Two-color SERS microscopy for protein co-localization in prostate tissue with primary antibody-protein A/G-gold nanocluster conjugates.

SERS microscopy is a novel staining technique in immunohistochemistry, which is based on antibodies labeled with functionalized noble metal colloids called SERS labels or nanotags for optical detection. Conventional covalent bioconjugation of these SERS labels cannot prevent blocking of the antigen recognition sites of the antibody. We present a rational chemical design for SERS label-antibody conjugates which addresses this issue. Highly sensitive, silica-coated gold nanoparticle clusters as SERS labels are non-covalently conjugated to primary antibodies by using the chimeric protein A/G, which selectively recognizes the Fc part of antibodies and therefore prevents blocking of the antigen recognition sites. In proof-of-concept two-color imaging experiments for the co-localization of p63 and PSA on non-neoplastic prostate tissue FFPE specimens, we demonstrate the specificity and signal brightness of these rationally designed primary antibody-protein A/G-gold nanocluster conjugates.

Rapid immuno-SERS microscopy for tissue imaging with single-nanoparticle sensitivity.

Immuno-SERS microscopy is a novel imaging technique in nano-biophotonics, which employs antibodies labeled with SERS-active nanoparticles in conjunction with Raman microscopy. Rapid data acquisition is of central importance for screening large areas of tissue specimens. Here, we first discuss the role of SERS labels with single-particle sensitivity in immuno-SERS microscopy, in particular with respect to false-negative results. In combined single-particle experiments (SERS microscopy/dark-field microscopy/HR-SEM), we then demonstrate that small glass-coated clusters (dimers and trimers) of gold nanospheres exhibit the desired single-particle SERS sensitivity, even at acquisition times as short as 30 msec per pixel, while monomers do not. The proof-of-concept for rapid immuno-SERS microscopy with 30 msec acquisition time per pixel for selective imaging of the p53 family member p63 in prostate tissue sections is demonstrated.

Systematic analysis of in vitro chemosensitivity and mib-1 expression in molecular breast cancer subtypes.

BACKGROUND: Molecular breast cancer subgroups show differences with regard to prognosis and response to chemotherapy. In vitro chemotherapy sensitivity and resistance assays (CSRAs) are a means to directly evaluate tumour cell response to a given drug. METHODS: Using tissue microarray (TMA) analysis, 550 invasive breast cancers were investigated for the expression of oestrogen receptor (ER), progesterone receptor (PR), vimentin, Ck5, Ck14, Ck19, HER2 and Mib-1/Ki-67. All carcinomas were subjected to in vitro CSRA analysis using three separate chemotherapy combinations. In vitro chemotherapy sensitivity was established using an adenosine triphosphate (ATP) bioluminescence assay. Results of immunohistochemical staining and in vitro response were correlated. RESULTS: Mib-1 expression was associated with sensitivity against Paclitaxel/Epirubicin (p = 0.014) and Docetaxel/Epirubicin (p = 0.014). Mib-1 expression was positively/negatively correlated with expression of basal/luminal markers, respectively. Hierarchical clustering revealed three subtypes, resembling luminal, basal-like and HER2-like breast cancer subtypes. In vitro response for Paclitaxel/Epirubicin was most frequently observed for cases in the basal category (40.3%) compared to HER2-like (25.8%) and luminal cases (28.6%). In multivariate analysis expression of Mib-1 was not a significant independent predictor of in vitro response to chemotherapy. CONCLUSION: Breast cancer molecular subclasses show differences regarding in vitro chemotherapy sensitivity. These may in part explain differences observed between breast cancer molecular subtypes in vivo.

Effects of hormone therapy on estrogen synthesis from E1S in the mammary gland of postmenopausal women.

OBJECTIVES: To investigate the effect of hormone therapy (HT) on the expression of hormone receptors and the ex vivo estrogen biosynthesis in the breast. METHODS: Comparative studies were carried out in breast tissue from 28 postmenopausal women undergoing breast surgery due to breast cancer (BC). Glandular breast tissue at least 1cm distant from the tumor was analyzed. Groups included patients having received HT (n=18), and non-user of HT (controls, n=10) prior to BC diagnosis. Steroid sulfatase (STS) activity was evaluated by incubating homogenized breast tissue with [3H]-estrone sulfate. Thin layer chromatography was performed to separate the products estrone (E1) and estradiol (E2). Histomorphometry for breast tissue composition and immunohistochemistry for expression of estrogen receptor (ER) alpha and beta as well as progesterone receptor (PR) were performed. RESULTS: In all groups, significantly more E2 than E1 was produced. Local E2 formation was higher in women having been treated with estrogen and progestogen (p< or =0.05). Local EZ formation was positively correlated with ER alpha expression (r(s)=0.5; p=0.03). Histomorphometrical and immunohistochemical outcomes did not differ between groups. However, the amount of vessels was higher in women having been treated with HT compared to controls (p=0.09). CONCLUSIONS: Long-term HT increases local estrogen formation in normal human breast tissue.

Cytokeratin 8/18 expression indicates a poor prognosis in squamous cell carcinomas of the oral cavity.

BACKGROUND: Intermediary filaments are involved in cell motility and cancer progression. In a variety of organs, the expression of distinct intermediary filaments are associated with patient prognosis. In this study, we seeked to define the prognostic potential of cytokeratin and vimentin expression patterns in squamous cell carcinomas (SCC's) of the oral cavity. METHODS: 308 patients with histologically proven and surgically treated squamous cell carcinomas of the oral cavity were investigated for the immunohistochemical expression of a variety of intermediary filaments including high- and low-molecular weight cytokeratins (Ck's), such as Ck 5/6, Ck 8/18, Ck 1, CK 10, Ck 14, Ck 19 and vimentin, using the tissue microarray technique. Correlations between clinical features and the expression of Cytokeratins and vimentin were evaluated statistically by Kaplan-Meier curves and multivariate Cox regression analysis. RESULTS: The expression of Ck 8/18 and Ck 19 were overall significantly correlated with a poor clinical prognosis (Ck 8/18 p = 0.04; Ck19 p < 0.01). These findings could also be reproduced for Ck 8/18 in primary nodal-negative SCC's and held true in multivariate-analysis. No significant correlation with patient prognosis could be found for the expression of the other cytokeratins and for vimentin. CONCLUSION: The expression of Ck 8/18 in SCC's of the oral cavity is an independent prognostic marker and indicates a decreased overall and progression free survival. These results provide an extended knowledge about the role of intermediary filament expression patterns in SCC's.

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.

First evidence supporting a potential role for the BMP/SMAD pathway in the progression of oestrogen receptor-positive breast cancer.

Oestrogen receptor expression is generally a sign of better tumour differentiation and comparatively good clinical outcome in invasive breast cancer. However, oestrogen receptor-positive, poorly differentiated carcinomas with a poor clinical outcome exist. The underlying genetic mechanisms and the genes involved remain obscure, even though chromosome 7p gains seem to be associated with these uncommon tumours. In this study, we compared two subsets of oestrogen receptor-positive breast cancers, which differed in tumour grade, cytogenetic instability, and tumour proliferation, for their differential gene expression in order to identify proteins involved in the progression of oestrogen receptor-positive breast cancers. We were able to show by means of subtractive suppression hybridization, real-time reverse transcriptase PCR, and tissue microarray analysis that expression of the bone morphogenetic protein receptor IB (BMPR-IB) is a major hallmark of the progression and dedifferentiation of breast cancer. Strong expression of BMPR-IB was associated with high tumour grade, high tumour proliferation, cytogenetic instability, and a poor prognosis in oestrogen receptor-positive carcinomas. Western blot analysis revealed that downstream signalling of this receptor is mainly mediated via phosphorylation of SMAD 1 in oestrogen receptor-positive breast cancer. Even though BMPR-IB was expressed in oestrogen receptor-positive and -negative breast cancers, an impact on tumour grade, proliferation, and cytogenetic instability, as parameters of tumour progression, could only be demonstrated in oestrogen receptor-positive carcinomas. This pro-proliferative effect was complemented by significant anti-apoptotic activity, indicated by XIAP and IAP-2 expression in BMPR-IB-positive carcinomas. These results show that the BMP/SMAD pathway is activated in breast cancer and may contribute to breast cancer progression and dedifferentiation in oestrogen receptor-positive breast cancer. The definition of this pathway characterizes a new potential target in the molecular treatment of invasive breast cancer.

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.

Allelic length of a CA dinucleotide repeat in the egfr gene correlates with the frequency of amplifications of this sequence--first results of an inter-ethnic breast cancer study.

Overexpression of the epidermal growth factor receptor (EGFR) is a common finding in invasive breast cancer and represents a potential target for new treatment options. However, little is known about the parameters that might indicate a potential clinical response for these anti-EGFR-based therapies. In order to gain further insights into the interplay between the length of a CA-SSR I repeat in intron 1 of egfr, copy numbers of this untranslated regulatory sequence, and protein expression, the present study investigated breast cancers from Germans and Japanese patients by microsatellite analysis, quantitative 5' nuclease assay by egfr enzyme-linked immunosorbent assay (ELISA), and comparative genomic hybridization (CGH). Japanese breast cancer patients displayed significantly longer alleles for the CA-SSR I repeat (p < 0.001), associated with significantly lower EGFR expression (mean 65 versus 36 fmol/mg membrane protein). Allelic imbalance (restricted to CA-SSR I) was observed in 55% of the informative Japanese breast cancers compared with only 34% of the German breast cancer reference group. Using a quantitative 5' nuclease assay for egfr, a significantly higher percentage of Japanese breast cancer patients revealed amplifications of the CA-SSR I repeat (p < 0.01). Japanese patients with these amplifications were characterized by a significantly higher EGFR content compared with the German breast cancer patients (p < 0.05). These data show, on the one hand, that the correlation of EGFR overexpression and an inherited CA repeat polymorphism within intron 1 of egfr is a general finding in breast cancer, as has been shown previously. On the other hand, the data demonstrate clearly for the first time an interaction between the length of a polymorphism in intron 1 of egfr as an inherited genetic factor and the frequency of egfr amplification, as an acquired genetic factor, both factors contributing to EGFR overexpression in breast cancer. This new knowledge about mechanisms of regulation of EGFR expression might serve as an additional basis for evaluating anti-EGFR-based therapies.

Poorly differentiated breast carcinoma is associated with increased expression of the human polycomb group EZH2 gene.

Polycomb group (PcG) genes contribute to the maintenance of cell identity, cell cycle regulation, and oncogenesis. We describe the expression of five PcG genes (BMI-1, RING1, HPC1, HPC2, and EZH2) innormal breast tissues, invasive breast carcinomas, and their precursors. Members of the HPC-HPH/PRC1 PcG complex, including BMI-1, RING1, HPC1, and HPC2, were detected in normal resting and cycling breast cells. The EED-EZH/PRC2 PcG complex protein EZH2 was only found in rare cycling cells, whereas normal resting breast cells were negative for EZH2. PcG gene expression patterns in ductal hyperplasia (DH), well-differentiated ductal carcinoma in situ (DCIS), and well-differentiated invasive carcinomas closely resembled the pattern in healthy cells. However, poorly differentiated DCIS and invasive carcinomas frequently expressed EZH2 in combination with HPC-HPH/PRC1 proteins. Most BMI-1/EZH2 double-positive cells in poorly differentiated DCIS were resting. Poorly differentiated invasive carcinoma displayed an enhanced rate of cell division within BMI-1/EZH2 double-positive cells. We propose that the enhanced expression of EZH2 in BMI-1(+) cells contributes to the loss of cell identity in poorly differentiated breast carcinomas, and that increased EZH2 expression precedes high frequencies of proliferation. These observations suggest that deregulated expression of EZH2 is associated with loss of differentiation and development of poorly differentiated breast cancer in humans.

Cytogenetic alterations and cytokeratin expression patterns in breast cancer: integrating a new model of breast differentiation into cytogenetic pathways of breast carcinogenesis.

The introduction of a concept proposing multiple cellular subgroups in the normal female breast, including cytokeratin 5/6 (Ck 5/6)-positive progenitor cells, offers a new explanation for the existence of highly aggressive breast cancers with and without Ck 5/6 expression. Using the tissue microarray technique, 166 breast cancer cases, all characterized by comparative genomic hybridization, were evaluated by immunohistochemistry, using 15 different antibodies (estrogen receptor, progesterone receptor, p53, Ki-67, c-erbB2, epidermal growth factor receptor, cyclins A, D1, and E, bcl-2, p21, p27, Ck 5/6, Ck 8/18, and smooth muscle actin) and chromogenic in situ hybridization for c-erbB2. Biomathematical cluster analysis was applied to confirm the conventional interpretation of the results by an independent approach. Ck 5/6-positive breast carcinomas were in general negative for estrogen receptor and progesterone receptor, were highly proliferating (as reflected by Ki67 and cyclin A), and were associated with specific protein expression patterns, such as expression of p53 and epithelial growth factor receptor (all related to more aggressive tumor behavior), which could further be demonstrated by biomathematical cluster analysis. In contrast Ck 5/6-negative breast carcinomas revealed a lower tumor proliferation rate, an increased expression of p21, p27, c-erbB2, and bcl-2, and a significantly lower number of genetic alterations, with losses of chromosomal material of 16q as the most common genetic alteration. Our data give the first hints to the hypothesis that different cellular subgroups in the female breast give rise to subgroups of breast carcinomas with differing protein expression and cytogenetic alteration patterns that may be related to clinical behavior.

Early placenta insulin-like growth factor (pro-EPIL) is overexpressed and secreted by c-erbB-2-positive cells with high invasion potential.

By differential-display PCR a subclone of the SKBR3 cell line with high in vitro transendothelial invasiveness was identified to express increased levels of the INSL-4 gene. This new member of the insulin-like growth factor family encodes for a peptide, designated early placenta insulin-like (EPIL), being expressed in the so-called "invasive" phase of the placental development. Immunohistochemistry on tissue microarrays revealed a heterogeneous expression of EPIL in breast cancer tissue and no expression in the surrounding stroma cells. A coexpression of pro-EPIL and c-erbB-2 could be observed predominantly in cell clusters at the infiltrating edge of the tumor. Our results give new suggestions for the presence of a signaling network of receptor tyrosine kinases underlying breast cancer invasion and metastasis.