HER2 overexpression in ER-positive ductal carcinoma in situ has not been previously evaluated in detail. To address this issue, we studied ER expression and HER2 overexpression in 148 cases of DCIS using a sensitive double immunostaining technique and assessed the frequency of ER expression and HER2 overexpression in relation to each other and in relation to DCIS grade. Overall, ER expression was seen in 114 cases (77%) and HER2 protein overexpression was seen in 42 cases (28%). Of 114 ER-positive ductal carcinoma in situ, 14 (12%) showed concurrent HER2 protein overexpression, and all 14 of these DCIS lesions were of high nuclear grade. In addition, in all 14 ER-positive DCIS cases that showed HER2 overexpression, double immunostaining demonstrated that ER and HER2 protein were coexpressed by the same neoplastic cells. We conclude that a subset of ER-positive DCIS show concomitant overexpression of HER2 protein. Whether or not HER2 overexpression is associated with a diminished response to tamoxifen in patients with ER-positive DCIS will require investigation in clinical outcome studies.     

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.     

Overexpression of laminin-5 gamma2 chain and its prognostic significance in urothelial carcinoma of urinary bladder: association with expression of cyclooxygenase 2, epidermal growth factor receptor [corrected] and human epidermal growth factor receptor [corrected] 2

Laminin-5 (LN-5) and cyclooxygenase 2 (COX-2) play important roles in many kinds of cancers. Recently, it has been reported that epidermal growth factor receptor [corrected] (EGFR) and/or human epidermal growth factor receptor [corrected] 2 (HER2) expressions are associated with LN-5 and/or COX-2 expressions in a few carcinoma cell lines and human tumor tissue. LN-5, COX-2, EGFR, and HER2 expressions were examined immunohistochemically in 67 patients with urothelial carcinomas (UCs), and associations among these 4 biomarkers and clinicopathologic characteristics were investigated. Patients were classified into transurethral resection group and cystectomy group based on clinical end points, and prognostic significances of increased expressions were evaluated. Overexpression of LN-5, COX-2, EGFR, and HER2 was observed in 16 (23.9%), 34 (50.7%), 42 (62.7%), and 15 (22.4%) of 67 patients, respectively. LN-5 overexpression was associated high-grade (P = .002), invasive (pTa+1 versus pT2-4, P = .011), and nonpapillary (P = .027) UCs. Concerning EGFR and HER2, high-grade (EGFR, P = .0009; HER2, P = .003) and nonpapillary (EGFR, P = .016; HER2, P = .0002) UCs had a significantly higher overexpression rate. UCs penetrating basal membrane (pT1-4) showed significantly higher overexpression rates than pTa UCs on all biomarkers. In transurethral resection group, LN-5 overexpression could be proved as an independent prognostic parameter for intravesical recurrence (P = .007), whereas in cystectomy group, nodal involvement was an independent prognostic parameter for cause-specific survival (P = .025). The current study showed that the 4 biomarkers were associated with aggressive behaviors of UCs. Above all, LN-5 overexpression was considered to play an important role in intravesical recurrence of superficial UCs.     

Macrophage infiltration is associated with VEGF and EGFR expression in breast cancer

Angiogenesis is esential for tumour growth and metastasis. Vascular endothelial growth factor (VEGF) is a potent endothelial cell mitogen and is an important component of the angiogenic stimulus in a range of human neoplasias. In addition to its mitogenic activities, VEGF has also been found to stimulate migration in macrophages via the flt-1 VEGF receptor. It has previously been shown that increased focal tumour macrophage infiltration is associated with increased angiogenesis and worsened relapse-free and overall survival in breast cancer. Macrophages are able to stimulate angiogenesis by their production of a range of factors including VEGF, tumour necrosis factor-alpha (TNF-alpha), and thymidine phosphorylase (TP). Thus, in breast cancer, VEGF could have a dual role in the regulation of angiogenesis, by direct mitogenic stimulation of endothelial cells, and also indirectly by attracting macrophages into avascular tumours. The purpose of this study was to localize VEGF protein in a series of 96 consecutive primary breast carcinomas and to determine its relationship to focal macrophage infiltration (macrophage index). These two variables were also compared with the pathological features of the tumours, as well as oestrogen receptor (ER), epidermal growth factor receptor (EGFR), microvessel density, macrophage index, and survival. An inverse relationship (p=0.0006) was noted between VEGF and EGFR, with high VEGF expression correlating with low EGFR levels. In the EGFR-negative group of cases (n=56), positive associations were observed between VEGF expression and macrophage index (p=0.005), ER (p=0.05), p53 (p=0. 006), tumour grade (p=0.02), and tumour necrosis (p=0.03). Macrophage counts were higher in EGFR-positive tumours (p=0.0006) and no associations were found between VEGF expression and increased microvessel density. These results show that in breast cancers there are two types of macrophage infiltrates, one associated with the presence of EGFR and low VEGF expression in tumours and the other with high VEGF expression in EGFR-negative tumours. VEGF expression may be an important factor in the recruitment of tumour-associated macrophages into breast carcinomas and may thus have an additional, indirect, pathway of angiogenic stimulation in this type of tumour.     

Loss of expression of transforming growth factor beta type II receptor correlates with high tumour grade in human breast in-situ and invasive carcinomas

AIMS: Loss of transforming growth factor beta type II receptor (TGFbeta-RII) expression has been associated with resistance to TGFbeta-mediated inhibition of cell proliferation and tumour progression. We investigated whether the expression of TGFbeta-RII is related to the progression of human breast cancer and whether there is a correlation between TGFbeta-RII expression and phenotypic markers of biological aggressiveness. METHODS AND RESULTS: Immunohistochemical methods were used to detect TGFbeta-RII in archival breast samples including benign proliferative lesions, ductal carcinoma in situ (DCIS) and invasive mammary carcinomas (IMC). Neoplastic cells showed reduced expression of TGFbeta-RII in comparison to the normal breast tissue and benign lesions. There was a significant inverse correlation between loss of TGFbeta-RII expression and tumour grade within both DCIS (P = 0.004) and IMC (P = 0.001) groups. There was an inverse correlation between TGFbeta-RII expression and both mitotic count (P = 0.001) and clinical stage (P = 0.004). Oestrogen receptor (P = 0.07) and lymph node status (P = 0.10) were not significantly associated with TGFbeta-RII expression. CONCLUSIONS: These data indicate that decreased expression of TGFbeta-RII may contribute to breast cancer progression and is related to a more aggressive phenotype in both in-situ and invasive carcinomas.     

Correlation of HER2 gene amplification with expression of the apoptosis-suppressing genes bcl-2 and bcl-x-L in ductal carcinoma in situ of the breast.

The protein product of the HER2 oncogene is overexpressed in an estimated 25 to 30% of breast carcinomas and is considered an indicator of poor clinical outcome. The bcl-2 and the bcl-x-L genes are the 2 main genes of the bcl-2 gene family that suppress tumor cell death/apoptosis. HER2 gene amplification is also described in a percentage of cases of ductal carcinoma in situ (DCIS) of the breast. However, the relationship of such overexpression with the apoptosis-suppressing genes is currently unknown. A total of 37 consecutive cases of DCIS were immunostained for HER2 overexpression (clone CB11, Ventana), and expression of bcl-2 and bcl-x-L. DCIS cases were graded using the criteria of Holland et al. HER2 overexpression was scored 0 to 3+; 0 and 1+ were considered negative staining and 2+ and 3+ were considered positive staining. HER2 gene amplification was also confirmed with fluorescent in situ hybridization (FISH). HER2 was positive in 22 of the 37 DCIS cases (60%) in accordance with previous reports. Immunohistochemical overexpression of HER2 was also highly correlated with HER2 amplification by FISH. HER2 overexpression (confirmed by FISH) was mostly seen in grade II (9 of 17) and grade III (9 of 12) DCIS lesions. Only 1 of the HER2-amplified cases was a grade I lesion. Furthermore, HER2 overexpression correlated with the presence of necrosis (P=0.003). Similarly, of the cases overexpressing HER2 at the highest level (3+), 90% were grade II or grade III lesions. A total of 73% of these cases also exhibited necrosis. Overexpression of HER2 3+ was also highly correlated with the presence of the apoptosis-suppressing gene bcl-x-L (coexpression in 87% of cases, P=0.01) but not with the prototype apoptosis-suppressing gene bcl-2 (coexpression in 50% of cases, P value not significant). First, in DCIS overexpression of HER2 the majority of grade II and grade III lesions is seen, and this correlates with the presence of necrosis. Second, HER2 overexpression is also highly correlated with the expression of the apoptosis-suppressing gene bcl-x-L, but not with the prototype apoptosis-suppressing gene bcl-2. These differences may prove useful in defining groups of DCIS lesions with enhanced tumor cell growth and propensity for progression to invasion.     

HER2 status in pure ductal carcinoma in situ and in the intraductal and invasive components of invasive ductal carcinoma determined by fluorescence in situ hybridization and immunohistochemistry

AIM: To determine the HER2 status of ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) of the breast. The increased prevalence of HER2 amplification and overexpression in DCIS is considered to be maintained in the intraductal component of IDC; however, HER2 amplification and overexpression are detected much less in IDC. METHODS AND RESULTS: Fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) were performed to detect HER2 in 270 IDCs with an intraductal component and in 50 pure DCIS samples; IHC was also performed in 116 metastatic nodes. HER2 was found to be amplified in 77 cases (28.5%) and overexpressed in 79 (29.3%) of the 270 IDCs. HER2 amplification was similar between intraductal and invasive components of the same tumour. The concordance for HER2 status between invasive and intraductal components of individual tumours was 98.5% and 99.3% by FISH and IHC, respectively. HER2 was amplified in 25 (50%) of the 50 pure DCIS samples. HER2 overexpression in metastatic nodes resembled the HER2 status in the primary tumour for 108 (93.1%) of 116 cases (kappa =0.831). CONCLUSION: Our study indicates that the intraductal component of IDC may differ biologically when compared with pure DCIS. HER2 appears to lack a critical role in the progression from DCIS to IDC and HER2 status is maintained in metastatic lesions.     

Identification of a basal-like subtype of breast ductal carcinoma in situ

Microarray profiling of invasive breast carcinomas has identified subtypes including luminal A, luminal B, HER2-overexpressing, and basal-like. The poor-prognosis, basal-like tumors have been immunohistochemically characterized as estrogen receptor (ER)-negative, HER2/neu-negative, and cytokeratin 5/6-positive and/or epidermal growth factor receptor (EGFR)-positive. The aim of this study was to determine the prevalence of basal-like ductal carcinoma in situ in a population-based series of cases using immunohistochemical surrogates. A total of 245 pure ductal carcinoma in situ cases from a population-based, case-control study were evaluated for histologic characteristics and immunostained for ER, HER2/neu, EGFR, cytokeratin 5/6, p53, and Ki-67. The subtypes were defined as: luminal A (ER+, HER2-), luminal B (ER+, HER2+), HER2 positive (ER-, HER2+), and basal-like (ER-, HER2-, EGFR+, and/or cytokeratin 5/6+). The prevalence of breast cancer subtypes was basal-like (n = 19 [8%]); luminal A, n = 149 (61%); luminal B, n = 23 (9%); and HER2+/ER-, n = 38 (16%). Sixteen tumors (6%) were unclassified (negative for all 4 defining markers). The basal-like subtype was associated with unfavorable prognostic variables including high-grade nuclei (P < .0001), p53 overexpression (P < .0001), and elevated Ki-67 index (P < .0001). These studies demonstrate the presence of a basal-like in situ carcinoma, a potential precursor lesion to invasive basal-like carcinoma.     

Expression of vascular endothelial growth factor and vascular endothelial growth factor receptors 1 and 2 in invasive breast carcinoma: prognostic significance and relationship with markers for aggressiveness

Dhakal H P, Naume B, Synnestvedt M, Borgen E, Kaaresen R, Schlichting E, Wiedswang G, Bassarova A, Holm R, Giercksky K‐E & Nesland J M
(2012) Histopathology  61, 350–364 Expression of vascular endothelial growth factor and vascular endothelial growth factor receptors 1 and 2 in invasive breast carcinoma: prognostic significance and relationship with markers for aggressiveness Aims: Vascular endothelial growth factor (VEGF), VEGF receptor 1 (VEGFR‐1) and VEGF receptor 2 (VEGFR‐2) play a role in breast cancer growth and angiogenesis. We examined the expression and relationship with clinical outcome and other prognostic factors. Methods and results: Tumour sections from 468 breast cancer patients were immunostained for VEGF, VEGFR‐1, and VEGFR‐2, and their relationships with tumour vascularity, disseminated tumour cells (DTCs) in bone marrow and other clinicopathological parameters were evaluated. VEGF, VEGFR‐1 and VEGFR‐2 immunoreactivities were observed in invasive breast carcinoma cells. VEGF expression was significantly associated with VEGFR‐1 and VEGFR‐2 expression (P < 0.001). High‐level cytoplasmic expression of VEGFR‐1 was associated with significantly reduced distant disease‐free survival (DDFS) (P = 0.017, log‐rank) and breast cancer‐specific survival (BCSS) (P = 0.005, log‐rank) for all patients, and for node‐negative patients without systemic treatment (DDFS, P = 0.03, log‐rank; BCSS, P = 0.009, log‐rank). VEGFR‐1 expression was significantly associated with histopathological markers of aggressiveness (P < 0.05). Significantly reduced survival was observed in DTC‐positive patients as compared with DTC‐negative patients in the combined moderate/high VEGFR‐1 group (P < 0.001 for DDFS and BCSS), and the same was true for DDFS in the moderate VEGFR‐2 group (P = 0.006). Conclusions: High‐level expression of VEGFR‐1 indicates reduced survival. Higher‐level expression of VEGFR‐1 or VEGFR‐2 in primary breast carcinomas combined with the presence of DTC selects a prognostically unfavourable patient group.