Antibody CB11 Research Articles

Abstract 2840: Evaluation of HER-2 RNA and protein levels in a large cohort of breast cancer specimens to support development of a diagnostic immunofluorescence assay quantified by AQUA® Technology

Abstract With the advent of multiple HER-2 targeting agents, the need for a diagnostic test that accurately predicts the levels of HER2 in newly diagnosed breast cancer patients has never been greater. The goal of this study was to develop an immunofluorescence based assay to objectively and reproducibly quantify HER-2 protein level in Formalin-Fixed Paraffin-Embedded (FFPE) specimens using AQUA Technology and to provide a binary readout, thus eliminating reflex testing and enabling quick treatment decisions. Methods: Three well known HER-2 antibody clones, A0485, CB11, and SP3 were evaluated across a three-log dilution series under four different antigen retrieval conditions on a tissue microarray (TMA) containing 80 breast cancer cases with known central HER2 immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) amplification status. The top six immunofluorescence assay combinations out of 108 conditions were quantified by AQUA scoring based on the their ability to accurately separate negative and positive cases using receiver operating characteristic (ROC) analysis. These top six assay conditions were then assessed on an independent TMA containing 94 breast cancer cases and their AQUA scores were compared to HER2 protein levels determined by reverse phase protein microarray (RPMA®), HER2 mRNA levels assessed by RNAscope® as well as to the current “gold standard” IHC and FISH amplification. Results: Top three conditions clearly segregating HER2 positive and HER2 negative breast cancer cases were selected and analyzed on an independent TMA. Conditions associated with both SP3 and A0485 antibodies demonstrated high correlations with both HER2 protein levels determined by RPMA analysis and HER2 mRNA levels assessed by RNAscope (spearman's rho >0.85). Conclusion: Assessment of HER2 status using AQUA Technology was confirmed using two expression platforms, including both protein (RPMA) and RNA (RNAscope) supporting the utilization of the AQUA methodology for diagnostic test development. Citation Format: Jennifer Bordeaux, Krupa Chandrasekaran, Sue Beruti, Mike Nerenberg, Corinne Ramos, David Rimm, Jelveh Lameh, Naveen Dakappagari. Evaluation of HER-2 RNA and protein levels in a large cohort of breast cancer specimens to support development of a diagnostic immunofluorescence assay quantified by AQUA® Technology. [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 2840. doi:10.1158/1538-7445.AM2014-2840

Central retesting of breast cancer with HER2 immunohistochemistry score of 0 or 1+ using silver-enhanced in situ hybridisation: a multicentre, prospective study in Greece

Abstract Newly diagnosed invasive breast cancers should be evaluated for Human Epidermal Growth Factor Receptor 2 (HER2) status by immunohistochemistry (IHC) and/or in situ hybridisation (ISH) to determine eligibility for trastuzumab or other HER2-targeted therapies. Previous reports of high discordance rates between IHC and ISH have raised concerns over the accuracy of HER2 testing, especially when IHC is conducted locally. This study aimed to determine the rate of false-negative IHC results at three pathology centres (one central, two local) in Greece by central retesting of 240 prospectively collected invasive breast cancers scored as IHC 0/1+ at initial testing. All samples were from female patients (median age 58.0 years). Initial IHC tests utilised either the CB11 (159/239; 66.5%) or 4B5 (80/239; 33.5%) antibodies and were scored as 0 in 105/240 cases (43.8%) and 1+ in 135/240 cases (56.3%). All samples were centrally retested by automated silver in situ hybridisation (SISH). Of 237 samples with SISH staining suitable for assessment, 223 (94.1%; 95% confidence interval 90.3–96.5%) were classed as SISH-negative (HER2:chromosome enumeration probe 17 (CEP17) <1.8). Eight tested SISH-positive (HER2:CEP17 >2.2), providing a false-negative rate of 3.4%. A further four samples (1.7%) exhibited equivocal amplification status (HER2:CEP17 1.8–2.2) and two (0.8%) showed polysomy of chromosome 17. The proportion of SISH-negative results did not significantly differ between the IHC 0 and 1+ subgroups (95.2% vs. 93.2%; p=0.505). In conclusion, the low observed rate of false-negative IHC results in this study supports the use of IHC for initial HER2 status assessment in local or central laboratories in Greece.

HER-2 expression is not prognostic in osteosarcoma; a Children's Oncology Group prospective biology study.

Since the initial reports of human epidermal growth factor receptor 2 (HER-2) expression as being prognostic in osteosarcoma, numerous small studies varying in the interpretation of the immunohistochemical (IHC) staining patterns have produced conflicting results. The Children's Oncology Group therefore embarked on a prospective biology study in a larger sample of patients to define in osteosarcoma the prognostic value of HER-2 expression using the methodology employed in the initial North American study describing an association between HER-2 expression and outcome. The analytic patient population was comprised of 149 patients with newly diagnosed osteosarcoma, 135 with localized disease and 14 with metastatic disease, all of whom had follow up clinical data. Paraffin embedded material from the diagnostic biopsy was stained with CB11 antibody and scored by two independent observers. Correlation of HER-2 IHC score and demographic variables was analyzed using a Fisher's exact test and correlation with survival using a Kaplan-Meier analysis. No association was found with HER-2 status and any of the demographic variables tested including the presence or absence of metastatic disease at diagnosis. No association was found between HER-2 status and either event free survival or overall survival in the patients with localized disease. HER-2 expression is not prognostic in osteosarcoma in the context of this large prospective study. HER-2 expression cannot be used as a basis for stratification of therapy. Identification of potential prognostic factors should occur in the context of large multi-institutional biology studies.

Abstract 5341: Clinical relevance of metastasis-related miRNAs in early breast cancer.

Abstract Introduction It was recently demonstrated that miRNAs control breast cancer metastasis by influencing multiple metastatic steps. In this study we firstly evaluated the clinical relevance of specific miRNAs -recently shown to correlate with metastasis in breast cancer by exploring their expression profile in FFPEs samples of early breast cancer patients in respect to the clinical outcome of the patients. Finally, we investigated the correlations between the expression of these selected miRNAs and other parameters such as ER and PR expression. Materials and Methods One hundred and twelve (112) FFPE blocks were obtained from breast tumors of early breast cancer patients (follow-up period of 11 years) and 13 mammoplasty specimens from healthy tissues which were used as controls. The median overall survival (OS) and disease free interval (DFI) were 84 and 68 months respectively. We have chosen to quantify the expression of six metastasis related miRNAs, namely: miR-10b, miR-2,1 miR-205, miR-210, miR-335 and let-7a by RT-qPCR in the LightCycler (Roche). HER2, ER and PR expression of the primary tumors was detected by immunohistochemistry with the monoclonal antibody CB11 and with monoclonal antibodies to ER and PR, respectively, using the OPTIMAX automated system. For all miRNAs relative expression was determined using the ΔCq approach and expression values were normalized to miR-191 which has been shown to be a suitable reference miRNA. Results Kaplan-Meier survival curves demonstrated that patients with high miR-21 expression (n=56) had a significant shorter DFI than those with low miR-21 expression (P=0.043). Moreover, patients with low expression of miR-205 (n=57) had both shorter DFI and OS time than those with high expression levels (P=0.040 and P=0.047 respectively). However, the expression levels of the other four miRNAs were not related with the DFI and OS. We also found that in the ER-negative subgroup (n=46), disease relapse was more common in patients where miR-10b was over-expressed (P=0.019), while in the PR-negative subgroup (n=47), disease relapse was more common in patients with miR-21 overexpression (P<0.001) or miR-10b overexpression (P =0.016). Finally, in the group of PR-negative patients we noticed that patients who overexpressed miR-21 had shorter OS (P=0.021). Conclusions Quantification of metastasis related miRNAs expression in primary tumors can give important prognostic information in patients with early breast cancer. Citation Format: Athina N. Markou, George M. Yousef, Yu Liang, E Stathopoulos, Vassilios Georgoulias, Evi S. Lianidou. Clinical relevance of metastasis-related miRNAs in early breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5341. doi:10.1158/1538-7445.AM2013-5341

Abstract 5577: HER2 cytoplasmic staining and penile cancer

Abstract BACKGROUND: Although rare in developed regions, penile carcinoma is a serious concern for undeveloped countries such as Brazil, where it can represent up to 10% of all malignancies. HER2 is a well-known growth factor membrane receptor that has its expression level usually associated with aggressive disease. Recently, some studies have shown that soluble fractions of HER2 intracellular domain are found in tumors cytoplasm and they still activate intracellular pathways related to cell proliferation and survival. MATERIAL AND METHODS: Immunohistochemistry against HER2 was carried out in 195 penile carcinoma samples selected from the files of AC Camargo Hospital, Brazil, using the monoclonal CB11 antibody (Novocastra™), which recognizes specifically HER2 intracellular domain. Cases were classified as positive or negative according to membrane and to cytoplasmic staining. All statistical analyses were performed by the SPSS for Windows 18.0, SPSS Inc. Comparison between category variables was performed by the Pearson chi-square test or the Fisher's exact test. Survival rates were calculated using the Kaplan-Meier method and the curves were compared by means of the log-rank test. In all statistical tests, the alpha error was set at 5%. RESULTS: None of the cases showed membrane staining for HER2. However, a clear and strong cytoplasm staining was observed in 28 cases (14,9%), as shown in figure 1. The staining was limited to the tumor cells and does not represent background or artifact. Univariate analyses showed that overexpression is associated with higher histologic grade (p <0,001) and lower overall survival (p=0,007). Multivariate analyses showed that HER2 in the cytoplasm is an independent risk factor for death (RR=2,966; IC 95% [1,6-5,2]; p <0,001). CONCLUSIONS: Corroborating with recent findings that cytoplasmic fragments of HER2 can be active kinases, the present study suggests that the presence of HER2 in tumor cytoplasm is a prognostic marker in penile carcinoma. We have also demonstrated that, although no membrane staining was seen, presence of HER2 in cytoplasm is an independent risk factor for death in this neoplasm. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5577. doi:1538-7445.AM2012-5577

P4-07-09: Automated Quantitative Assessment of HER2 Expression of Circulating Tumor Cells (CTC) in Metastatic Breast Cancer (IC 2006–04 Study).

Abstract Background Beyond quantitative CTC analysis for outcome assessment, their qualitative analysis could predict drug response and resistance. Classification of CTC and quantification of treatment targets on CTC can be subjective as they are morphologically heterogeneous. Current HER2 fluorescence assessment methods rely on a visual comparison between CTC images and external controls (cells lines) and are poorly reproducible. We report an automated classification and quantification of HER2 expression on CTC in a large cohort of metastatic breast cancer (MBC) patients (pts). Materials and Methods: CTC enumeration and HER2 assessment was performed with the CellSearch® system in pts with MBC included in the prospective IC 2006–04 study (Pierga, Ann Oncol 2011), before first line chemotherapy. The FITC conjugated antibody CB11 was used to identify HER2. For quantitative assessment of HER2 the breast cancer lines SKBR3 and MCF7 with known HER2 status were spiked in blood of normal donors and processed with the CellSearch system. Digital images of Cytokeratin-PE, DAPI, CD45-APC, and HER2−FITC from these samples were stored. An algorithm, previously developed in prostate cancer (Ligthart, AACR 2011), was applied on these images to automate identification of CTC. The fluorescence of the HER2 FITC channel for each CTC as well as the CD45+, DAPI+ leukocytes contained within each sample were also quantified using this algorithm. HER2 fluorescence levels of CTC were compared to HER2 status of status of pts. Results: Among 267 pts included in this study, 103 had at least 1 CTC/7.5 ml and HER2 fluorescence assessment. In these pts, correlation analysis between automated (aCTC) and manual (mCTC) CTC counts showed an excellent correlation (R2 =0.978). HER2 fluorescence of CTC, varied greatly within and between pts, whereas leukocyte fluorescence was more homogeneous. The vast majority of CTC exhibited less fluorescence than SKBR3 cells, even in HER2+ pts. Primary tumor was HER2+ and HER2− in 36 and 58 pts respectively. Percentage of HER2+ CTC was higher in pts with primary HER2+ status. Although a rare event, the presence within a sample of at least 1 CTC whose HER2 expression is superior to SKBR3 cells was also strongly associated with HER2+ primary tumor (N=15/36 vs. 3/58; p<0.001). Conclusion: This study is the first to assess HER2 staining intensity in each CTC isolated from a large number of MBC pts. This was made possible by the aCTC count that was highly correlated with the mCTC count in our cohort and permitted HER2 quantification. Our study shows that HER2 staining is highly heterogeneous among CTC within each pt. These findings demonstrate the feasibility of real time quantitative assessment of treatment targets on CTC and opens a path towards personalized treatment. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-07-09.

HER2 assessment on core biopsy specimens using monoclonal antibody CB11 accurately determines HER2 status in breast carcinoma

HER2 status is a prognostic factor in breast carcinoma and predicts response to trastuzumab therapy. Trastuzumab is effective in the neoadjuvant, adjuvant and advanced disease settings. Knowledge of HER2 status early in the diagnostic and therapeutic process is vital for treatment planning. HER2 analysis is usually carried out by immunohistochemistry or fluorescence in situ hybridization (FISH). The aim of this study was to establish whether HER2 immunohistochemistry using monoclonal antibody CB11 and carried out on the initial diagnostic core biopsy specimen accurately predicts HER2 amplification status. This is the largest study to date in which HER2 protein expression has been assessed by CB11 immunohistochemistry on the diagnostic core biopsy specimen and correlated with the result of FISH. Using FISH as the definitive HER2 status, we studied 568 invasive breast cancers using CB11 immunohistochemistry on core biopsy. This analysis had a sensitivity of 99.4%, specificity of 93.9%, false-positive frequency of 3.9% and false-negative frequency of 1.1%. These data are as good as those obtained from analysing resection specimens alone in UK national reference centres. CB11 immunohistochemistry accurately predicts HER2 amplification status and can be reliably carried out on core biopsy specimens of breast carcinoma.

Abstract C25: Enabling in situ quantitative biomarker analysis in the context of tissue morphology

Abstract Modern personalized medicine makes use of individual molecular biomarkers to subdivide tumors into subsets that behave differently from each other. A large number of clinical trials are evaluating several novel therapeutic targets and relevant biomarkers. Tissue biomarkers are gaining prominent position in drug discovery and development programs and are commonly included in clinical trial testing to identify patients that would profit from particular treatments, measure drug efficacy and/or monitor the success of therapeutic interventions. The demands on accuracy and reliability of tests are very high. The quality of tissue biomarker testing can be affected by various pre-analytic factors (time to fixation, time of fixation, type of fixation, method of tissue processing), analytic factors (assay validation, equipment validation, test reagents, standardized control materials) and post-analytic factors (interpretation criteria, use of image analysis, quality assurance procedures). In this poster we will focus on analytic and post-analytic aspects, and more specifically - imaging cytometry as a method of choice in automated analysis of tissue and tissue microarray specimens. Materials and Methods: 1. Standard cytometric QC materials - for within-run precision of fluorescence measurements, Flow Check beads (Beckman-Coulter), for run-to-run precision of fluorescence measurements, CaliBrite Beads (B.D. Biosciences), for run-to-run precision of chromatic staining, negative and positive punch cores of breast tissue labeled for HER2/Neu with CB11 antibody developed with DAB and counterstained with Hematoxylin. 2. Tissue microarrays - whole mouse, rat and major human organ TMAs (Invitrogen).Arrays stained with dual IHC protocols for CD31/CD34, cytokeratin 7/CD31, S100/GFAP, kappa/lambda (in situ hybridization) and Ki67/CD68. 3. Specimens were analyzed on an iCys® Imaging Cytometer (CompuCyte Corporation, Westwood, MA). Quality control measures and Results: a) within-run precision of fluorescence measurement - a Coefficient of Variation (CV) of fluorescence intensity of less than 3% measured over a large bead population; b) run-torun precision of fluorescence measurements, the mean of fluorescence intensity measurements within +/− 2 Standard Deviations (SDs) as established by baseline runs; c) run-to-run precision of chromatic staining - the mean DAB laser light loss measurements within +/− 2 Standard Deviations (SDs) as established by baseline runs. TMA analysis: instrument automatically identified core elements, performed spectral deconvolution to isolate markers of interest. Chromatic measurements by laser-light-loss and fluorescence measurements were obtained simultaneously and combined to quantify the expression of the markers of interest. High resolution images ofthe cores were obtained simultaneously with the quantitative analytical data. Quantitative end-points on a per-core basis included: percent positive marker expression, mean expression level, total marker expression level and co-localization of markers. The technique of “image masking” effectively reduced the background levels in tissue elements. Summary: LSC technology provides accurate and reproducible measurements of tissue biomarkers in unbiased, highly quantitative manner. It is well suited for tissue-based biomarker discovery and clinical trials. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C25.

HER2 Carboxyl-terminal Fragments Regulate Cell Migration and Cortactin Phosphorylation

A group of breast cancer patients with a higher probability of developing metastasis expresses a series of carboxyl-terminal fragments (CTFs) of the tyrosine kinase receptor HER2. One of these fragments, 611-CTF, is a hyperactive form of HER2 that constitutively establishes homodimers maintained by disulfide bonds, making it an excellent model to study overactivation of HER2 during tumor progression and metastasis. Here we show that expression of 611-CTF increases cell motility in a variety of assays. Since cell motility is frequently regulated by phosphorylation/dephosphorylation, we looked for phosphoproteins mediating the effect of 611-CTF using two alternative proteomic approaches, stable isotope labeling with amino acids in cell culture and difference gel electrophoresis, and found that the latter is particularly well suited to detect changes in multiphosphorylated proteins. The difference gel electrophoresis screening identified cortactin, a cytoskeleton-binding protein involved in the regulation of cell migration, as a phosphoprotein probably regulated by 611-CTF. This result was validated by characterizing cortactin in cells expressing this HER2 fragment. Finally, we showed that the knockdown of cortactin impairs 611-CTF-induced cell migration. These results suggest that cortactin is a target of 611-CTF involved in the regulation of cell migration and, thus, in the metastatic behavior of breast tumors expressing this CTF.

Validation of the 4B5 rabbit monoclonal antibody in determining Her2/neu status in breast cancer

HER2 overexpression in breast cancer is associated with worse clinical outcome. To select patients for anti-Her2-based therapy immunohistochemistry is commonly performed as a first step to assess Her2 status. However, interobserver and interlaboratory variability can significantly compromise adequate assessment of Her2 status. In addition, immunohistochemistry does not always result in an unambiguous test result requiring additional testing for Her2 gene amplification. This study aimed to improve the reliability of Her2 immunohistochemistry by using rabbit monoclonal antibody 4B5 as an alternative to mouse monoclonal antibody CB11 routinely used in our laboratory. Therefore, 283 breast adenocarcinomas were included in a tissue microarray. Immunohistochemistry using the 4B5 and CB11 antibodies, and fluorescence and chromogenic in situ hybridization (FISH or CISH) were performed. Immunohistochemistry was scored by two independent investigators. We found that 4B5 staining was more distinct than CB11 staining. For CB11 staining, there were 12% (BV) and 5% (JW) 2+ scores compared with 4% (BV) and 2% (JW) for 4B5. There was a strong trend towards higher interobserver agreement for 4B5 compared with CB11 (4B5: κ 0.87, 95% CI 0.79–0.96; CB11: κ 0.77, 95% CI 0.66–0.88). There were no significant differences in sensitivity, specificity and predictive values between CB11 and 4B5. Our results indicate that the 4B5 antibody provides more robust assessment of immunohistochemical Her2/neu status and will reduce the number of gene amplification tests compared with CB11. However, for tumours with a 2+ score additional gene amplification measurement using FISH or CISH remains necessary.

Shorter survival-times following adjuvant endocrine therapy in oestrogen- and progesterone-receptor positive breast cancer overexpressing HER2 and/or with an increased expression of vascular endothelial growth factor

To investigate the possible correlation between expression of HER2 and vascular endothelial growth factor (VEGF), and to determine the predictive value of these factors in patients receiving adjuvant endocrine therapy including the group with a breast cancer (BC) positive for both oestrogen receptor (ER) and progesterone receptor (PgR). By enzyme immuno-sorbent assays (ELISA) tumour levels of HER2 and VEGF proteins were determined in 679 consecutive primary BC patients, median age 63 years, median follow-up time 92 months. A total of 404 patients received adjuvant endocrine therapy, mainly tamoxifen, out of them 295 had an ER and PgR positive BC. In 160 patients, HER2 status was also determined by immunohistochemistry (IHC) using the monoclonal antibody CB11. Overexpression of HER2 by IHC was found in 15% of the patients. Overexpression of HER2 by ELISA correlated with HER2 by IHC (P < 0.001) and a higher VEGF expression (P = 0.004). Patients receiving adjuvant endocrine therapy with high VEGF (RFS P = 0.0087, BCCS P = 0.0012) or over-expressing HER2 (RFS P = 0.0116, BCCS P = 0.0036) had significantly shorter survival. Factors retaining statistical significance in multivariate analyses for recurrence-free survival (RFS) were nodal status (P < 0.001), tumour size (P = 0.005) and VEGF (P = 0.032) and for breast cancer corrected survival (BCCS) nodal status (P < 0.001), tumour size (P = 0.001), ER status (P = 0.022), and VEGF (P = 0.016). Both factors were significantly correlated with survival in the group with a BC positive for both ER and PgR; VEGF (RFS P = 0.0177, BCCS P = 0.0321) and HER2 (RFS P = 0.0143, BCCS P = 0.0311). In multivariate analyses, nodal status (P < 0.001) and VEGF (P = 0.021) were independent factors for RFS. Nodal status (P < 0.001) and tumour size (P = 0.016) retained independent factors for BCCS. Combined analysis identified a high-risk group (HER2 positive and high VEGF) with significantly reduced survival. The results from this retrospective analysis suggest that overexpression of HER2 and higher VEGF expression may add information on patient's outcome after adjuvant endocrine therapy in ER and PgR positive BC.

Intralaboratory reproducibility of HER2 testing in breast cancer by immunohistochemistry and comparison of results obtained by different assays

To investigate the intralaboratory reproducibility of immunohistochemistry (IHC) testing for HER2 status in breast cancer, and to evaluate the factors which influence the reproducibility. The concordance between monoclonal antibody CB11 and HercepTest was also assessed. HER2 overexpression on paraffin sections from thirty-seven cases of breast invasive ductal carcinoma was evaluated using CB11 and the evaluation procedure had been repeated for five times scored the tests together according to the HercepTest and new American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) grading schemes by 2 experienced pathologists together. Reproducibility rates of the five rounds were assessed using Kappa statistic, and the results from two scoring systems were compared. HercepTest kit was applied to the same cases afterward and the results were compared with CB11. Substantial intralaboratory reproducibility was achieved among 5 rounds tests. Excluding the influence effect of changing antibody lots, the intralaboratory reproducibility was closed to the perfect threshold (Kappa = 0.7858, HercepTest scheme). The results derived from the two grading schemes had an almost perfect agreement (Kappa = 0.8549). The concordance (positive vs. negative) between CB11 and HercepTest was 83.78%. Laboratory work with strict supervision and more experience will ensure a reliable testing consistency. Reproducibility analysis could be adopted to evaluate the intralaboratory staining quality on HER2 testing. Different antibody lots bring some influence to the intralaboratory reproducibility, but not significant. CB11 could be accepted to screen HER2 status in routine practice after testing validation.

C‐erbB‐2 expression and prognostic significance in osteosarcoma

C-erbB-2 is postulated as a prognostic biological marker for osteosarcoma (OST). We investigated c-erbB-2 expression status in osteosarcomas and its prognostic significance. Archival pre-treatment tumor biopsies of 79 cases were stained by immunohistochemical (IHC) methods with CB11 antibody for cell-membrane specific c-erbB-2 expression. Relation with different variables was examined and survival rates were calculated. Median age was 13 years (7-17). Distant metastases were detected in 8 (10.1%) cases at initial diagnosis. 36/79 (45.6%) patients had positive c-erbB-2 staining by IHC. There was no significant difference between c-erbB-2 expression and presence of distant metastases at initial diagnosis or during follow-up, gender, age groups, tumor size, tumor sites, serum alkaline phosphatase, and lactate dehydrogenase levels. At a median follow-up of 67.4 months (9.2-246.1), 36 cases died, 20 were alive, and 23 abandoned treatment. For 58 cases without initial metastases who underwent definitive surgery, 5-year event-free (EFS) and overall survival (OS) rates were 50% and 62.1% in cases negative for c-erbB-2 expression, and 22.4% and 32.3% in cases positive for c-erbB-2 expression, respectively (EFS, P = 0.044; OS, P = 0.029). Five-year EFS and OS rates were significantly higher in cases with a tumor size <10 cm. Cox regression analyses revealed that only size persisted as a prognostic factor for EFS. C-erbB-2 expression status didn't appear as an independent prognostic factor. Overexpression of c-erbB-2 in OST was not correlated with metastatic potential. No independent prognostic significance was detected. The results do not support directed therapies for c-erbB-2 in OST.

Comparative analysis of c‐erbB‐2 (HER‐2/neu) in squamous cell carcinoma of the tongue: does over‐expression exist? And what is its correlation with traditional diagnostic parameters?

Over-expression of the proto-oncogene c-erbB-2 (HER-2/neu) has been shown to be a prognostic marker in many kinds of cancer, whereas conflicting data exist about the prevalence of HER-2/neu over-expression in squamous cell carcinoma (SCC) of the tongue. The status of Her-2/neu was evaluated in a series of SCC of the tongue to verify the frequency of over-expression of HER-2/neu and evaluate the correlation with traditional diagnostic parameters of this neoplasm. Fourty patients with SCC of the tongue were investigated for over-expression of the protein through immunohistochemistry using CB11 antibody, the Hercep Test kit and FISH. Data obtained using the Hercep Test differ from published reports concerning the over-expression of Her-2/neu and there was no correlation between levels of expression of Her-2/neu and other clinico-pathological and/or prognostic parameters. Of the 40 specimens, using CB11 we obtained results in line with published reports; however, with the Hercep Test we found only 1 positive case (2.5%) (score 3+). These data, confirmed by FISH, suggest that Her-2/neu is not a suitable marker that could play a primary role in the clinical-therapeutic management of SCC of the tongue.

Prognostic significance of Her2/neu overexpression in patients with muscle invasive urinary bladder cancer treated with radical cystectomy

The aim of the study was to evaluate the status of Her2/neu protein expression in patients with muscle-invasive urothelial carcinomas of the bladder treated with radical cystectomy and to determine its prognostic significance. We retrospectively analyzed the data of 90 patients who had undergone cystectomy for invasive transitional cell carcinoma of the urinary bladder. Immunohistochemical analysis for Her2/neu was done on paraffin-fixed tissues with CB11 antibodies (BioGenex, San Ramon, CA, USA). Sections with grade 2 and grade 3 staining were considered positive for Her2/neu. Over a median follow-up period of 46 months (24-96 months) 46 patients are living without disease recurrence and six with recurrent disease either at the local site or with distant metastases. The remaining 38 patients have died. The median overall survival time was 50 months, and median disease-free survival time was 40 months. The Her2/neu status was significantly related to the tumor stage (P = 0.001), lymph node involvement (77% in N+ vs 23% in N0; P = 0.001) and the grade of the disease (32% of grade 2 vs 71% of grade 3; P = 0.037). Kaplan-Meier curves showed a significantly worse disease-related survival period (log rank P = 0.011) for patients with Her2 overexpressing tumors than for those without overexpression. In addition to tumor stage [P = 0.001; relative risk (RR) = 2.62] and lymph node status (P = 0.0001; RR = 2.95), Her2 status (P = 0.020; RR = 2.22) was identified as an independent predictor for disease-related survival in a multivariate analysis. These results suggest that Her2 expression might provide additional prognostic information for patients with muscle-invasive bladder cancer. Future studies on Her2 expression with chemosensitivity and the efficacy of Her2-targeted therapies in urothelial carcinomas are warranted.

The effects of antibody clone and pretreatment method on the results of HER2 immunostaining in cytologic samples of metastatic breast cancer: A query and a review of the literature

The standardization and use of heat-induced epitope retrieval (HIER) is particularly important with immunohistochemical markers that direct the course of cancer treatment, such as Herceptin therapy. Increasingly, many laboratories are performing immunohistochemical analysis using various antibodies and methodologies for HER2/neu. We attempted to determine the effects of antibody clone and pretreatment methods on the interpretation of HER-2/neu staining in cytologic samples. Cell block sections from 54 cases of metastatic breast cancer (24 FNAs, 30 effusions) were analyzed for HER2 expression using antibodies to CB-11, TAB250, and A0485. Antibodies were analyzed with and without HIER. One pathologist using the FDA-approved scoring system for the HercepTest reviewed all slides in a blinded fashion. Five of fifty-four cases (9%) using CB-11 showed a significant increase in HER2 immunoreactivity using HIER (i.e. from 0/1+ to 2-3+). However, in twenty-nine of fifty-four cases (54%), the cytoplasmic background was significantly higher after HIER. With the A0485 antibody, two of fifty four cases (4%) showed a significant increase in immunoreactivity using HIER, while seventeen of fifty-four cases (31%) exhibited only more pronounced cytoplasmic staining. HIER pretreatment did not increase HER2 staining in any TAB250 stained sample, rather four of fifty-four cases (7%) showed a significant decrease in staining with HIER. We conclude that HIER may enhance membrane staining with the CB-11 and A0485 antibodies, but also increases cytoplasmic background. Loss of antigenicity is seen when HIER is used with TAB250.

HER2 status of bone marrow micrometastasis and their corresponding primary tumours in a pilot study of 27 cases: a possible tool for anti-HER2 therapy management?

Discrepancies have been reported between HER2 status in primary breast cancer and micrometastatic cells in bone marrow. The aim of this study was to assess HER2 gene status in micrometastatic cells in bone marrow and corresponding primary tumour. Micrometastatic cells were detected in bone marrow aspirations in a prospective series of 27 breast cancer patients by immunocytochemistry (pancytokeratin antibody). HER2 status of micrometastatic cells was assessed by fluorescence in situ hybridisation (FISH), respectively in 24 out of 27. Primary tumour HER2 status was assessed by immunohistochemistry (CB11 antibody) and by FISH in 20 out of 27 of the cases. HER2 was amplified or overexpressed in five out of 27 (18.5%) primary tumours and in four out of 27 (15%) micrometastatic cells. In two cases, HER2 was overexpressed and amplified in primary tumour, but not in micrometastatic cells, whereas, in one case, HER2 presented a low amplification rate (six copies) in micrometastatic cells not found in the primary tumour. We demonstrated that negative and positive HER2 status remained, in the majority of the cases, stable between the bone marrow micrometastasis and the primary tumour. Therefore, the efficiency of anti-HER2 adjuvant therapy could be evaluated, in a clinical trial, by sequential detection of HER2-positive micrometastatic cells within the bone marrow, before and after treatment.

HER2 evaluation using the novel rabbit monoclonal antibody SP3 and CISH in tissue microarrays of invasive breast carcinomas

Background: Laboratory methods for HER2 assessment currently include immunohistochemical (IHC) methods (measuring protein overexpression) and fluorescence in situ hybridisation (FISH) (measuring gene amplification). The measure of HER2 protein by IHC...

Detection of HER-2/neu gene amplification in breast carcinomas using quantitative real-time PCR — A comparison with immunohistochemical and FISH results

The aim of our study was to evaluate the value of quantitative real-time-PCR (qPCR) in the determination of HER-2/neu amplification status of human breast carcinomas by comparing qPCR, FISH and immunohistochemistry results from the same samples. A total of 210 breast carcinomas were examined. Ready-to-use CB11 antibody was applied to detect HER-2/neu oncoprotein expression. In 76 out of 210 cases FISH was performed, and 162 cases were investigated with qPCR. Seventy-five tumors were 2+ or 3+ positive with immunohistochemistry, while 135 samples were either completely negative or 1+. In 45 cases results from all three methods were available. Out of these, in twenty negative and sixteen positive cases both FISH and qPCR led to similar results. The mean qPCR amplification ratio in the concordant positive cases was 5.424 while in the qPCR+/FISH- group the mean ratio was 2.765. Out of 121 samples with scores of 0 or 1+ immunohistochemical result, analyzed also with qPCR, 26 showed HER-2/neu gene amplification. In these cases the mean amplification ratio was 2.53. Comparison of FISH and qPCR together with immunohistochemistry shows that qPCR is more sensitive to detect HER-2/neu gene amplification in tumors scored as 2+ with immunohistochemistry, but the diagnostic cut-off ratio should be defined above 2.7 to avoid high number of false positive cases. Amongst the immunohistochemistry score 2+ cases, 10 of 18 showed gene amplification by qPCR while 10 of 26 by FISH. In conclusion, a well calibrated HER-2/neu qPCR assay may serve as useful alternative to FISH in breast cancer patients.

Evaluation of HER-2 in serum and tissue: correlation with clinical-pathological parameters and outcome in early stage breast cancer (BC) patients (pts)

20066 Background: HER2 is amplified and/or overexpressed in approximately 20–30% of invasive BC and is associated with poor prognosis. It is also a predictive marker of response to trastuzumab. The extracellular domain of the HER2 protein (sHER2) is frequently cleaved and released into the circulation where it can be detected by ELISA in up to 45% of advanced BC. We evaluated HER2 expression in paired serum and tissue samples of operable BC pts to analyze 1) the correlation between sHER2 and HER2 tumor status 2) their relationship with clinical-pathological parameters and 3) their impact on the outcome. Methods: 188 consecutive stage I-III BC pts were included in this study from May 2000 to July 2005. sHER2 was measured by ELISA (manual Kit “Oncogene Science Diagnostics” and automated version “ADVIA Centaur”) before the local treatment. Tumor tissue was analyzed by IHC with CB11 antibody and scored with Dako Hercept-test. HER2 amplification was determined using the Ventana FISH assay in patients with 2+ by IHC. Chi-squared test was used to evaluate the association between HER2 and patients’ clinical-pathological features. Survival outcomes were analyzed using Cox’s model. Results: Median age was 56.6 years; 122 pts received adjuvant chemotherapy, 54 endocrine therapy and 83 both. Forty-three pts (23%) had HER2 overexpression/amplification in tumor tissue and 25 pts (13%) had sHER2 levels ≥15 ng/ml (cut-off level) with a concordance of 85%. Both high sHER2 levels and HER2 tumor expression were associated with high histological grade (p = .02 and p &lt; .0001 respectively) and negativity of ER (p = .0035 and p &lt; .0001) and PgR (p = .0056 and p =. 002). At a median follow-up of 2.4 years we observed 8 deaths and 19 relapses. At univariate analysis high sHER2 levels (evaluated as continuous variable) were significantly correlated with shorter DFS (p = 0.0002) even if at multivariate analysis high sHER2 levels, after adjustment for stage and ER status, were associated with a shorter DFS with borderline significance (p = 0.06). Conclusion: Our preliminary data indicate that the evaluation of sHER2 at diagnosis defines a small subgroup of early BC pts with a poor prognosis even if it needs to be confirmed in a longer follow up. No significant financial relationships to disclose.