Pia Wülfing

HER2-Positive Circulating Tumor Cells Indicate Poor Clinical Outcome in Stage I to III Breast Cancer Patients

Purpose: Early metastasis in node-negative breast cancer indicates that breast cancer cells obviously can bypass the lymph nodes and disseminate directly hematogenous to distant organs. For this purpose, we evaluated the prognostic value of blood-borne, HER2-positive circulating tumor cells (CTC) in the peripheral blood from 42 breast cancer patients with a median follow-up of 95 months. Experimental Design: Cells were isolated by the patented combined buoyant density gradient and immunomagnetic separation procedure and analyzed by immunocytochemistry. Results: We detected one to eight CTCs in the peripheral blood of 17 of 35 patients (48.6%) presenting no overt metastasis. As a positive control, 7 of 7 (100%) patients with metastatic disease presented positive. Healthy persons and patients (n = 32) operated for nonmalignant diseases presented negative for CTCs. The presence and frequency of HER2-positive CTCs correlated with a significantly decreased disease-free survival (P < 0.005) and overall survival (P < 0.05). Interestingly, in 12 patients with HER2-positive CTCs, the primary tumor was negative for HER2 as assessed by immunohistochemical score and fluorescence in situ hybridization. Conclusions: This study provides some evidence of a prognostic effect of HER2-positive CTCs in stage I to III breast cancer. Future studies have to determine the outcome of patients treated with HER2-targeting therapies with respect to HER2-positive CTC levels because it is not unlikely that high levels of HER2-positive CTCs reflect the activity of the tumor and may predict response to trastuzumab.

The origin of vimentin expression in invasive breast cancer : epithelial-mesenchymal transition, myoepithelial histogenesis or histogenesis from progenitor cells with bilinear differentiation potential?

Vimentin expression is a rather rare finding in invasive breast cancer, and is associated with high tumour invasiveness and chemoresistance. It is currently explained by two different biological theories: direct histogenetic derivation from myoepithelial cells, and epithelial–mesenchymal transition (EMT) reflecting the end‐stage of breast cancer dedifferentiation. In this study we aimed to obtain further insights into the biological hallmarks of these vimentin‐expressing breast cancers. We applied immunohistochemistry for vimentin and 15 other differentiation markers to a series of 364 invasive breast cancer cases, using tissue microarray technology. 7.7% of all tumours expressed vimentin. Almost all of these cases (19/21) were Grade 3 invasive ductal carcinomas, and the majority (13/21) of these were associated with a ductal in situ component. Vimentin expression was also seen in the respective in situ components and correlated positively with the expression of SMA, CD10, CK 5, p53, Mib‐1 and EGFR. A negative correlation was seen for the expression of CK 8/18 and the oestrogen receptor. Vimentin‐expressing carcinomas revealed a significantly higher average absolute number of cytogenetic alterations per case, but a significantly lower frequency of chromosome 16q losses compared to vimentin‐negative cases. Our present results demonstrate that, despite analogies between vimentin‐positive breast cancers and myoepithelial cells in their expression of differentiation‐related proteins, neither myoepithelial histogenesis nor EMT can exclusively explain the biology of these distinct tumours. This is mainly supported by the significantly higher incidence of vimentin‐expressing breast cancers compared to any other myoepithelial breast tumours and the fact that vimentin is already observed in ductal in situ components. We therefore propose the alternative hypothesis that vimentin‐expressing breast carcinomas may derive from breast progenitor cells with bilinear (glandular and myoepithelial) differentiation potential. Copyright

Histone deacetylase-1 and -3 protein expression in human breast cancer: a tissue microarray analysis

SummaryImpaired histone acetylation was recognized to be involved in carcinogenesis. Furthermore, histone deacetylase (HDAC) inhibitors induce differentiation of breast cancer cells and inhibit tumour growth. These results prompted us to study HDAC-1 and -3 expression in breast tumours to establish their potential therapeutic and prognostic significance.HDAC-1 und HDAC-3 protein expression was analyzed immunohistochemically on a tissue microarray (TMA) containing 600 core biopsies from 200 patients. HDAC-1 and -3 expression was correlated to steroid hormone receptor-, Her2/neu- and proliferation status of tumours as well as to overall and disease free survival.Moderate or strong nuclear immunoreactivity for HDAC-1 was observed in 39.8% and for HDAC-3 in 43.9% of breast carcinomas. HDAC-1 and -3 expression correlated significantly with oestrogen and progesterone receptor expression (both p< 0.001). HDAC-1 expression predicted significantly better disease free survival (DFS: p=0.044), in particular, in patients with small tumours of all differentiation types (DFS: p=0.016). Multivariate analysis demonstrated that HDAC-1 is an independent prognostic marker.Our data suggest that evaluation of HDAC-1 protein expression enables a more precise assessment of the prognosis of breast cancer patients. Thus, HDAC-1 expression analysis might be clinically useful to facilitate an individual, risk-directed, adjuvant systemic therapy in breast cancer patients.

Differential roles for membrane-bound and soluble syndecan-1 (CD138) in breast cancer progression.

The heparan sulfate proteoglycan syndecan-1 (Sdc1) modulates cell proliferation, adhesion, migration and angiogenesis. Proteinase-mediated shedding converts Sdc1 from a membrane-bound coreceptor into a soluble effector capable of binding the same ligands. In breast carcinomas, Sdc1 overexpression correlates with poor prognosis and an aggressive phenotype. To distinguish between the roles of membrane-bound and shed forms of Sdc1 in breast cancer progression, human MCF-7 breast cancer cells were stably transfected with plasmids overexpressing wild-type (WT), constitutively shed and uncleavable forms of Sdc1. Overexpression of WT Sdc1 increased cell proliferation, whereas overexpression of constitutively shed Sdc1 decreased proliferation. Fibroblast growth factor-2-mediated mitogen-activated protein kinase signaling was reduced following small-interfering RNA (siRNA)-mediated knockdown of Sdc1 expression. Constitutively, membrane-bound Sdc1 inhibited invasiveness, whereas soluble Sdc1 promoted invasion of MCF-7 cells into matrigel matrices. The latter effect was reversed by the matrix metalloproteinase inhibitors N-isobutyl-N-(4-methoxyphenylsufonyl) glycyl hydroxamic acid and tissue inhibitor of metalloproteinase (TIMP)-1. Affymetrix microarray analysis identified TIMP-1, Furin and urokinase-type plasminogen activator receptor as genes differentially regulated in soluble Sdc1-overexpressing cells. Endogenous TIMP-1 expression was reduced in cells overexpressing soluble Sdc1 and increased in those overexpressing the constitutively membrane-bound Sdc1. Moreover, E-cadherin protein expression was downregulated in cells overexpressing soluble Sdc1. Our results suggest that the soluble and membrane-bound forms of Sdc1 play different roles at different stages of breast cancer progression. Proteolytic conversion of Sdc1 from a membrane-bound into a soluble molecule marks a switch from a proliferative to an invasive phenotype, with implications for breast cancer diagnostics and potential glycosaminoglycan-based therapies.

Expression and prognostic impact of the protein tyrosine phosphatases PRL-1, PRL-2, and PRL-3 in breast cancer

The aim of this study was to investigate the expression of the protein tyrosine phosphatases (PTP) PRL-1, PRL-2, and PRL-3 in human breast cancer and to evaluate its clinical and prognostic significance. PRL-PTP mRNA expression was examined in malignant (n=7) and nonmalignant (n=7) cryoconserved breast tissue samples as well as in eight breast cancer cell lines by RT–PCR. Furthermore, protein expression of PRL-3 was analysed semiquantitatively by immunohistochemistry in ductal breast carcinoma in situ (n=135) and invasive breast cancer (n=147) by use of tissue microarray technology (TMA). In 24 lymph node-positive patients we selected the corresponding lymph node metastases for analysis of PRL-3 expression, and a validation set (n=99) of invasive breast cancer samples was examined. Staining results were correlated with clinicopathological parameters and long-term follow-up. PRL-3 mRNA expression was significantly higher in malignant compared to benign breast tissue. For PRL-1 and PRL-2 expression no significant differences were observed. Staining of TMAs showed PRL-3 expression in 85.9% ductal carcinoma in situ and 75.5% invasive breast carcinomas. Analysis of survival parameters revealed a shorter disease-free survival (DFS) in patients with PRL-3-positive carcinomas, and in particular a significantly shorter DFS in nodal-positive patients with PRL-3 overexpressing tumours as compared to PRL-3-negative breast carcinomas (66±7 months (95% CI, 52–80) vs 97±9 months (95% CI, 79–115); P=0.032). Moreover, we found a more frequent expression of PRL-3 in lymph node metastases as compared to the primary tumours (91.7 vs 66.7%; P=0.033). Our results suggest that PRL-3 might serve as a novel prognostic factor in breast cancer, which may help to predict an adverse disease outcome.

Analysis of cyclooxygenase-2 expression in human breast cancer: high throughput tissue microarray analysis

PurposeThe objective of this study was to evaluate breast carcinomas for the expression of cyclooxygenase-2 (Cox-2) using a tissue microarray (TMA) and to determine its clinical and prognostic relevance.MethodsWe analyzed Cox-2 expression in 600 samples from 200 breast carcinomas immunohistochemically performing TMA technology and semiquantitative analysis. Results were correlated with various clinicopathological variables and follow-up data. Expression of estrogen receptor, progesterone receptor, Ki-67, and Her-2/neu-oncogene was analyzed and correlated with Cox-2 status.ResultsWe observed a moderate or strong cytoplasmic staining for Cox-2 in 78 (40.6%) of breast carcinomas. Increased Cox-2 expression corresponded to higher pT stage (P=0.038), amplification of Her-2/neu (P=0.032), lymphovascular invasion (P=0.006), a high MIB-1 labeling index (LI) (P<0.001), and histological grading (P=0.013). We also observed an inverse relationship between strong Cox-2 expression and estrogen and progesterone receptor content of tumors (P=0.037 and P=0.010). However, we could not demonstrate a significant association between Cox-2 staining and overall survival or disease free survival time.ConclusionsThese results suggest that Cox-2 expression is significantly associated with less differentiated and more aggressive breast carcinomas and might therefore be a useful prognostic indicator as well as a target for therapy.

An expression signature of syndecan-1 (CD138), E-cadherin and c-met is associated with factors of angiogenesis and lymphangiogenesis in ductal breast carcinoma in situ

IntroductionHeparan sulphate proteoglycan syndecan-1 modulates cell proliferation, adhesion, migration and angiogenesis. It is a coreceptor for the hepatocyte growth factor receptor c-met, and its coexpression with E-cadherin is synchronously regulated during epithelial-mesenchymal transition. In breast cancer, changes in the expression of syndecan-1, E-cadherin and c-met correlate with poor prognosis. In this study we evaluated whether coexpression of these functionally linked prognostic markers constitutes an expression signature in ductal carcinoma in situ (DCIS) of the breast that may promote cell proliferation and (lymph)angiogenesis.MethodsExpression of syndecan-1, E-cadherin and c-met was detected immunohistochemically using a tissue microarray in tumour specimens from 200 DCIS patients. Results were correlated with the expression patterns of angiogenic and lymphangiogenic markers. Coexpression of the three prognostic markers was evaluated in human breast cancer cells by confocal immunofluorescence microscopy and RT-PCR.ResultsCoexpression and membrane colocalization of the three markers was confirmed in MCF-7 cells. E-cadherin expression decreased, and c-met expression increased progressively in more aggressive cell lines. Tissue microarray analysis revealed strong positive staining of tumour cells for syndecan-1 in 72%, E-cadherin in 67.8% and c-met in 48.6% of DCIS. E-cadherin expression was significantly associated with c-met and syndecan-1. Expression of c-met and syndecan-1 was significantly more frequent in the subgroup of patients with pure DCIS than in those with DCIS and a coexisting invasive carcinoma. Levels of c-met and syndecan-1 expression were associated with HER2 expression. Expression of c-met significantly correlated with expression of endothelin A and B receptors, vascular endothelial growth factor (VEGF)-A and fibroblast growth factor receptor-1, whereas E-cadherin expression correlated significantly with endothelin A receptor, VEGF-A and VEGF-C staining.ConclusionSyndecan-1, E-cadherin and c-met constitute a marker signature associated with angiogenic and lymphangiogenic factors in DCIS. This coexpression may reflect a state of parallel activation of different signal transduction pathways, promoting tumour cell proliferation and angiogenesis. Our findings have implications for future therapeutic approaches in terms of a multiple target approach, which may be useful early in breast cancer progression.

Endothelin-1-, Endothelin-A-, and Endothelin-B-Receptor Expression Is Correlated with Vascular Endothelial Growth Factor Expression and Angiogenesis in Breast Cancer

Purpose: Endothelin-1 (ET-1) and its receptors (ETAR and ETBR), referred to as the endothelin (ET) axis, are overexpressed in breast carcinomas, and influence tumorigenesis and tumor progression by various mechanisms, including angiogenesis. The objective of the study was to clarify if expression of the ET axis participates in angiogenesis of breast carcinoma Experimental Design: We analyzed expression of ET-1, ETAR, ETBR, and vascular endothelial growth factor (VEGF) immunohistochemically in 600 tissue array specimens from 200 paraffin-embedded breast carcinomas performing tissue microarray technology. Microvessel density (MVD) was determined by counting microvessels (identified by factor VIII) in each core specimen. Results: Moderate or strong immunostaining was observed for ET-1 in 25.4%, for ETAR in 43.7%, and for ETBR in 22.2% of breast carcinomas. Of all cases, 44.7% showed significant expression of VEGF. MVD varied between different tumor specimens (range, 0–80; median, 17). We observed a statistically significant correlation between MVD and ET expression status with higher MVD in ET-positive tumors. Moreover, expression of VEGF was found more frequently in tumors with overexpression of the ET axis (each P < 0.001). Staining of VEGF was correlated positively with MVD Conclusions: These results indicate that increased ET-1, ETAR, and ETBR expression is associated with increased VEGF expression and higher vascularity of breast carcinomas and, thus, could be involved in the regulation of angiogenesis in breast cancer. Our findings provide evidence that the expression pattern of the ET-axis and in particular of ETAR may have clinical relevance in future antiangiogenic targeted therapies for breast cancer.

Amplifications of the epidermal growth factor receptor gene (egfr) are common in phyllodes tumors of the breast and are associated with tumor progression

Phyllodes tumors of the breast are rare biphasic tumors with the potential for invasion and metastatic spread. An important role of the epidermal growth factor receptor (EGFR) in phyllodes tumors has been proposed. However, detailed pathogenetic mechanisms remained unclear. We investigated 58 phyllodes tumors of the breast (40 benign, 10 borderline and eight malignant) by means of egfr fluorescence in situ hybridization (FISH) and gene dosage PCR for a regulatory sequence within intron 1 of egfr. Immunohistochemical staining was performed for EGFR, p16, p21, p27, p53, c-myc, Cyclin A, Cyclin D1, Cyclin E, c-kit and Ki67. Immunopositivity for EGFR was detected in 19% of phyllodes tumors (75% of all malignant tumors) in stromal tumor cells but not in the epithelial component. Whole-gene amplifications were seen by FISH in 15.8% (in stromal cells only) and intron 1 amplifications by gene dosage PCR in as much as 41.8% of all phyllodes tumors. Significant correlations were seen between tumor grade on the one hand and EGFR overexpression (P=0.001) and intron 1 amplifications (P<0.05) on the other. EGFR overexpression further correlated positively with immunohistochemical staining for p53, p16, Cyclin A, Cyclin E, Ki67 and c-kit. Presence of intron 1 amplifications correlated with p16 (P<0.01), p21 (P=0.009) and p53 immunoreactivity (P<0.001). Neither EGFR overexpression nor whole-gene amplification was observed in a control series of 167 fibroadenomas and only one of 43 (2.3%) exhibited intron 1 amplification in gene dosage PCR. In conclusion, our results show for the first time that activating mutations in and overexpression of egfr are associated with the progression in grade of phyllodes tumors of the breast. The observed association between intron 1 amplification and overexpression of EGFR provides further insight into regulation mechanisms of EGFR overexpression.

Secretory carcinoma of the breast: a distinct variant of invasive ductal carcinoma assessed by comparative genomic hybridization and immunohistochemistry

Secretory carcinomas (SCA) are distinguished from infiltrating ductal carcinomas (IDC) of the breast by their characteristic histomorphology and more favorable prognosis and by the expression of a chimeric tyrosine kinase that is encoded by the ETV6-NTRK3 fusion gene. On this basis, we evaluated 13 SCAs (12 of them with ETV6-NTRK3 gene fusion) by molecular and immunohistochemical (IHC) methods. DNA was obtained from 8 of 13 microdissected SCAs and was analyzed for genetic alterations (GA) by comparative genomic hybridization (CGH). IHC staining was performed for estrogen receptor (ER), progesterone receptor (PR), HER2/neu, and Ki-67 (MIB1) in all 13 cases. Molecular and immunohistochemical results in SCAs were compared with previous data regarding immunohistochemical and molecular characteristics of IDCs. An average of 2.0 GAs (range: 0 to 6) were detected, including recurrent gains of chromosome 8q (37.5%) and 1q (25%) and losses of 22q (25%). Four of 13 (31%) SCAs were positive for ER, and 2 were positive for PR. The mean MIB1-labeling index was 11.4% (range: <1 to 34%). Her-2/neu protein overexpression was detected in 2 cases, including 1 with strong (score 3+) and 1 with weak HER2/neu expression (score 2+). Fluorescence in situ hybridization analysis of the latter case showed no evidence of HER-2/neu-gene amplification. Compared with previous findings in IDCs, SCAs are characterized by a relatively low number of GAs, a low proliferative rate, infrequent HER2/neu protein overexpression, decreased steroid hormone receptor expression, and expression of ETV6-NTRK3 fusion gene. These results support the hypothesis that SCAs have immunohistochemical and genetic features that distinguish them from IDCs of the usual type.