Detection Of HER2 Research Articles

Highly sensitive detection of cancer antigen human epidermal growth factor receptor 2 using novel chicken egg yolk immunoglobulin

Human epidermal growth factor receptor 2 (HER2) is an important biomarker that plays a crucial role in therapeutic decision-making for breast cancer patients. Ensuring the accuracy and reproducibility of HER2 assays by enzyme-linked immunosorbent assay (ELISA), western blot and immunohistochemistry (IHC) requires high sensitive and specific antibodies. Immunoglobulin Y (IgY) is a kind of avian antibody usually isolated from chicken egg yolks. Generation and use of IgY is of increasing interest in a wide variety of applications within the life sciences. In this study, IgY antibodies against two different truncated proteins of the extracellular domain (ECD) of human HER2 were produced, their sensitivity and specificity were evaluated. Specific IgYs were produced by hens immunized with the ECD proteins of human HER2 in long-standing immunization response and were isolated from yolks with a purity of 90% by water dilution, salt precipitations and ultrafiltration. The anti-HER2 IgYs were analytically validated for specificity by ELISA, western blot, immunocytochemistry and IHC. The IgYs bound desired targets in cells and fixed tissues and showed high affinity to HER2. The results demonstrated the viability of detection of HER2 with IgYs and showed promise for the using of IgYs in strict clinical validation.

A novel antibody–antigen based impedimetric immunosensor for low level detection of HER2 in serum samples of breast cancer patients via modification of a gold nanoparticles decorated multiwall carbon nanotube-ionic liquid electrode

A highly sensitive impedimetric immunosensor based on a gold nanoparticles/multiwall carbon nanotube-ionic liquid electrode (AuNPs/MW-CILE) was developed for the determination of human epidermal growth factor receptor 2 (HER2). Gold nanoparticles were used to enhance the extent of immobilization and to retain the immunoactivity of the antibody Herceptin on the electrode. Cyclic voltammetry and electrochemical impedance spectroscopy were employed for characterization of various layers coated onto the AuNPs/MW-CILE. The impedance measurements at different steps were based on the charge transfer kinetics of the [Fe(CN)6]3−/4− redox pair. The immobilization of antibody and the corresponding antigen–antibody interaction at the electrode surface altered the interfacial electron transfer. The interactions of antibody with various concentrations of antigen were also monitored via the change of impedance response. The results showed that the charge transfer resistance increases linearly with increasing concentrations of HER2 antigen. The linear range and limit of detection were found as 10–110ngmL−1 and 7.4ngmL−1, respectively. The sensitivity and specificity of the immunosensor were validated. The results showed that the prepared immunosensor is a useful tool for screening of trace amounts of HER2 in serum samples of breast cancer patients.

Hormone receptor expression in breast carcinoma at our hospital: An experience

Background: Breast carcinoma is the most common cancer among women in the urban Indian population and second only to cervical cancer in the rural population based on cancer registry data. Prognosis and management of breast cancer are influenced by classic variables such as histologic type, grade, tumor size and lymph node status. More recently hormone receptor (HR) status of estrogen receptor (ER), progesterone receptor (PR), and HER-2/neu expression have opened a new gateway in the field of adjuvant hormonal and/or chemotherapeutic regimen. Objective: The objective of this study was to assess the ER, PR, and HER-2/neu reactivity pattern in breast carcinomas at our hospital. Materials and Methods : A study of 100 patient samples of breast carcinoma was carried out from June 2011 to June 2014 in the Department of Pathology, ESIC Medical College and PGIMSR, ESIC Model Hospital, Rajaji Nagar, Bengaluru. Brief demographic and clinical data were obtained. Immunohistochemistry (IHC) was done by peroxidase antiperoxidase technique for detection of ER, PR, and HER-2/neu receptor status. Details regarding histopathological diagnosis, pathological grading, staging, and HR status of breast carcinoma were collected. Obtained parameters were evaluated using descriptive statistical analysis and presented in terms of percentage. Results : The age of the patients ranged from 24 to 75 years. Majority of tumors were infiltrating ductal carcinomas-not otherwise specified and predominantly histological grade 2. By IHC 52% were ER+/PR+, 25% were HER-2/neu positive and 20% of triple negatives. Conclusion : Detection of hormone expression is of paramount importance since these are one among the classic variables needed for providing suitable adjuvant hormonal and/or chemotherapeutic options, targeted treatment, and predicting prognosis.

Abstract 3742: Detection of EGFR, HER2, HER3 and HER4 homodimerization and heterodimerization using EFC-based cellular assays

Abstract Ligand-induced receptor dimerization is the first functional step in receptor signaling, representing the most proximal, functional read-out for receptor activation. Here we present a novel application of the Enzyme Fragment Complementation system to monitor receptor-receptor interactions at the surface of intact cells, applicable to diverse receptor types such as RTKs, Interleukin receptors, BMP receptors and cytokine receptors, amongst others. For the purpose of this poster, we will focus on the HER family of receptors. It is well understood that the HER family proteins can dimerize with the other members of its family leading to a complicated oncogenic signaling cascade. Surprisingly, existing cellular assays have been unable to faithfully monitor these interactions proximally in a drug discovery setting. We present EFC-based cellular assays that monitor EGFR homodimerization, EGFR-ErbB2 and ErbB2-ErbB3 heterodimerization events at the receptor. The high signal to noise ratio, serum tolerance and reproducibility make these assays ideal for a diverse range of applications for the identification and development of therapeutic small molecules and biologics, including screening, functional characterization, QC lot release assays and neutralizing antibody studies. Citation Format: Jane Lamerdin, Abhishek Saharia, Jennifer Lin-Jones, Mimi Nguyen, Hyna Fabionar, Sangeetha Gunturi, Abha Srivastava, Tom Wehrman. Detection of EGFR, HER2, HER3 and HER4 homodimerization and heterodimerization using EFC-based cellular assays. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3742. doi:10.1158/1538-7445.AM2014-3742

Abstract 32: Heterogeneous expression of Her2, estrogen and progesterone receptors in human breast cancer metastasized to the brain

Abstract Assessment of hormone (estrogen (ER) and progesterone (PR)) and human epidermal growth factor-2 (Her2) receptors expression in breast cancer has been an accepted standard to predict clinical outcome. Importantly, expression of these receptors in primary breast cancer has been an important predictor of visceral organ metastasis. Although many studies of breast cancer have reported risk factors for brain metastasis, including Her2 positivity and ER negativity, currently it is not possible to accurately predict brain metastasis. One possible reason for this impasse could be the expression of the above receptors in different combinations influencing metastasis to the brain (brain mets). To address this possibility, we analyzed six different human brain mets for the expression of Her-2, ER and PR by the fluorescent in situ hybridization (FISH) and immunohistochemistry procedures. To further validate this data, eight different human primary breast tumors were similarly analyzed. The immunohistochemistry analysis showed that brain mets are heterogenous in the expression of these receptors: five were Her2 positive and one negative; four were ER positive and two were negative; five were PR positive and one negative. However, expression of these receptors in their combination is also heterogenous: three brain mets were positive for all of the Her2, ER and PR; one for Her2 positive but negative for ER/PR; one positive for PR but negative for Her2/ER. Similar heterogenous expression of these receptors in primary tumors were also observed using immunohistochemistry procedure. While this data is mostly corroborative to the FISH analysis, the FISH identified receptor positivity could not be established by immunohistochemistry. Additionally, tumors characterized by immunohistochemistry as PR negative were found to express this receptor, albeit in tens of cells among thousands in brain mets. Such differences in results using these two procedures were noted in the Her2 expression in both brain mets and primary tumors. Undoubtedly, FISH is highly sensitive and identifies gene amplification, whereas, immunohistochemistry allows the detection of receptor expression which is heavily dependent on several factors including accessibility of antigen and affinity of the antibody. In conclusion, our analysis suggests that the currently utilized Her2, estrogen and progesterone receptor detection to predict brain mets needs further refinement to allow for better understanding of the mechanisms of metastasis to the central nervous system and treatment of such brain mets. Citation Format: Morgan Labhart, Prema S. Rao, Seshadri Thirumala, Subrahmanyeswara U. Rao. Heterogeneous expression of Her2, estrogen and progesterone receptors in human breast cancer metastasized to the brain. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 32. doi:10.1158/1538-7445.AM2014-32

Detection of Her-2/neu expression in gastric cancer: Quantitative PCR versus immunohistochemistry.

The aim of this study was to compare quantitative polymerase chain reaction (qPCR) with immunohistochemistry (IHC) for the detection of Her-2 in gastric cancer, and to investigate the correlation between the expression levels of human epidermal growth factor receptor 2 (Her-2) and clinical features. Clinical data from 426 cases of gastric cancer were collected. Her-2 expression levels in cancerous tissue were detected using IHC, and the Her-2/neu gene expression levels were determined by qPCR. The correlation between the expression level of Her-2 and clinical features was investigated. The positive expression rate of Her-2 in cancerous tissue detected using qPCR and IHC was 11.17% (46/412) and 13.38% (57/426), respectively. The positive expression of the Her-2 protein/gene was significantly correlated with the depth of invasion and lymphatic metastasis, as well as the TNM stage (P<0.05). No significant correlation was identified between positive expression of the Her-2 protein/gene and tumor location, age, gender, differentiation degree and Lauren classification (P>0.05). The diagnostic consistency was good between the two methods (κ=0.828). The results indicate that the expression of Her-2/neu is closely associated with the development of gastric cancer. qPCR is a convenient, objective and efficient method, which may be used as an alternative to IHC or fluorescence in situ hybridization for the detection of Her-2/neu gene.

Mutually exclusive FGFR2, HER2, and KRAS gene amplifications in gastric cancer revealed by multicolour FISH

Gastric cancer (GC) is a major cause of global cancer mortality. Previous genomic studies have reported that several RTK-RAS pathway components are amplified in GC, with individual tumours often amplifying one component and not others (“mutual exclusivity”). Here, we sought to validate these findings for three RTK/RAS components (FGFR2, HER2, KRAS) using fluorescence in situ hybridisation (FISH) on a series of gastric tumours, cell lines and patient-derived xenografts. Applying dual-colour FISH on 137 gastric tumours (89 FFPE surgical resections and 48 diagnostic biopsies), we observed FGFR2 amplification in 7.3% and HER2 amplification in 2.2% of GCs. GCs exhibiting FGFR2 amplification were associated with high tumour grade (p = 0.034). In FISH positive tumours, striking differences in copy number levels between cancer cells in the same tumour were observed, suggesting intra-tumour heterogeneity. Using a multicolour FISH assay allowing simultaneous detection of FGFR2, HER2, and KRAS amplifications, we confirmed that these components exhibited a mutually exclusive pattern of gene amplification across patients. The FISH data were also strongly correlated with Q-PCR levels and at the protein level by immunohistochemistry. Our data confirm that RTK/RAS components are mutually exclusively amplified in GC, and demonstrate the feasibility of identifying multiple aneuploidies using a single FISH assay. Application of this assay to GC samples, particularly diagnostic biopsies, may facilitate enrollment of GC patients into clinical trials evaluating RTK/RAS directed therapies. However, the presence of intra-tumour heterogeneity may require multiple biopsy samples to be obtained per patient before a definitive diagnosis can be attained.

The expression and significance of five types of miRNAs in breast cancer.

BackgroundThis study aimed to investigate the expression and significance of 5 types of miRNAs in breast cancer to provide a theoretical and practical foundation for using these miRNAs in the diagnosis and treatment of breast cancer, thereby improving medical services.Material/MethodsStem-loop real-time RT-PCR was used to detect the expression levels of miR-145, miR-21, miR-10b, miR-125a, and miR-206 in 35 cases of breast cancer and adjacent normal breast tissues, and to analyze the relationship of miRNAs expression with clinicopathological features of breast cancer. The expression levels of estrogen receptor (ER) and progesterone receptor (PR) were examined by immunohistochemistry. Fluorescence in situ hybridization was used for the detection of HER-2 and TOP 2A.ResultsThe expression levels of miR-145, miR-125a, and miR-206 in breast cancer were lower than those in adjacent normal tissues. MiR-145 was negatively correlated with tumor size, lymph node metastasis, ER, HER-2, and TOP 2A (P<0.05), regardless of age, menstruation, and PR. MiR-125a was correlated with negative node status, negative HER-2 status (P<0.05), whereas tumor size, age, menstruation, ER, and PR were independent factors. MiR-206 expression was correlated with negative ER status, negative PR status, and negative HER-2 status (P<0.05), regardless of age, menstruation, lymph node metastasis, and TOP 2A. MiR-21 and miR-10b expression in breast cancer tissues was significantly higher than that in adjacent tissues (P<0.05). MiR-21 in post-menstrual patients with lymph node metastasis was highly expressed (P<0.05), and had no correlations with tumor size, ER, PR, and TOP 2A expression. MiR-10b expression was positively correlated with breast cancer tumor size, lymph node metastasis, and TOP 2A status (P<0.05), but had no correlations with age, menstruation, ER, PR, and HER-2.ConclusionsMiR-145, miR-21, miR-10b, miR-125a, and miR-206 may play important roles in breast cancer development and invasion.

Positron Emission Tomography Imaging with 18F-Labeled ZHER2:2891 Affibody for Detection of HER2 Expression and Pharmacodynamic Response to HER2-Modulating Therapies

Expression of HER2 has profound implications on treatment strategies in various types of cancer. We investigated the specificity of radiolabeled HER2-targeting ZHER2:2891 Affibody, [(18)F]GE-226, for positron emission tomography (PET) imaging. Intrinsic cellular [(18)F]GE-226 uptake and tumor-specific tracer binding were assessed in cells and xenografts with and without drug treatment. Specificity was further determined by comparing tumor localization of a fluorescently labeled analogue with DAKO HercepTest. [(18)F]GE-226 uptake was 11- to 67-fold higher in 10 HER2-positive versus HER2-negative cell lines in vitro independent of lineage. Uptake in HER2-positive xenografts was rapid with net irreversible binding kinetics making possible the distinction of HER2-negative [MCF7 and MCF7-p95HER2: NUV60 (%ID/mL) 6.1 ± 0.7; Ki (mL/cm(3)/min) 0.0069 ± 0.0014] from HER2-positive tumors (NUV60 and Ki: MCF7-HER2, 10.9 ± 1.5 and 0.015 ± 0.0035; MDA-MB-361, 18.2 ± 3.4 and 0.025 ± 0.0052; SKOV-3, 18.7 ± 2.4 and 0.036 ± 0.0065) within 1 hour. Tumor uptake correlated with HER2 expression determined by ELISA (r(2) = 0.78), and a fluorophore-labeled tracer analogue colocalized with HER2 expression. Tracer uptake was not influenced by short-term or continuous treatment with trastuzumab in keeping with differential epitope binding, but reflected HER2 degradation by short-term NVP-AUY922 treatment in SKOV-3 xenografts (NUV60: 13.5 ± 2.1 %ID/mL vs. 9.0 ± 0.9 %ID/mL for vehicle or drug, respectively). [(18)F]GE-226 binds with high specificity to HER2 independent of cell lineage. The tracer has potential utility for HER2 detection, irrespective of prior trastuzumab treatment, and to discern HSP90 inhibitor-mediated HER2 degradation.

RETRACTED: Correlations of β-catenin, Ki67 and Her-2/neu with gastric cancer

To study the clinical pathologic characteristics of β-catenin, Ki67 and Her-2/neu in gastric cancer and the correlation of β-catenin and Ki67 to the protein expression and gene conditions of Her-2/Neu. The protein expression of β-catenin, Ki67 and Her-2/Neu was detected by immunohistochemistry in 101 cases of gastric cancer and the gene conditions of Her-2/Neu by fluorescence in situ hybridization (FISH). The protein expression of β-catenin, Ki67 and Her-2/Neu had close relationship with the clinical pathologic characteristics of gastric cancer. The β-catenin and Ki67 had obvious correlation to the differentiation, infiltration and lymphatic metastasis of the gastric cancer (P<0.05). The Ki67 had close relationship with the tumor-node-metastasis staging staging of gastric cancer (P<0.05). Her-2/Neu had close relationship with the differentiation and tumor-node-metastasis staging of gastric cancer (P<0.05) but had no relationship with the infiltration and lymphatic metastasis of the gastric cancer (P<0.05). The protein expression of Ki67 had significantly positive correlation to the protein expression and gene amplification conditions of Her-2/Neu (r=0.567, P<0.05 for protein; r=0.304, P<0.05 for gene). Combined detection of β-catenin, Ki67 and Her-2/Neu can be used as a reliable method to help the observation of biological behavior, diagnosis and prognosis of gastric cancer, and Ki67 can be used to serve the preliminary screening of Her-2/Neu gene state.

A novel method combining gene and protein platforms on one slide for the detection of HER2 in gastric cancer: Final results.

19 Background: The evaluation of gene amplification and/or protein overexpression of HER2 in gastric cancer is a prerequisite to establish an adequate treatment strategy. Gastric tumors are heterogeneous and separate evaluations lead to uncertainties in localizing distinct clones and are time consuming. The aim of this study was to evaluate the feasibility of gene-protein platform in comparison to single staining methods. Methods: Immunohistochemistry (IHC) plus silver in situ hybridization (SISH) (IHC/SISH) and the new gene-protein platform (gene/protein) method for HER2 were performed in randomly collected 100 cases of gastric carcinoma. Evaluation was performed by two observers, in discrepant cases a third observer was consulted to make a decision by consensus. Results of IHC and SISH were compared with gene/protein staining. Rüschoff criteria were applied. Tumors showing HER2 expression 3+ or amplification were considered HER2 positive. Results: 96 of 100 samples were eligible. Amplification was observed in 14.6% by both, conventional SISH and gene/protein platform. 70.8% by IHC and gene/protein had no expression (0) and 10.4%/11.5% (IHC vs. gene/protein) had weak (1+) HER2 expression. Moderate expression (2+) was observed in 9.4% by IHC and 7.3% by gene/protein. Rate of overexpression (3+) was similar in IHC (9.4%) and gene/protein (10.4%). There were complete concordances (100%; κ=1) in IHC assessment of cases with score 0 and SISH amplified tumors. High concordance are shown in score 1+ (98.96%; κ=0.947) and 3+ (96.88%; κ=0.825) cases. Concordance in cases with score 2+ was found in 95.83% (κ=0.728) of the observations. After third observation, there were 5 discordant cases with most discrepancies in assessment of IHC score 2+ or 3+. Conclusions: Gene-protein platform has been tested for first time in gastric carcinoma. Results showed that this novel platform can be a feasible alternative to single methods. Discrepancies in cases with moderate HER2 expression are a result of observer variability.

Detection of circulating tumor cells during follow-up of patients with early breast cancer: Clinical utility for monitoring of therapy efficacy

Introduction. Circulating tumor cells (CTCs) detection prior to and during therapy is considered as an independent and strong prognostic marker. The present study was designed to isolate and characterize CTCs in peripheral blood of an early breast cancer (BC) patient as a biomarker for monitoring treatments efficacy. Materials and methods. In total, 54 early breast cancer patients undergoing neoadjuvant and/or adjuvant chemotherapy regimens were enrolled into a prospective study. CTC detection in blood was performed by AdnaTest BreastCancer™ (AdnaGen AG, Germany), which is based on the detection of EpCAM, HER2 and MUC1 specific transcripts in enriched CTC-lysates. Additionally, cDNA from isolated CTCs and PBMC was used for qPCR gene expression analysis of TOP1, TOP2A, CTSD, ST6, CK19 and reference gene actin. Results. We found that CTCs can be detected in the peripheral blood of approximately 31% of early stage breast cancer patients. The presence of CTCs was detected in 36% ER positive, 32% PR positive and 30% HER2 positive patients. We found no correlation between CTCs and tumor size, tumor grade, histological grade and receptor status. Only 7% of all patients remained CTCs positive after adjuvant therapy. Gene expression analysis revealed a particular heterogeneity of the studied genes. Conclusions. In conclusion, CTC detection may be a promising early marker of disease progression potentially enhancing the difficult therapeutic decisions. Further studies should, however, clearly demonstrate its utility for both the prediction of outcome and monitoring the effect of treatment.

Abstract P1-04-13: The role of excision repair cross-complementation 1 enzyme (ERCC1)-positive circulating tumor cells in primary and metastatic breast cancer

Abstract Background: Multi-drug resistance to chemotherapeutic agents, as well as resistance to radiation therapy is a major cause of treatment failure in breast cancer. The expression of the excision repair cross-complementation 1 (ERCC1) enzyme in tumor tissue has been associated with resistance to platinum based chemotherapy but also with radiation therapy in different cancer diseases. However, studies on ERCC1 in breast cancer are limited. Furthermore, after primary tumor resection, there is no validated clinical parameter to predict the sensitivity to radio/chemotherapy. Circulating Tumor Cells (CTCs), as potential surrogate markers for minimal residual disease, would be an ideal ‘surrogate tissue’ to identify such biomarkers. Here we evaluated ERCC1 expression in CTCs of primary and metastatic breast cancer patients as a potential biomarker for radio/chemoresistance and correlated these findings with the presence of HER2-positive and stemness like CTCs (slCTCs), including cells being able to perform epithelial-mesenchymal transition (EMT). Patients and Methods: 2 × 5 ml blood from primary (n = 110) and metastatic (n = 56) breast cancer patients were analyzed for CTCs with the AdnaTest BreastCancer (AdnaGen AG, Hannover, Germany) for the detection of EpCAM, MUC-1, HER-2, and beta-Actin transcripts. The recovered c-DNA was additionally tested for the presence of a) ERCC1 b) slCTCs using the AdnaTest TumorStemCell for the expression of ALDH1 (both using single-plex RT-PCR) and c) CTCs in EMT applying the AdnaTest EMT (multiplex RT-PCR for TWIST, AKT2, PI3K). The analysis of PCR products was performed by capillary electrophoresis on the Agilent Bioanalyzer 2100. Results: In primary breast cancer, the overall detection rate for CTCs was 27% (30/110 patients) with the expression rate of 83% for HER2. ERCC1 was detected in 51/110 patients (46%), slCTCs in 27/110 patients (25%) and CTCs in EMT in 47/110 patients (43%), respectively. ERCC1 expression was significantly associated with the presence of HER2-positive CTCs (p = 0.001), slCTCs (p = 0.014) and CTCs in EMT (p = 4×10E-8), respectively. In metastatic breast cancer, the positivity rate for CTCs was 36% (20/56 patients) with the expression rate of 40% for HER2. ERCC1 was detected in 15/56 patients (27%), slCTCs in 16/56 patients (29%) and CTCs in EMT in 12/56 patients (22%), respectively. ERCC1 expression was significantly associated with the presence of HER2-positive CTCs (p = 0.0019), slCTCs (p = 0.00014) and CTCs in EMT (p = 0.00037), respectively. Conclusion: The tumor stem cell phenotype and EMT are widely discussed to be correlated with increasing cell survival and chemotherapy resistance including increased DNA repair capacity characterized by the overexpression of genes like ERCC1. Using CTCs as a potential surrogate marker for radio/chemoresistance, we could show a significant correlation of the ERCC1 profile and CTCs displaying tumor stem cell or EMT characteristics but also a HER2-positive phenotype. Thus, the presence as well as the molecular profile of CTCs might become an important tool to predict radio/chemoresistance in breast cancer patients. The clinical relevance on prognosis and therapy response has to be evaluated in a prospective trial. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-04-13.

HER2 mRNA Status Contributes to the Discrepancy Between Gene Amplification and Protein Overexpression in Gastric Cancer

Human epidermal growth factor receptor 2 (HER2) is an important proto-oncogene of prognostic use in gastric cancer (GC). Fluorescence in-situ hybridization (FISH) and immunohistochemistry (IHC) are the main clinical methods of detection of HER2, but consistency between the methods is poor and the cause of the discrepancy is unclear. To investigate the involvement of HER2 mRNA status in the disparity between gene amplification and protein overexpression. We investigated HER2 gene, mRNA, and protein profiles in gastric precancer and cancer tissues by use of the molecular approaches FISH, real-time polymerase chain reaction, and IHC. The relationships between HER2 and matrix metalloproteinase 9 (MMP9) and Smad7 expression were analyzed and the involvement of HER2 in the interaction between tumor cells and lymphocytes was investigated by coculturing GC cell lines with peripheral blood mononuclear cells (PBMCs). HER2 protein expression was significantly increased in cancer compared with precancer (P=0.003), and the corresponding mRNA levels were significantly lower in precancer and cancer tissues than in normal tissues (κ=0.290, P=0.025). HER2 mRNA levels were significantly higher in tumor than in peritumor tissue (P=0.028), and were positively correlated with MMP9 and Smad7 mRNA levels in tumor tissues. HER2 mRNA expression in GC cell lines was increased by coculture with PBMCs. Different HER2 mRNA profiles, possibly in relation to contact between tumor cells and lymphocytes, might help to explain the discrepancy between gene amplification and protein overexpression results.

Abstract B140: Positron emission tomography imaging of HER2 expression and pharmacodynamic response to HSP90 inhibition with the next-generation ZHER2:2891 Affibody molecule [18F]GE-226.

Abstract Accurate assessment of HER2 status remains a clinical challenge, with up to 20% of patients being potentially withdrawn from therapy or exposed to unnecessary toxicity. Non-invasive imaging is widely seen as a viable alternative to current methods, in particular in the setting of locoregional and distant recurrences not amenable to biopsy. A next-generation HER2-targeting Affibody-based radiotracer has been developed, [18F]GE-226, with enhanced pharmacokinetic characteristics and improved properties for large-scale and GMP grade synthesis. Kinetic modeling gave insights into Affibody-HER2 interactions. Intrinsic affinity to HER2 (KD = 76 pM) resulted in 11 to 67-fold higher [18F]GE-226 uptake in ten HER2 positive versus negative cell lines in vitro independent of lineage. Uptake correlated with HER2 protein expression but was independent of presence of other targets like EGFR. Blocking with [19F]GE-226 and HER2 siRNA treatment reduced uptake by 96.8 ± 2.6% and 81.7 ± 9.2%, respectively. Uptake in HER2 positive xenografts was rapid with steady state net irreversible binding kinetics making possible the distinction of HER2 negative (MCF7 (n = 6) and MCF7-p95HER2 (n = 3): NUV60 (normalized uptake value at 60 min; %ID/mL) 6.1 ± 0.7; Ki (irreversible uptake rate; mL/cm3/min) 0.0069 ± 0.0014) from HER2 positive tumors (NUV60 and Ki: MCF7-HER2, 10.9 ± 1.5 and 0.015 ± 0.0035; MDA-MB-361, 18.2 ± 3.4 and 0.025 ± 0.0052; SKOV-3, 18.7 ± 2.4 and 0.036 ± 0.0065; all n = 6) within 1 h. Tumor uptake correlated with HER2 expression determined by ELISA (r2=0.78). Specificity was further determined by comparing tumor localization of a fluorescently labeled tracer analogue with DAKO HercepTest. Affibody signal co-localized with HER2 expression at the cellular level independent of spatial heterogeneity. Tracer binding was not influenced by short-term or continuous exposure to trastuzumab in SKOV-3 xenografts (n=6) in keeping with differential epitope binding. Inhibition of the chaperone HSP90– of which HER2 is a client protein– by the therapeutic development candidate NVP-AUY922 caused dose-dependent HER2 degradation and consequently reduced tracer uptake in SKOV-3 cells in vitro and xenografts in vivo (area under the curve, AUC0-60: 618.4±90.1 and 446.7±42.8 %ID/mL*min for vehicle (n=4) and drug (n=5), respectively; P=0.043). In conclusion, [18F]GE-226 differentiates HER2 negative from HER2 expressing tumors. The tracer has potential utility for HER2 detection, irrespective of prior trastuzumab treatment and to monitor response to HSP90 inhibition. Lineage-independence of these results extends application beyond breast cancer. Due to the specific annotation to HER2, enhanced pharmacokinetic properties and completion of initial preclinical toxicology testing, [18F]GE-226 is now transitioning into clinical development. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B140. Citation Format: Sebastian Trousil, Susan Hoppmann, Quang-Dé Nguyen, Maciej Kaliszczak, Giampaolo Tomasi, Peter Iveson, Duncan Hiscock, Eric O. Aboagye. Positron emission tomography imaging of HER2 expression and pharmacodynamic response to HSP90 inhibition with the next-generation ZHER2:2891 Affibody molecule [18F]GE-226. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B140.

Consistency of gene amplification and its expression at protein level of human epidermal growth factor receptor 2 in gastric carcinoma

Objective To investigate the consistency of gene amplification and its expression at protein level of human epidermal growth factor receptor 2 (HER2) in gastric carcinoma.Methods From 2010 to 2012,120 gastric cancer specimens of patients with gastric cancer were collected,of which 100 were surgical specimens and 20 were specimens from biopsy under gastroscope.The protein expression of HER2 in 120 specimens was detected by immunohistochemistry (IHC).According to the results of IHC,the positive parts of HER2 expression of IHC slices were developed into tissue microarrays for fluorescence in situ hybridization (FISH) to test the gene amplification of HER2.The different parts with different color intensity of focal (+ + +) (≤ 10％ tumor cell strongly staining) specimens detected by IHC detection were compared with the results of FISH.Kappa test was performed for statistical analysis.Results Among 120 gastric cancer specimens,the results of IHC indicated that 77 specimens were positive with different staining intensity including 16 strong positive (+++),six focal positive (+++),37 moderate positive (++) and 18 weak positive (+).The positive rate of HER2 protein expression detected by IHC was 18.3％ (22/120).The results of FISH showed 41 specimens were positive and the rate of gene amplification was 34.2％.Among which,21 were moderate positive (++) detected by IHC,15 were strong positive (+ + +) and five were focal positive (+ ++).The positive rate of HER2 was 35.0％ (42/120) with IHC and FISH combined detection.The consistent rate of IHC and FISH was 91.6 ％ (76/83).Kappa coefficient was 0.960 (P＜0.01).In five positive specimens detected by FISH and which were focal positive (+ + +) by IHC,the different parts with different color intensity were compared with the results of FISH and gene amplification was found in all specimens.Conclusion Tissue microarray technology is consistent with IHC in HER2 detection in gastric cancer specimens and could help to improve the detection rate. Key words: Stomach neoplasms; Receptor, epidermal growth factor; Immunohistochemistry; In situ hybridization, fluorescence; Gene amplification

Manually Created Tissue Microarrays for Immunohistochemical Detection of Her-2, ER, and PR in Carcinoma Breast

Breast cancer is the most common cancer in women worldwide. HER-2 and ER/PR are the commonest molecular markers implicated in its prognosis and management. The new molecular classification of breast cancer is based on the expression of HER-2, estrogen receptor (ER), and progesterone receptor (PR). Tissue microarray is a technique by which many tissue …

Parallel Visualization of Multiple Protein Complexes in Individual Cells in Tumor Tissue

Cellular functions are regulated and executed by complex protein interaction networks. Accordingly, it is essential to understand the interplay between proteins in determining the activity status of signaling cascades. New methods are therefore required to provide information on different protein interaction events at the single cell level in heterogeneous cell populations such as in tissue sections. Here, we describe a multiplex proximity ligation assay for simultaneous visualization of multiple protein complexes in situ. The assay is an enhancement of the original proximity ligation assay, and it is based on using proximity probes labeled with unique tag sequences that can be used to read out which probes, from a pool of probes, have bound a certain protein complex. Using this approach, it is possible to gain information on the constituents of different protein complexes, the subcellular location of the complexes, and how the balance between different complex constituents can change between normal and malignant cells, for example. As a proof of concept, we used the assay to simultaneously visualize multiple protein complexes involving EGFR, HER2, and HER3 homo- and heterodimers on a single-cell level in breast cancer tissue sections. The ability to study several protein complex formations concurrently at single cell resolution could be of great potential for a systems understanding, paving the way for improved disease diagnostics and possibilities for drug development.

Detection of Truncated HER2 Forms in Formalin-Fixed, Paraffin-Embedded Breast Cancer Tissue Captures Heterogeneity and Is Not Affected by HER2-Targeted Therapy

Truncated forms of HER2, previously identified in subsets of HER2-positive breast cancer, originate from proteolytic extracellular domain (ECD) cleavage or alternative translation initiation. They lack ECD but may retain intracellular domain functionality, potentially associated with unfavorable prognosis, metastasis, and decreased sensitivity to antibody-based HER2-targeted therapy. To study the distribution of truncated HER2 in breast cancer, we detected loss of membrane-bound ECD independently of its molecular origin in paraffin sections, combining multispectral unmixing of chromogenic duplex IHC for HER2 ECD and intracellular domain with advanced image analysis. HER2 C-terminal fragment 611-transfected MCF7 and 4-aminophenylmercuric acetate-treated SKBR3 cell lines were used as controls. Applying a prototype work flow to whole sections, paired surgical resection/core needle biopsy samples, and paired samples from 69 patients of a phase 2 neoadjuvant clinical trial, we observed unexpected heterogeneity of ECD loss at the single-cell level, and in different areas of individual tumors, indicating that extent and localization of HER2 ECD loss add relevant information to averaging truncated HER2 across whole sections. We show acceptable run-to-run variation (coefficient of variation, <0.15), image analysis results in moderate agreement with conventional slide assessment (Cohen's κ = 0.59), and no obvious interference with previous HER2-ECD-targeted therapy. We conclude that duplex IHC and digital image processing extend current approaches of truncated HER2 detection.

Evaluation of risk recurrence in early breast cancer assessed by Oncotype DX and tumor cell dissemination to blood and bone marrow.

e11516 Background: The OncotypeDX assay is a validated genomic test that predicts the likelihood of breast cancer (BC) recurrence, patients’ (pts) survival within 10 years of diagnosis and benefit of chemotherapy in early-stage, N0, ER-pos BC. In addition, disseminated tumor cells (DTC) in the bone marrow (BM) and circulating tumor cells (CTCs) in blood, especially stemness like tumor cells (slCTCs) including cells being able to perform epithelial-mesenchymal transition (EMT), have been associated with worse outcome in BC. Here we use OncotypeDX as well as the presence of DTCs, CTCs and slCTCs to evaluate the risk for recurrence in early BC pts. Methods: In total, 90 pts with newly diagnosed HER2-neg early stage BC received breast conserving surgery and sentinel lymphonodectomy. 17 pts were ER-/PR-, 12 pts ER+/PR-, 3 pts ER-/PR+ and 59 pts were ER+/PR+. Analysis of OncotypeDX and KI67 were performed. In case of G2 tumors, UPA and PAI1 were determined as further proliferation markers in tissue samples. Two BM aspirates from these patients were analyzed by immunocytochemistry for DTCs using the pan-cytokeratin antibody A45-B/B3. Furthermore, 2 x5 ml blood were analyzed for CTCs with the AdnaTest BreastCancer for the detection of EpCAM, MUC-1, HER-2, and beta-Actin transcripts. The recovered c-DNA was additionally multiplex tested for the presence of slCTCs using the AdnaTest EMT (multiplex RT-PCR for TWIST, AKT2, PI3K), and the AdnaTest TumorStemCell (ALDH1). Results: OncotypeDX was performed in 68/91 cases. 30/68 pts (44%) had a low RS, 29/68 pts (43%) an intermediate RS and 9/68 pts (13%) a high RS, respectively. BM aspiration could be performed in 70/91 pts with a positivity rate of 34% (24/70) for DTCs. CTCs were detected in 16/68 (25%) evaluable pts and slCTCs in 27/62 (44%) pts, respectively. In preliminary statistical analysis, KI67, PR and grading are showing association to RS as well as the presence of slCTCs. Tissue samples of patients with G2 tumors (N=65) underwent evaluation for UPA/PAI1analysis. Conclusions: Final statistical analysis for the complete data set including correlations to RS with all evaluable parameter will be available for presentation at the ASCO.