Human Epidermal Growth Factor Receptor-2 Gene Amplification Research Articles

Her two Gene Amplification by Fish Technique in Ovarian Cancer Patients

Human Epidermal Growth Factor Receptor-2 (HER2) gene triggers a cascade of cellular responses leading to cell proliferation, survival, and metastasis in malignant cells. Therapies targeting HER2 gene are emerging as a promising treatment option for patients with HER2 gene amplification. Hence, the present study aims to investigate the HER2 gene amplification and relationship with clinicopathological features in ovarian cancer patients.

Amplification and clinicopathological significance of HER-2 in Kazakh esophageal squamous cell carcinoma.

Amplification and overexpression of the human epidermal growth factor receptor-2 (HER-2) gene accelerates cell division and proliferation, and promotes tumor growth and metastasis in various malignant tumors. However, there are few reports on its influence and mechanism in esophageal cancer. The aim of the present study was to investigate the gene amplification and clinicopathological significance of HER-2 in Kazakh esophageal squamous cell carcinoma (ESCC). HER-2 gene amplification was detected in 70 esophageal cancer tissues using fluorescence in situ hybridization. The association between the HER-2 gene amplification and the clinicopathological characteristics of patients with esophageal cancer was also analyzed. The amplification rate of the HER-2 gene in patients with esophageal cancer was 54.2% (38/70). The results also revealed a positive association between the amplification rate of the HER-2 gene in esophageal squamous cell carcinomas and the level of tissue differentiation, increasing gradually and significantly among the highly, moderately and poorly differentiated tissues (P<0.05). The amplification rate of the HER-2 gene in patients with lymph node metastasis was higher than those without (P<0.05). There was no significant association between the amplification rate of the HER-2 gene and any of the clinic pathological parameters, such as sex, age, depth of invasion and 3-year survival, among patients (P>0.05). In conclusion, the amplification rate of the HER-2 gene in patients with Kazakh ESCC was high. There was an association with various prognostic factors, including cancer differentiation and lymph node metastasis. HER-2 gene expression levels may be considered as an indicator of poor prognosis in patients with ESCC in the clinical setting, and this may provide a basis of treatment for individualized targeted therapies.

HER2 status in patients with residual rectal cancer after preoperative chemoradiotherapy: the relationship with molecular results and clinicopathologic features.

The specific role of human epidermal growth factor receptor-2 (HER2) status in rectal cancers remains unclear. This study therefore aimed to explore clinicopathologic and molecular characteristics, and prognostic value of HER2-positivity in residual mid- and/or low-rectal cancers after preoperative chemoradiotherapy (CRT). Surgical specimens from 145 patients with residual rectal cancer after preoperative CRT between January 2006 and January 2011 were used to evaluate HER2 status. HER2 protein expression and gene amplification were determined using immunohistochemistry (IHC) and silver in situ hybridization (SISH) on whole tissue sections, respectively. Polymerase chain reaction was used to analyze molecular characteristics, including microsatellite instability (MSI) and mutations in KRAS exon 2 (codon 12 and 13) and BRAF V600E mutation. Of 139 eligible patients, 8 (5.8%) had HER2 overexpression (IHC 2+ and 3+) that was not associated with clinicopathologic characteristics and patient survival, except positive circumferential resection margin (CRM) (P = 0.012). SISH was performed on 24 patient samples with IHC 1+ (n = 16), 2+ (n = 6), and 3+ (n = 2). HER2 amplification was identified in 3 patients (2.2%); however, this was also associated with positive CRM (P = 0.009) but not survival (all P > 0.05). Moreover, HER2 overexpression and amplification had no relationship with KRAS or BRAF mutations, and MSI status (all P > 0.05). HER2 positivity was found in a minority of rectal cancer patients and was not significantly associated with clinicopathologic and molecular characteristics. Our findings can be helpful in understanding the clinicopathologic bases of HER2 status in rectal cancers.

A novel HER2 gene body enhancer contributes to HER2 expression

The transcriptional regulation of the human epidermal growth factor receptor-2 (HER2) contributes to an enhanced HER2 expression in HER2-positive breast cancers with HER2 gene amplification and HER2-low or HER2-negative breast cancers following radiotherapy or endocrine therapy, and this drives tumorigenesis and the resistance to therapy. Epigenetic mechanisms are critical for transcription regulation, however, such mechanisms in the transcription regulation of HER2 are limited to the involvement of tri-methylated histone 3 lysine 4 (H3K4me3) and acetylated histone 3 lysine 9 (H3K9ac) at the HER2 promoter region. Here, we report the identification of a novel enhancer in the HER2 3’ gene body, which we have termed HER2 gene body enhancer (HGE). The HGE starts from the 3’ end of intron 19 and extends into intron 22, possesses enhancer histone modification marks in specific cells and enhances the transcriptional activity of the HER2 promoters. We also found that TFAP2C, a known regulator of HER2, binds to HGE and is required for its enhancer function and that DNA methylation in the HGE region inhibits the histone modifications characterizing enhancer and is inversely correlated with HER2 expression in breast cancer samples. The identification of this novel enhancer sheds a light on the roles of epigenetic mechanisms in HER2 transcription, in both HER2-positive breast cancer samples and individuals with HER2-low or HER2-negative breast cancers undergoing radiotherapy or endocrine therapy.

Abstract P1-03-10: HER2 expression in clinical breast cancer samples: A novel detection methodology for HER2 protein quantitation using fluorescent nanoparticles

Abstract Background: The human epidermal growth factor receptor-2 (HER2) is a member of a family of transmembrane tyrosine kinase receptors that play an important role in regulated normal cell growth and differentiation. The over-expression of HER2 in a subset of 15-20% of invasive breast cancers has an important bearing on prognosis, as HER2-positive tumors are associated with an aggressive clinical course and poor outcome. Targeting HER2-overexpression has been shown to be a remarkably effective therapeutic modality; however testing of tumor samples to assess the HER2 status of the patient's breast cancer is required. Clinical assays to assess the HER2 status in patients being considered for targeted therapy include immunohistochemistry (IHC), which detects protein over-expression, or fluorescence in situ hybridization (FISH), which detects gene amplification. Both the IHC and FISH methodologies have limitations. Given that the target of the currently approved drugs is the receptor protein, novel detections systems that could more accurately and quantitatively detect HER2 protein in clinical samples over a broad dynamic range would be advantageous and may be clinically helpful. Material and Methods: A novel detection technology using streptavidin-coated Phosphor Integrated Dot fluorescent nanoparticles (PID) has been developed that can be visualized by fluorescence microscopy and used for quantitative immunofluorescence detection of protein in clinical samples using computer assisted image analysis. In the current study, PID- nanoparticles were used to analyze HER2 protein expression in breast cancer cell lines and 120 well characterized breast cancer samples. These results have been compared with HER2 IHC and HER2 FISH analysis. Results: The expression levels of HER2 protein from 8 breast cancer cell lines was evaluated by antibody-binding capacity with FACS analysis. Formalin fixed paraffin embedded cell pellets for these cell lines were prepared and used for quantitative HER2 analysis by PID. The PID score/cell for each of these cell lines showed a strong linear correlation with antibody-binding capacity sites/cell by FACS analysis (R2 = 0.94). For the 120 breast cancer samples, PID score/cell was measured and compared against HER2 IHC membrane intensity measure by image analysis (Aperio) and HER2 FISH results. The HER2 PID score/cell showed a correlation coefficient of R2=0.72 versus the average HER2 copy number per cell by FISH, compared with a correlation coefficient of R2=0.41 for HER2 IHC membrane intensity measured by Aperio. For the HER2/CEP17 ratio, the correlation coefficient for the PID score/cell was R2=0.79 compared with a correlation coefficient of R2=0.32 for the HER2 IHC membrane intensity. Conclusions: PID-nanoparticles demonstrate great potential for the quantitative measurement of protein of clinical interest in routine clinical samples with morphologic confirmation of the tissue being studied. Further studies looking for PID-score thresholds for HER2 gene amplification and correlations with clinical outcome data are warranted and ongoing. Citation Format: Hicks DG, Goda H, Zhai H, Okada H, McMahon L, Sullivan N, Tang P, Nakano Y. HER2 expression in clinical breast cancer samples: A novel detection methodology for HER2 protein quantitation using fluorescent nanoparticles [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-03-10.

Development of a quantitative PCR assay to evaluate HER2 status of Gastric carcinoma in a cohort of Sri Lankan patients

Introduction Human epidermal growth factor receptor2 (HER2) protein over expression and/or HER2gene amplification are/is linked to dismal outcome of Gastric carcinoma (GCa). Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) are key-methods to identify patients for HER2 targeted therapy. Drawbacks of both methods warrant novel-tests. Objective To determine whether quantitative Polymerase Chain Reaction (qPCR) could serve as a supplementary-method to evaluate HER2 status of GCa in a cohort of Sri Lankan patients and investigate correlation between HER2 assessed by different methods and clinicopathological features. Methods Twenty GCa-patients with known IHC-HER2 scores were evaluated. qPCR was performed for HER2 gene and Ameloid precursor protein (reference gene) in Formalin fixed paraffin embedded GCa tissue. Threshold values (Ct) were analyzed using Pfaffl-method to detect HER2 gene amplification. Results HER2positivity by IHC (protein) and qPCR (gene) were 20% and 35% respectively. Sensitivity and specificity of qPCR was 67% and 76% respectively and results were reproducible. HER2 protein positivity was correlated with Tumour TNM-stage and Lauren-histological types (P Discussion & Conclusion Discrepancies between HER2 positivity by IHC and qPCR were possibly due to transcription activation by other genes in the absence of HER2 gene amplification and the aberrant form of HER2 protein not detected by IHC. Studies also indicate a higher-proportion of IHC negative, but HER2gene amplified GCa by FISH. qPCR may be used as a supplementary-method for detection of HER2 status of GCa in local setting.

Application of a quantitative PCR assay to evaluate Human Epidermal Growth Factor Receptor - 2 status of Gastric carcinoma in a cohort of Sri Lankan patients

Introduction Human epidermal growth factor receptor2 (HER2) protein over expression and/or HER2 gene amplification is linked to dismal outcome of gastric carcinoma (GCa). Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) are key methods to identify patients for HER2 targeted therapy. Draw backs of both methods warrant novel tests. The objectives were to determine whether quantitative Polymerase Chain Reaction (qPCR) could serve as a supplementary method to evaluate HER2 status of GCa and investigate the correlation between HER2 assessed by different methods and clinicopathological features. Methodology Twenty GCa patients with known IHC-HER2 scores were evaluated. qPCR was performed for HER2 gene and Amyloid precursor protein (reference gene) in formalin fixed paraffin embedded GCa tissue. Threshold values (Ct) were analyzed using Pfaffl-method to detect HER2 gene amplification. Results HER2 positivity by IHC (protein) and qPCR (gene) were 20% and 35% respectively. Sensitivity and specificity of qPCR was 67% and 76% respectively and results were reproducible. HER2 protein positivity was correlated with TNM-stage and Lauren histological types (P Discussion Discrepancies between HER2 positivity by IHC and qPCR were possibly due to transcription activation by other genes in the absence of HER2 gene amplification and the aberrant form of HER2 protein not detected by IHC. Studies also indicate a higher proportion of IHC negative, but HER2 gene amplified GCa by FISH. Conclusion qPCR may be used as a supplementary method for evaluation of HER2 status in gastric carcinoma proven to be HER2 negative by immunohistochemistry.

Genetic Heterogeneity of HER2 Amplification and Telomere Shortening in Papillary Thyroid Carcinoma.

Extensive research is dedicated to understanding if sporadic and familial papillary thyroid carcinoma are distinct biological entities. We have previously demonstrated that familial papillary thyroid cancer (fPTC) cells exhibit short relative telomere length (RTL) in both blood and tissues and that these features may be associated with chromosome instability. Here, we investigated the frequency of HER2 (Human Epidermal Growth Factor Receptor 2) amplification, and other recently reported genetic alterations in sporadic PTC (sPTC) and fPTC, and assessed correlations with RTL and BRAF mutational status. We analyzed HER2 gene amplification and the integrity of ALK, ETV6, RET, and BRAF genes by fluorescence in situ hybridization in isolated nuclei and paraffin-embedded formalin-fixed sections of 13 fPTC and 18 sPTC patients. We analyzed BRAFV600E mutation and RTL by qRT-PCR. Significant HER2 amplification (p = 0.0076), which was restricted to scattered groups of cells, was found in fPTC samples. HER2 amplification in fPTCs was invariably associated with BRAFV600E mutation. RTL was shorter in fPTCs than sPTCs (p < 0.001). No rearrangements of other tested genes were observed. These findings suggest that the association of HER2 amplification with BRAFV600E mutation and telomere shortening may represent a marker of tumor aggressiveness, and, in refractory thyroid cancer, may warrant exploration as a site for targeted therapy.

HER-2 expression and clinical significance in esophagogastric junction adenocarcinoma (Siewert type Ⅱ)

To investigate the human epidermal growth factor receptor-2 (HER-2) expression in esophagogastirc junction adenocarcinoma(Siewert type Ⅱ)and its clinical significance. A total of 180 patients with esophagogastric junction adenocarcinoma (Siewert type Ⅱ) were included in this study. The HER-2 expression was detected by immunohistochemistry (IHC), and fluorescence in situ hybridization (FISH) analysis was performed to assess the HER-2 gene amplification in the IHC-positive and IHC-weak positive cases. HER-2 overexpression (3+ ), weak positive (2+ ) and negative (1+ /0) was 11.7%(21/180), 8.9%(16/180), and 79.4%(143/180), respectively. The FISH analysis showed HER-2 gene amplification in 95.2%(20/21) of HER-2(3+ ) cases and 18.8% (3/16) of HER-2(2+ ) cases. The concordance rate between IHC and FISH was 95.2%. Overexpression of HER-2(3+ ) was associated with the tumor differentiation (P<0.05), and irrelevant to age, sex, pT stage, pN stage, pM stage and pTNM stage (P>0.05). The median overall survival time (OS) was 13 months in HER-2(3+ ) patients, significantly shorter than the 21 months in HER-2(2+ ) and HER-2(+ /-) cases (P<0.01). Approximately 11.7% of the patients with esophagogastric junction adenocarcinoma (Siewert type Ⅱ) are HER-2-positive on IHC. HER-2 overexpression is associated with the tumor differentiation. IHC can be used as a screening test for the positive expression of HER-2 in the esophagogastirc junction adenocarcinoma (Siewert type Ⅱ). However, FISH detection can be used as a more reliable detection method.

Correlations between HER2 Expression and Other Prognostic Factors in Breast Cancer: Inverse Relations with the Ki-67 Index and P53 Status.

Overexpression or amplification of human epidermal growth factor receptor-2 (HER2) is associated with grade of malignancy and a poor prognosis in breast cancer (BC). The aim of this study was to evaluate of value of HER2 as a prognostic marker, and to analyze associations with common histopathological parameters in BC cases. Between of 2007 to 2014, 260 patients with BC referred to Oncology Clinic provided cancer tissue samples which underwent immunohistochemistry (IHC) for markers. ER and PR positivity was defined as ≥10% positive tumor cells with nuclear staining. HER2-positive was defined as either HER2 gene amplification by fluorescent in situ hybridization (FISH) or scored as 3+ by IHC. For HER2 (2+), FISH was performed to determine HER2 positivity. The mean age at diagnosis for the patients with HER2-negative was significantly higher than in HER2-positive cases. Also, there were significant correlations between histological grade, nuclear grade, lymph node metastasis, tumor size, ER status, PR status, p53 overexpression and Ki-67 index with HER2 expression. HER2-negative lesions were of higher grade and more likely to be ER-negative, PR-negative, p53-positive, lymph node metastasis, with a tumor size<2cm and also Ki-67≥20% as compared to the HER2-positive group. Contrary to the results of other studies, HER2-positive tumors in our study had a lower Ki-67 index and were p53-positive. Also, Ki-67 proliferation index ≥20% in more studies was associated with p53-positive.Therefore, tumors which are HER2-positive and have a Ki-67≥20% had a more aggressive behavior compared to HER2-positive and Ki-67<20% lesions.

Comparison of HER2 status in breast cancer patients using CISH and immunohistochemical protocols

The evaluation of human epidermal growth factor receptor-2 (HER2) status in invasive breast carcinoma has been proven to be of prognostic value for the patients. HER2 overexpression is found in about 20% of the breast cancer cases and is thought to be associated with a poor clinical outcome. The test can be performed by different molecular methods, including in situ hybridization or immunohistochemistry. We evaluated the accuracy of our immu-nohistochemical protocol for HER2 breast cancer testing in comparison to the chromogenic in situ hybridization method (CISH). A total of 19 cases were analyzed. Using the HercepTest from Dako, three cases were initially evaluated as 3+ strong positive (with concomitant hormone receptor positivity). Fifteen cases were evaluated as 2+ weak positive and one triple negative case as 1+ negative. The results from CISH confirmed HER2 gene amplification in all three 3+ positive cases, whereas 46.7% of the 2+ cases showed gene amplification. The triple negative 1+ case did not reveal HER2 gene amplification. These results indicate that our immunohistochemical protocol has satisfactory sensitivity for detecting HER2 3+ positive cases and can reliably be used to select the HER2 negative cases. Nevertheless, CISH protocol should be used to clarify all ambiguous cases.

HER-2 gene amplification in human breast cancer without concurrent HER-2 over-expression

BackgroundTesting for human epidermal growth factor receptor-2 (HER-2) in breast cancer is performed by either immunohistochemistry (IHC) or in situ hybridization (ISH). The growth factor receptor-bound protein-7 (GRB7) gene is in close proximity to HER-2 on chromosome 17q11-12 and codes a signal transduction molecule shown to be an independent adverse marker in breast cancer.MethodsHER-2 and GRB7 protein expression from 613 frozen breast tumors was determined by Western analysis. HER-2 protein results were confirmed with IHC. Commercial HER-2 FISH was performed on a subset of tumors with multi-probe FISH used to assess the extent of HER-2 gene amplification. mRNA expression was determined by Multi-plex RT-PCR.ResultsSeven tumors with GRB7 protein over-expression scored HER-2 FISH amplified but had no HER-2 protein over-expression. Four of the 7 tumors showed elevated GRB7 but not HER-2 mRNA over-expression. The breast cancer cell line HCC3153 did not over-express HER-2 protein but showed HER-2 FISH amplification of a limited segment around the HER-2 gene. Ten breast cancer tumors from the TCGA database had gene copy number increases around HER-2 without HER-2 mRNA or protein over-expression.ConclusionsA subset of human breast cancers that test positive with FISH for HER-2 gene amplification do not over-express HER-2 protein. One mechanism for this discordance is the incomplete amplification of the smallest HER-2 region of chromosome 17q11-12, which includes GRB7. HER-2 gene amplification without protein over-expression is clinically significant because patients with such tumors are unlikely to benefit from HER-2 targeted therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/2193-1801-2-386) contains supplementary material, which is available to authorized users.

PTU-174 Systematic review and meta-analysis of the influence of human epidermal growth factor receptor 2 (HER2) expression and amplification on survival in patients with oesophageal cancer

IntroductionSurvival and recurrence rates following oesophagectomy remain poor despite the use of neoadjuvant therapy. Human epidermal growth factor receptor-2 (HER2) overexpression is associated with poorer survival in patients with gastric...

Human epidermal growth factor receptor-2 gene amplification in gastric cancer using tissue microarray technology

To assess human epidermal growth factor receptor-2 (HER2)-status in gastric cancer and matched lymph node metastases by immunohistochemistry (IHC) and chromogenic in situ hybridization (CISH). 120 cases of primary gastric carcinomas and 45 matched lymph node metastases from patients with full clinicopathological features were mounted onto multiple-punch and single-punch tissue microarrays, respectively, and examined for HER2 overexpression and gene amplification by IHC and CISH. Twenty-four tumors (20%) expressed HER2 immunohistochemically. An IHC score of ≥ 2+ was observed in 20 tumors (16.6%). HER2 amplification was detected by CISH in 19 tumors (15.8%) and in their matched lymph node metastases. A high concordance rate was found between HER2 positivity (as detected by IHC) and HER2 gene amplification (as detected by CISH), since 19 of the 20 IHC positive cases were amplified (95%). All amplified cases had 2+ or 3+ IHC results. Amplification was associated with intestinal phenotype (P < 0.05). No association with grading, staging or survival was found. In gastric cancer, HER2 amplification is the main mechanism for HER2 protein overexpression and is preserved in lymph node metastases.

HER2 gene amplification occurs frequently in the micropapillary variant of urothelial carcinoma: analysis by dual-color in situ hybridization

Human epidermal growth factor receptor-2 (HER2) is a well-recognized growth-promoting factor in cancer, although its application to urothelial carcinoma has been limited because of a low frequency of gene amplification. We evaluated HER2 protein expression and gene amplification in micropapillary carcinoma, a rare but highly aggressive variant of urothelial carcinoma by dual-color in situ hybridization. Gene amplification was defined by a HER2:CHR17 ratio of ≥2.2; low and high levels of amplification were further defined as <2.5 and ≥2.5, respectively. Immunohistochemistry was used to determine HER2 protein expression using the American Society of Clinical Oncology/College of American Pathologists Guidelines of HER2 staining. Protein expression, gene amplification, and chromosome 17 aneusomy were compared by Jonchkeere–Terpstra and Cochran–Armitage trend tests. In all, 19 of the 20 micropapillary carcinoma samples yielded usable dual-color in situ hybridization and immunohistochemistry results for evaluation. Overall, 68% (n=13) demonstrated HER2 protein expression of 2+ to 3+ staining. Gene amplification was present in 42% of samples (n=8), with 100% correlation with 2+ and 3+ protein expression. Gene amplification and protein expression were significantly associated (P=0.01). Overall, 53% of samples (n=10) had aneusomy of chromosome 17. Chromosome 17 aneusomy was present in approximately half of the samples evaluated, suggesting inherent genomic instability in this variant of urothelial carcinoma. However, increased HER2:CHR17 ratios demonstrate increased HER2 expression due to amplification in the majority of micropapillary carcinomas. These results suggest that HER2-targeted therapy may be successful on the genomic level in patients with this disease.

Lipid-rich carcinoma of the breast clinicopathologic analysis of 17 cases

Lipid-rich carcinoma of the breast is a rare variant of breast cancer, accounted for <1% of all breast malignant tumors. We retrospectively analyzed the clinicopathologic characteristics of lipid-rich carcinomas of the breast. A panel of estrogen receptor, progesterone receptor, human epidermal growth factor receptor-2 (HER-2), cytokeratin (CK) 5/6, CK14, and P63 was prepared for detection of lipid-rich carcinoma. Fluorescence in situ hybridization and electron microscope assays were performed for detecting HER-2 gene amplification and ultrastructure. Survival analysis were carried out using Kaplan-Meier and Cox regression methods. Receiver operating characteristic test was also performed. Estrogen receptor, CK5/6, CK14, and P63 were negative. Progesterone receptor (1/17) and HER-2 (17/17) were positive. HER-2 gene amplification was detected in all included cases (ratio values >2.2). Ultrastructure showed fat droplet and electron-dense material in the cytoplasm. Statistical differences were detected among survival and age (P = .033), histologic grade (P = .004), lymph node involvement (P = .001), and HER-2 expression (P = .002), respectively, using Kaplan-Meier methods. Statistical differences were also detected (P = .017) using Cox regression methods. Receiver operating characteristic test displayed significant statistical differences; the prognosis had a correlation between HER-2 expression "+" and HER-2 expression "++" (P = .004). Lipid-rich carcinoma of the breast was an HER-2 overexpressing subtype of the breast carcinoma. Survival of lipid-rich carcinoma might be associated with age, histologic grade, lymph node involvement, and HER-2. The HER-2 expression, however, might play an important role in predicting the prognosis of lipid-rich carcinoma of the breast.

Quantification of HER2 autoantibodies in the amplification phenomenon of HER2 in breast cancer

Gene amplification of HER2 (human epidermal growth factor receptor 2) is a well-known phenomenon in various cancers. However, little is known about the mechanism of the gene amplification phenomenon itself. Autoantibodies to cellular receptors have been described in several cancer types. We hypothesised that autoantibodies against HER2 might have a stimulatory capacity and could be the cause of the HER2 gene amplification phenomenon. To investigate this, we developed a test for the detection of autoantibodies against HER2 in serum (S-HER2Ab). Blood and tissue samples were collected from 311 women consecutively admitted for surgical treatment of primary breast cancer. Paraffin embedded tissue sections were analysed by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). HER2 protein concentrations in tissue were determined in 115 patients. Circulating extracellular domain of HER2 (S-HER2) was measured using the Advia Centaur (Siemens AG, Munich, Germany). Analysis for autoantibodies was developed on an ImmunoCAP 100 (Phadia AB, Uppsala, Sweden) with an automated Fluorescent Enzyme Immuno Assay. Of 311 women, 55 (17.7%) had HER2Ab and 51 (16.4%) showed amplification of the HER2 gene determined by IHC/FISH. Eleven women had detectable S-HER2Ab as well as HER2 gene amplification, but no statistically significant correlation was found between the two phenomena. A significantly higher level of S-HER2Ab was found both in HER2 gene-amplified and non-amplified breast cancer patients compared to an age-matched healthy control group. No statistically significant difference in presence or concentration of S-HER2Ab was found in HER2 gene-amplified vs. non-amplified breast cancer. S-HER2Ab can be measured accurately with the ImmunoCAP 100. There is an increased prevalence and concentration of S-HER2Ab in breast cancer patients but no correlation with HER2 gene amplification. We conclude that autoantibodies against HER2 do not seem to be the cause of HER2 gene amplification.

Abstract P4-08-03: False Positive HER-2 Testing by Fluorescence In Situ Hybridization in Human Breast Cancer

Abstract Background: Testing for human epidermal growth factor receptor-2 (HER-2) is routinely performed after breast cancer diagnosis by either immunohistochemistry (IHC) or fluorescence in situ hybridization (FISH). FISH is more accurate and reproducible, often considered the gold standard, and a more reliable predictor of a patient's response to HER-2 directed therapies in clinical practice. The growth factor receptor-bound protein-7 gene (GRB7) encodes a multi-domain signal transduction molecule located in close proximity to HER-2 on chromo some 17q11-12 and has been shown to be an independent adverse prognostic marker in breast cancer. Methods: We performed western blotting analysis of protein extracts from 563 annotated frozen breast tumors, collected from 1988 - 1998. GRB7 and HER-2 bands were assigned low or high values compared to specific protein controls, and tubulin bands. HER-2 FISH was performed on a subset of these tumors using an FDA approved Path Vysion kit on 4 μm frozen sections. HER-2 and chromosome 17 centromere (CEP17) signals were enumerated according to ASCO/CAP guidelines. Results: Sixty-six tumors (11.5%) over-expressed HER-2 and GRB7 proteins by Western analysis, 66 (11.5%) over-expressed HER-2 but not GRB7, and 30 (5.5%) over-expressed GRB7 but not HER-2. All 32 tumors (32/32) submitted to FISH analysis that over-expressed both HER-2 and GRB7 proteins demonstrated HER-2 gene amplification, while only one out of thirty-five (1/35) tumors that over-expressed HER-2 without GRB7 protein over-expression were HER-2 amplified. Interestingly, 7 of 28 tumors over-expressing GRB7 but not HER-2 protein were amplified for the HER-2 gene. Clinical laboratory HER-2 IHC testing confirmed the absence of HER-2 protein over-expression in all 7 of these tumors. Conclusions: Our study demonstrates that about ten percent of human breast cancers that test positive with FISH for HER-2 gene amplification do not over-express HER-2 protein, which may influence sensitivity to HER-2 directed therapy. All tumors that show false positive HER-2 gene amplification over-express GRB7 protein, suggesting the presence of GRB7 driven gene amplification in these tumors. Given that the HER-2 FISH probe contains GRB7 sequences, it is possible that HER-2 FISH false positivity may reflect GRB7 gene amplification. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-08-03.

Pathological complete response and survival according to the level of HER-2 amplification after trastuzumab-based neoadjuvant therapy for breast cancer

Background:We analysed whether the level of human epidermal growth factor receptor-2 (HER-2) amplification significantly influenced either pathological complete response (pCR) or recurrence-free survival (RFS) and overall survival (OS) after trastuzumab-based neoadjuvant therapy.Methods:In all, 99 patients with an HER-2-amplified breast tumour treated with trastuzumab-based neoadjuvant therapy were included. Tumours were classified as low amplified (LA; 6–10 signals per nuclei) or highly amplified (HA; >10 signals). Pathological response was assessed according to Chevallier's classification (pCR was defined as grade 1 or 2). Median follow-up lasted 46 months (6–83). Cox uni- and multivariate analyses were performed.Results:In all, 33 tumour samples were LA and 66 were HA. The pCR in HA tumours was significantly higher than in LA tumours (55% vs 24%, P=0.005), whereas no association was found between the pCR rate and tumour stage, grade or hormone receptor status. In multivariate analysis, the pathological nodal status (P=0.005) and adjuvant trastuzumab (P=0.037) were independently associated with RFS, whereas the level of HER-2 amplification nearly reached statistical significance (P=0.057). There was no significant difference between LA and HA tumours for OS (P=0.22, log-rank).Conclusion:The level of HER-2 gene amplification significantly influenced pCR but not RFS or OS in non-metastatic breast cancer treated with trastuzumab-based neoadjuvant therapy. However, RFS in patients with HA tumours tended to be shorter.

Abstract 4154: Intratumoral heterogeneity of HER2/neu expression in diffuse-gastric carcinoma of European patients and its relevance for patients’ management

Abstract Background: This study assessed the human epidermal growth factor receptor-2 (HER2) protein expression, its relationship with gene amplification and its intra-tumoral heterogeneity in diffuse-gastric carcinoma, a subset of gastric carcinoma that might benefit from Trastuzumab in case of HER2 amplification according to recently reported ToGA trial (J Clin Oncol 27:18s, 2009 (suppl; abstr LBA4509) Patients and methods: The study included 51 European consecutive patients who underwent surgical resection in our institution between January 1991 and December 2007 for diffuse gastric carcinoma. A tissue-microarray was constructed using paraffin-embedded tissues of gastrectomy and matched lymph nodes. HER2 were evaluated by immunohistochemistry (IHC), using the same criteria than for breast cancer. All samples with a 2+ or 3+ HER2 overexpression were evaluated by FISH. Results: HER2 overexpression was observed in 2 (4%) tumors and there was intratumoral heterogeneity. The first tumour shows 2+/3+ immunostaining in lymph nodes with negative immunostaining in the primary tumor. The second tumor shows a focal 3+ HER2 immunostaining in the primary tumor without evidence of positive immunostaining in lymph nodes and peritoneal metastasis. Using FISH, HER2 3+ tumour areas had a gene amplification, whereas no amplification was found in 2+ tumor. The 2 HER2 positive tumors showed the same heterogeneous pattern, with adjacent 3+ positive and negative areas by both IHC and FISH. There was a 100% concordance between TMA and the tissue-sections. Despite complete resection, the 2 patients with HER2 expression died within 15 months. Conclusions: This study showed that 4% of resected diffuse gastric carcinoma had a heterogeneous distribution of HER2 gene amplification. These findings raise the question whether HER2 testing should be realized systematically in these patients and whether Trastuzumab may benefit in tumours with heterogeneous HER2 expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4154.