Neu Monoclonal Antibody Research Articles

Reckoning the Status of HER2/neu Protein and Oncogene in Breast Cancer Specimens: Comparison of IHC Assay with FISH Assay at a Tertiary Care Centre, West India

Introduction: Breast carcinoma is the most prevalent malignant neoplasm among women with more than one million new cases per year. The benefit of humanised anti-Human Epidermal growth factor Receptor 2 (HER2)/neu monoclonal antibody trastuzumab (Herceptin) in HER2/neu-positive breast cancers has been well documented. Immunohistochemistry (IHC) for protein overexpression and Fluorescence In Situ Hybridisation (FISH) for detecting HER2/neu gene amplification is widely used. Some laboratories use IHC as primary test with FISH for subset of cases while using FISH as primary investigation followed by IHC if needed is done by others. Aim: To evaluate IHC assay in breast carcinoma cases for HER2/neu as screening test before FISH. Materials and Methods: A cross-sectional descriptive type of observational study was conducted in the Department of Pathology and Multi-Disciplinary Research Unit, SMS Medical College and attached hospitals Jaipur, India, between April 2020 and December 2021. A total 122 samples of invasive breast carcinoma were included in study for IHC and FISH analysis. On paraffin embedded breast tumour tissue sections, IHC was performed using mouse monoclonal antibody targeting the intracellular domain of HER2/neu protein and FISH was implemented by dual colour probes targeting the HER2/neu gene on chromosome 17. Cases were classified according to HER2/neu status on IHC interpretation and FISH interpretation as per American Society of Clinical Oncology/ the College of American Pathologists (ASCO/CAP) HER2/neu-2018 testing guidelines. Chi-square test, kappa coefficient and Z-test were applied for statistical analysis. The p-value<0.05 was considered significant. Results: Of the 122 patients of Invasive Duct Carcinoma (IDC), the mean age was 50.3±12.7 years with a age range of 25-70 years. HER2/neu IHC score 3+ (over expression) was seen in 19 (15.6%) cases while 2+ (equivocal) result was obtained in 84 (68.8%) cases and 19 (15.6%) cases showed negative (1+/0) HER2/neu expression. Out of 19 IHC positive cases, all cases were amplified by FISH and all 19 IHC negative cases, were non amplified by FISH. There were 84 IHC equivocal cases of which 37 (44%) cases were amplified, 45 (53.6%) non amplified and 2 (2.4%) cases were equivocal when analysed by FISH. Also, 11 (9%) cases were found Centromere Enumeration Probe (CEP) amplified in the study, they were negative or equivocal on IHC and none was IHC positive. Conclusion: The study concluded that combined FISH and IHC methodologies could optimise information on HER2/neu status in breast cancer patients. Also, testing algorithm is emphasised where laboratories may use IHC as a screening method and FISH can be used as accurate and specific method in IHC (2+) equivocal cases. Thus, patients with HER2/neu status positive of IHC (3+) or FISH (gene amplified) can be proposed to be treated with herceptin (trastuzumab).

Combined treatment with anti-HER2/neu and anti-4-1BB monoclonal antibodies induces a synergistic antitumor effect but requires dose optimization to maintain immune memory for protection from lethal rechallenge.

Human epidermal growth factor receptor type 2 (HER2)-positive breast cancer that is treated with anti-HER2/neu monoclonal antibody (mAb) is not free from late recurrences. Addition of anti-4-1BB mAb to anti-HER2/neu mAb has been demonstrated to strengthen the cytotoxic antitumor response. Our study expands on this by revealing the influence of anti-4-1BB mAb addition on the immune memory of anti-HER2/neu mAb. We designed murine breast cancer models by implanting TUBO and TUBO-P2J cell lines in mice, which were then treated with anti-HER2/neu and/or anti-4-1BB mAb. After complete surgical and/or chemical regression of the tumor, the mice were rechallenged with a second injection of cancer cells. Notably, anti-HER2/neu and anti-4-1BB mAb combination therapy had a synergistic antitumor effect at the initial treatment. However, the combination therapy did not evoke immune memory, allowing the tumors to thrive at rechallenge with reduced CD44+ expression in CD8+ T cells. Immune memory was also impaired when anti-4-1BB mAb was administered to naive CD8+ T cells but was sustained when this was administered to activated CD8+ T cells. In an attempt to resist the loss of immune memory, we controlled the dose of anti-4-1BB mAb to optimize the stimulation of activated CD8+ T cells. Immune memory was achieved with the dose regulation of anti-4-1BB mAb to 1mg/kg in our model. Our study demonstrates the importance in understanding the adaptive immune mechanism of anti-HER2/neu and anti-4-1BB mAb combination therapy and suggests a dose optimization strategy is necessary to ensure development of successful immune memory.

Predicting Schwannoma Growth in a Tumor Model Using Targeted Imaging.

Vestibular schwannoma (VS) is a common pathology encountered in neurotology clinics. Many patients are observed with a "wait and scan" approach. Previous efforts to determine radiographic indicators of future growth have been unsuccessful. Using a mouse subcutaneous tumor model, we seek to determine if fluorescent imaging with directed immunotargets could be used to predict schwannoma growth rate. Anti-VEGFR2 and anti-Her2/Neu monoclonal antibodies were covalently linked to a near-infrared probe (IRDye800). Immunodeficient mice underwent subcutaneous injections with a rat-derived schwann (R3) cell line. When tumor growth was evident, either Anti-VEGFR2-IRDye800, anti-Her2/Neu-IRDye800, or Immunoglobulin G (IgG) Isotype-IRDye800 (control) were injected via tail vein. The mice were serially imaged in a closed field near-IR device. Fluorescent data were analyzed for tumor signal and correlated with tumor sie and growth rate. Heterogeneity of fluorescent tumor signal was also assessed. In both anti-VEGFR2 and anti-Her2/Neu groups, there were strong correlations between day 1 mean tumor fluorescence and eventual maximum tumor volume (p = 0.002, 0.001; r2 = 0.92, 0.86). There was also strong correlation with maximum tumor signal on day 1 and maximum tumor volume (p = 0.003, 0.008; r2 = 0.90, 0.91). There was no such correlation in the control group (p = 0.99, 0.75; r2 = 0.0002, 0.028). Given the potential morbidity in VS intervention, observation is an appropriate approach for patients with slow-growing or stagnant tumors. We seek to identify immunotargets in a murine model that show promise in predicting schwannoma growth with advanced imaging techniques. Both Her2/Neu and VEGFR2 correlated strongly wth tumor size and growth rates and are promising targets that merit further investigation.

English

BACKGROUND Human epidermal growth factor receptor-2 HER2 / neu, is a trans membrane tyrosine kinase receptor of epidermal growth factor receptor (EGFR) family and is involved in the pathogenesis of urinary bladder cancer. In this study we attempted to evaluate the HER2 / neu expression in urothelial carcinoma of bladder and its association with tumour grading. METHODS This was a cross sectional study with a sample size of 75. Routine 4 micrometre thick sections of formalin fixed paraffin embedded tissue blocks stained with haematoxylin & eosin were reviewed. Tumour grade was determined by using the World Health Organization (WHO) / International Society of Urological Pathologist criteria (ISUP). Immunohistochemistry was done by using HER2 / neu monoclonal antibody and its expression were observed. The membrane staining intensity and pattern were studied and scored. RESULTS In our study 75 cases of urothelial carcinoma were studied, of which 49 cases were papillary urothelial carcinoma low grade, 26 cases were papillary urothelial carcinoma high grade. Among these, 19 cases were infiltrating urothelial carcinoma. HER2 / neu positivity were observed in 27 (36 %) cases and overexpression in 8 (10 %) cases. Low grade urothelial carcinoma showed HER2 / neu positivity in 11 (22 %) cases and overexpression in 1 (2 %) case. High grade urothelial carcinoma showed HER2 / neu positivity in 16 (64 %) cases, among which 7 (28 %) cases showed overexpression. HER2 / neu positivity was seen in 13 (68 %) cases of infiltrating urothelial carcinoma with 4 (21 %) cases showing overexpression. A statistically significant difference in HER2 / neu expression was noted in high grade and invasive urothelial carcinoma compared to low grade and non-invasive urothelial carcinoma. CONCLUSIONS Urothelial carcinomas show overexpression of HER2 / neu and this over expression increases with increasing grade of tumour and muscle invasiveness. KEYWORDS Urothelial Carcinoma, HER2 / neu, Overexpression, Tumour Grade, Trastuzumab

Immunohistochemical expression and prognostic value of epidermal growth factor receptor 1 and human epidermal growth factor receptor 2/neu in primary colorectal carcinoma

Objectives Colorectal carcinoma (CRC), a highly heterogeneous disease, is the seventh most common cancer in Egypt. Human epidermal growth factor receptor 1 (EGFR1) and human epidermal growth factor receptor 2 (HER2/neu) are biomarkers that have been introduced recently for patient stratification, prediction of prognosis, and implementation of personalized therapy. Therefore, this study aimed to investigate the immunohistochemical expression of EGFR1 and HER2/neu in primary CRC patients and their relations with clinicopathological parameters and overall survival (OS). Patients and methods Sixty-six patients with primary CRC from the National Cancer Institute of Egypt were enrolled in this study. Paraffin blocks were collected and sectioned for histopathological evaluation. Other sections were stained using anti-EGFR1 and anti-HER2/neu monoclonal antibodies. Results Cytoplasmic and membranous EGFR1 was expressed in 81.8% of cases, while complete membranous HER2/neu-positive staining (>10% of tumor cells) was detected in 2/3 of cases. The EGFR1 expression was significantly associated with advanced stage and positive lymph node status. None of the other parameters, including overall survival, showed a significant relation with the expression of both markers. Conclusion In conclusion, EGFR1 and HER2/neu proteins were highly expressed in the CRC cases investigated. EGFR1 expression was significantly associated with indicators of poor prognosis (advanced tumor stage and nodal positivity). Both markers showed a tendency toward worse survival.

PRMT5 Associates With the FOXP3 Homomer and When Disabled Enhances Targeted p185erbB2/neu Tumor Immunotherapy.

Regulatory T cells (Tregs) are a subpopulation of T cells that are specialized in suppressing immune responses. Here we show that the arginine methyl transferase protein PRMT5 can complex with FOXP3 transcription factors in Tregs. Mice with conditional knock out (cKO) of PRMT5 expression in Tregs develop severe scurfy-like autoimmunity. In these PRMT5 cKO mice, the spleen has reduced numbers of Tregs, but normal numbers of Tregs are found in the peripheral lymph nodes. These peripheral Tregs that lack PRMT5, however, display a limited suppressive function. Mass spectrometric analysis showed that FOXP3 can be di-methylated at positions R27, R51, and R146. A point mutation of Arginine (R) 51 to Lysine (K) led to defective suppressive functions in human CD4 T cells. Pharmacological inhibition of PRMT5 by DS-437 also reduced human Treg functions and inhibited the methylation of FOXP3. In addition, DS-437 significantly enhanced the anti-tumor effects of anti-erbB2/neu monoclonal antibody targeted therapy in Balb/c mice bearing CT26Her2 tumors by inhibiting Treg function and induction of tumor immunity. Controlling PRMT5 activity is a promising strategy for cancer therapy in situations where host immunity against tumors is attenuated in a FOXP3 dependent manner.

Profile of human epidermal growth factor receptor 2 expression in epithelial ovarian carcinomas: A multicenter study from Southeast Nigeria

Background: Human epidermal growth factor receptor 2 (HER2/neu) in epithelial ovarian cancers (EOCs) has been proposed in targeted oncological therapy as a way forward in the treatment of ovarian cancer. Currently, there is a paucity of data on the expression of HER2 in EOC in Nigeria. Objective: This study, therefore, aims to carry out an immunohistochemical study of histologically diagnosed EOC in Nnewi, Southeast Nigeria. Materials and Methods: The hematoxylin and eosin slides and formalin-fixed-paraffin-embedded tissue blocks of histologically diagnosed cases of epithelial ovarian carcinomas in the two histopathology laboratories in Nnewi, Anambra state, over a 12-year period were retrieved from the archives. Archival formalin-fixed-paraffin-embedded tissue blocks of histologically normal ovaries in these laboratories were also retrieved to serve as controls. Sections were made from the tissue blocks and stained with HER2 immunostain. Results: Fifty EOC specimens and 20 histologically normal ovaries were stained with HER2 immunostain. There was statistically significant difference in HER2 expression between normal ovaries and EOC (P = 0.016). HER2/neu- positive staining was not observed in the histologically normal ovaries. However, nine (18%) of the EOC showed HER2/neu- positive staining. HER2 positivity within EOC was independent of age (P = 0.134), tumor grade (P = 0.141), histological subtyping (P = 0.080), and molecular subtyping (P = 0.168). However, there was more HER2 positivity among those older than 50 years, low-grade carcinomas, mucinous EOC, and Type 1 EOC. Conclusion: Overall, these data support the dualistic model of ovarian carcinogenesis in Nigerian patients and therefore recommend further studies to determine the efficacy of anti-HER2/neu monoclonal antibodies in the treatment of EOC in these patients.

PRMT5 is a novel cofactor of Foxp3 and can be a target for tumor immunotherapy

Abstract Regulatory T cells (Tregs) are a subpopulation of T cells specialized for suppressing host immune systems, whose dysfunction can lead to severe autoimmunity and inflammatory disease in humans and animals. In a tumor bearing host, an enhanced activity of Tregs may limit host immunity against tumor antigens. Our laboratory has studied the Foxp3 transcriptional complex at the molecular and atomic levels. We identified a protein arginine methyl transferase 5 (PRMT5) as a binding partner of Foxp3. Mice whose expression of PRMT5 was deleted in Tregs developed severe autoimmunity, scurfy like phenotypes and died at ~3 weeks of age. A limited number of Tregs were found in the spleen, but there were normal numbers of Tregs in the peripheral lymph nodes. Tregs that lacked PRMT5 displayed limited suppressive function when compared to wild type cells. Two types of PRMT5 inhibitors, EPZ004777 and EPZ015666, were studied to determine if inhibition of PRMT5 reduces human Treg functions. While EPZ004777 could inhibit human Treg functions in vitro, EPZ015666 had limited ability to limit these Treg activities. EPZ004777 could also inhibit the methylation of Foxp3. Injection of EPZ004777 to p185/erbB2/neu tumor bearing mice enhanced the effect of anti-erbB2/neu monoclonal antibody therapy. Furthermore, treatment with EPZ004777 reduced the numbers of tumor infiltrating Tregs, indicating that EPZ004777 functions by inhibiting Treg functions and/or migration of Tregs into p185 expressing tumors. These studies support the concept that controlling PRMT5 activity is a promising strategy in a p185 targeted monoclonal antibody therapy in which host immunity against tumors is augmented in a Foxp3 dependent manner.

Disabling of the erbB Pathway Followed by IFN-γ Modifies Phenotype and Enhances Genotoxic Eradication of Breast Tumors.

Reversion of the malignant phenotype of erbB2-transformed cells can be driven by anti-erbB2/neu monoclonal antibodies (mAbs), which disrupt the receptor's kinase activity. We examined the biologic effects of IFN-γ alone or after anti-erbB2/neu mAb treatment of erbB2-positive cells. IFN-γ had no effect on its own. Treatment of the tumors with anti-erbB2/neu mAbs followed by IFN-γ led to dramatic inhibition of tumor growth in vitro and in vivo with minimal mAb dosing. Sequential therapy enhanced the effects of chemotherapy. Moreover, IFN-γ with mAb treatment of mice with IFNγR knockdown tumors did not demonstrate marked synergistic eradication effects, indicating an unexpected role of IFN-γ on the tumor itself. Additionally, mAb and IFN-γ treatment also induced immune host responses that enhanced tumor eradication. Biochemical analyses identified loss of Snail expression in tumor cells, reflecting diminution of tumor-stem-cell-like properties as a consequence of altered activity of GSK3-β and KLF molecules.

Human epidermal growth factor receptor 2/neu overexpression in urothelial carcinoma of the bladder and its prognostic significance: Is it worth hype?

Aims:In urothelial tumors of the urinary bladder, human epidermal growth factor receptor 2 (HER-2)/neu expression has been reported over 10 years, but there is no clear correlation between prognosis and recurrence rate. The present study evaluates prognostic implication of HER-2/neu expression.Subjects and Methods:In this study, 100 formalin-fixed paraffin-embedded specimens of primary transitional cell carcinoma of the bladder were processed. HER-2/neu monoclonal antibody immunohistochemistry staining procedure used for the study.Results:A total of 70 (70%) patients were positive for overexpression of HER-2/neu. HER-2/neu was positive in patients with 42 (70%) superficial tumor, 28 (70%) muscle invasive tumor, 41 (75.9%) high-grade tumor, 29 (63%) low grade tumor, 31 (68.9%) recurrent tumor, and 6 (66.6%) had positive lymph nodes.Conclusions:Human epidermal growth factor receptor 2/neu over expression was not correlated with the tumor stage, lymphnode metastasis or recurrence of the disease. HER-2/neu overexpression was statistically insignificantly correlated with the differentiation grade (P < 0.161) as compared to previous studies. Future studies on HER-2 expression with chemo-sensitivity and efficacy of HER-2-targeted therapies in urothelial carcinomas is needed.

The Characterization of Anti-HER-2/neu Monoclonal Antibody using Different in vivo Imaging Techniques

Recently, specific antibodies have been used extensively to diagnose and treat various diseases. It is essential to assess the efficacy and specificity of antibodies, especially the in vivo environment. Anti-HER-2/neu mAb was evaluated as a possible transporting agent for radioimmunotherapy. The monoclonal antibody was successfully radio-labeled with 131I. In vitro binding assays were performed to confirm its targeting ability using another radio-iodine, 125I. Binding percentage of 125I labeled anti-HER-2/neu mAb in HER-2/neu expressing CT-26 cells was found to be 4.5%, whereas the binding percentage of 125I labeled anti-HER-2/neu mAb in wild-type CT-26 was only 0.45%. In vivo images were obtained and analyzed through γ-camera and an optical fluorescent modality, IVIS-200. γ-camera images showed that 131I labeled anti-HER-2/neu mAb accumulated in HER-2/neu CT-26 tumors. Optical imaging based on near infrared fluorescence labeled anti-HER-2/neu mAb showed higher fluorescence intensities in HER-2/neu CT-26 tumors than in wild-type CT-26 tumors. Anti-HER-2/neu mAb was found to specifically bind to its receptor expressing tumor. Our study demonstrates that in vivo imaging technique is a useful method for the evaluation of an antibody’s therapeutic and diagnostic potentials.

Abstract A115: Withaferin A downregulates her-2/neu protein expression in human breast cancer cells.

Abstract HER-2/neu protein is over-expressed in approximately 20-30% of breast cancers. Overexpression of this oncoprotein results in a more aggressive tumor phenotype and poor prognosis for patients. Current treatment strategies include passive administration of anti-HER-2/neu monoclonal antibody Trastuzumab as well as the small molecule inhibitor lapatinib. Both of these molecules function at the receptor level to block HER-2/neu signaling resulting in inhibition of cell growth and tumor regression in the majority of patients. However, a significant number of patients relapse or develop progressive disease due to acquired mechanisms of resistance to current therapy. Alternative approaches, that utilize multiple modes of action, are therefore necessary to combat this severe form of breast cancer. Withaferin A (WA) is a natural compound isolated from the Indian ayurvedic medicinal plant Withania Somnifera. Natural compounds are notorious for their multiple mechanisms of action, which make them ideal therapeutics for complex diseases such as cancer. Recent research has demonstrated that WA is effective in inducing cell death in several cancer cell lines by inhibiting molecular pathways such as Akt and MAPK proliferative cascades which are downstream of her-2/neu activation. Additionally, WA has also been shown to inhibit function of the molecular chaperone heat shock protein 90 (HSP90) in pancreatic carcinoma cells. Since HER-2/neu is a client protein of HSP90 and the chaperone functions to stabilize HER-2/neu protein expression on the cell surface, our goal was to determine whether WA would directly affect HER-2/neu expression. We initially established the cytotoxic activity of WA on HER-2/neu expressing human breast cancer cell lines such as SKBr3, MCF-7her-2neu and BT474 by MTT assay. We report that WA exhibits potent cytotoxic activity in these cells with IC50 concentrations of 1.6, 2.1 and 0.6 μM respectively after 48hr treatment. Comparison of WA sensitivity in a low HER-2/neu expressing cell line MCF-7 generated an IC50 of 5.1μM, indicating that cell lines that overexpress HER-2/neu may be more susceptible to the compound. Analysis of HER-2/neu expression both at the cell surface and intracellular localization were next performed in response to WA. WA treatment completely downregulated the expression of HER-2/neu protein from cell surface by 24hr and was both time and concentration dependent. Abrogation of intracellular protein expression also correlated to the downregulation from the cell surface. We determined that WA does not alter transcription of HER-2/neu, as there was no change in the gene expression with WA treatment compared to untreated cells. Further experiments are ongoing to address whether WA specifically targets signaling pathways that alter HER-2/neu signaling. Understanding the mechanisms by which WA induces down-regulation of HER-2/neu would serve as a means of developing WA as an alternative therapeutic strategy for HER-2/neu cancers not amenable to standard treatments. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A115. Citation Format: Ananlise Smith, Sarah Poliquin, Darlah Lopez-Rodriguez, Samita Andreansky. Withaferin A downregulates her-2/neu protein expression in human breast cancer cells. [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 A115.

Targeted Therapies for Gastric Cancer

Gastric cancer represents one of the most common cancers internationally. Unfortunately the majority of patients still present at an advanced stage, and despite advances in diagnostic and treatment strategies, outcomes still remain poor with high mortality rates despite a decline in incidence. Whilst the utility of classical chemotherapy agents has been explored thoroughly (and continues to be investigated, alone or in various combinations), advances have been slow and the efficacy of these agents has reached a plateau. As such, the focus of recent study has shifted toward developing a greater understanding of the molecular biology of carcinogenesis and the cancer cell phenotype, and, in turn, the development of rationally designed drugs that target molecular aberrancies in signal transduction pathways specific to gastric cancer. These targets include circulating growth and angiogenic factors, cell surface receptors, and other molecules that comprise downstream intracellular signalling pathways, including receptor tyrosine kinases. Therapeutic advances in this area significantly lag behind other solid organ malignancies such as breast and colorectal cancer. This article reviews the role of targeted therapies in gastric cancer, including rationale and mechanism of action, current and emerging data, as single-agent therapy or in combination regimens. A recently published randomized phaseIII trial supporting the use of trastuzumab, an anti-human epidermal growth factor receptor 2 (HER2)/neu monoclonal antibody, in a selected population of patients is discussed. Therapies that have been evaluated in phase II trials are also reviewed, as well as promising new therapies currently being investigated in preclinical or phase I studies. There is optimism that targeted therapies, whether as single-agent therapy or in combination with traditional therapies, including chemotherapy, radiotherapy and surgery, may yet have an impact on improvement of the overall prognosis of gastric cancer.

Combination of epitope-optimized DNA vaccination and passive infusion of monoclonal antibody against HER2/neu leads to breast tumor regression in mice

HER2/neu is an oncogene amplified and over-expressed in 20-30% of breast adenocarcinomas. Treatment with the humanized monoclonal antibody trastuzumab has shown efficacy in combination with cytotoxic agents, although resistance occurs over time. Novel approaches are needed to further increase antibody efficacy. In this study, we provide evidence in a mouse breast cancer therapeutic tumor model that the combination of active immunization with a modified HER2/neu DNA vaccine and passive infusion of an anti-HER2/neu monoclonal antibody leads to significant regression of established tumors. Our data indicate that combination therapy with a HER2/neu DNA vaccine and trastuzumab may have clinical activity in breast cancer patients.

Prevalence of HER2/neu overexpression/amplification in a Hispanic population with gastric adenocarcinoma.

25 Background: In Europe, Asia, and North America, studies have identified that approximately 20% of patients with gastric adenocarcinoma exhibit overexpression of HER2/neu. This group of patients has been shown to benefit from combination therapy with the anti-HER2/neu monoclonal antibody trastuzumab. These findings have not been studied in Hispanics who may possess a distinct gastric cancer genotype. The aim of this study is to determine the rate of HER2/neu positivity in a completely Hispanic population with gastric adenocarcinoma and to analyze the clinical factors associated with HER2/neu. Methods: We conducted a retrospective study in three different hospitals in Monterrey, Mexico. The study population consisted of Hispanic patients with gastric adenocarcinoma who have had a gastric resection and for whom there were adequate amounts of tissue available in paraffin blocks. Tissue was tested for HER2/neu using both Immunohistochemistry (IHC) and the fluorescent in situ hybridization (FISH) techniques. Results: From 2000 to 2010, we initially evaluated 27 gastric cancer tumor samples. Sex distribution was 15 males and 12 females with a mean age of 57±15 (SD) years. Anatomic distribution of tumor was 4% in the cardias, 40% in the body and 56% in the antrum. Lauren's histological type distribution was 52% diffuse and 48% intestinal. Fifty-three percent of the tumors were high grade and nodal positivity was present in 69%. Overexpression of Her-2/neu (IHC ++ and +++) was found in five (17.4%) of the samples, being moderate (++) in two (7.4%) and strong (+++) in three (11%) tumor samples. FISH amplification for the HER2/neu gene was found in two (7.4%) tumors and was only seen in samples with a strong (+++) IHC result. There was no association identified between HER2/neu status with age, gender, degree of differentiation, T stage, or nodal status. Conclusions: In a homogeneous Hispanic population, 17.4% of gastric cancer patients were found to overexpress HER2/neu by IHC. FISH did not identify any additional HER2/neu positive tumors. It is important to consider IHC testing for HER2/neu in Hispanic gastric cancer patients as they could benefit from a chemotherapy regimen containing trastuzumab. No significant financial relationships to disclose.

Radioimmunotherapy of breast cancer metastases with alpha-particle emitter 225Ac: comparing efficacy with 213Bi and 90Y.

alpha-Particles are suitable to treat cancer micrometastases because of their short range and very high linear energy transfer. alpha-Particle emitter (213)Bi-based radioimmunotherapy has shown efficacy in a variety of metastatic animal cancer models, such as breast, ovarian, and prostate cancers. Its clinical implementation, however, is challenging due to the limited supply of (225)Ac, high technical requirement to prepare radioimmunoconjugate with very short half-life (T(1/2) = 45.6 min) on site, and prohibitive cost. In this study, we investigated the efficacy of the alpha-particle emitter (225)Ac, parent of (213)Bi, in a mouse model of breast cancer metastases. A single administration of (225)Ac (400 nCi)-labeled anti-rat HER-2/neu monoclonal antibody (7.16.4) completely eradicated breast cancer lung micrometastases in approximately 67% of HER-2/neu transgenic mice and led to long-term survival of these mice for up to 1 year. Treatment with (225)Ac-7.16.4 is significantly more effective than (213)Bi-7.16.4 (120 microCi; median survival, 61 days; P = 0.001) and (90)Y-7.16.4 (120 microCi; median survival, 50 days; P < 0.001) as well as untreated control (median survival, 41 days; P < 0.0001). Dosimetric analysis showed that (225)Ac-treated metastases received a total dose of 9.6 Gy, significantly higher than 2.0 Gy from (213)Bi and 2.4 Gy from (90)Y. Biodistribution studies revealed that (225)Ac daughters, (221)Fr and (213)Bi, accumulated in kidneys and probably contributed to the long-term renal toxicity observed in surviving mice. These data suggest (225)Ac-labeled anti-HER-2/neu monoclonal antibody could significantly prolong survival in HER-2/neu-positive metastatic breast cancer patients.

Evaluating the potential of 188Re-SOCTA–trastuzumab as a new radioimmunoagent for breast cancer treatment

Radioimmunotherapy, which utilizes monoclonal antibodies and therapeutic radioisotopes against antigen-expressing tumor tissues, is an attractive therapeutic approach for cancer therapy. Trastuzumab (Herceptin) is a humanized anti-HER-2/neu monoclonal antibody for breast cancer treatment. In this paper, we introduce a new radioimmunoagent, (188)Re-trastuzumab, via a bifunctional ligand, succinimidyl 3,6-diaza-5-oxo-3-[2-((triphenylmethyl)thio)ethyl]-8-[(triphenylmethyl)thio]octanoate (SOCTA), and evaluate its potential to be a therapeutic radiopharmaceutical for breast cancer treatment. Equimolar amounts of SOCTA and trastuzumab were selected to react, and the conjugation ratio of SOCTA-trastuzumab was evaluated by the MALDI-TOF method. The immunoreactivity of SOCTA-trastuzumab was compared with nonconjugated trastuzumab in HER-2/neu overexpressing human breast cancer cell BT-474. Biodistribution experiment and microSPECT/CT images of (188)Re-SOCTA-trastuzumab being administered intravenously to SCID mice bearing xenografted BT-474 breast cancer were investigated to evaluate the tumor-targeting capability. The covalent attachment of SOCTA to trastuzumab (at 1:1 molar ratio) resulted in the averaged conjugation ratio of 0.27+/-0.06 (n=3). The complex could easily be labeled with (188)Re and achieve 95% radiochemical purity (RCP) after 1 h of reaction at room temperature. The in vitro stability study also revealed that the RCP of (188)Re-SOCTA-trastuzumab was at a value of more than 85% after 48 h of incubation with human serum. The immunoreactivity evaluation showed that SOCTA-trastuzumab and nonconjugated trastuzumab had similar binding capacity (B(max)) to HER-2/neu receptor in BT-474 cells. The animal experiments showed that (188)Re-SOCTA-trastuzumab accumulated more intensively in the tumor site as compared to normal tissue. We suggest that (188)Re-SOCTA-trastuzumab could be a potential candidate for radioimmunotherapy.

229 POSTER Combination effects of SF1126, a vascular targeted PI3 kinase inhibitor, with herceptin in Her-2 overexpressing breast cancer cells

Targeted adenoviral gene delivery using human epidermal growth factor receptor 2 (HER2/neu) is one of the promising strategies for enhancing the transduction efficacy of PEGylated adenovirus (PEG–ADV). The viral capsid of adenovirus carrying the green fluorescent protein (GFP) was conjugated with bifunctional polyethylene glycol (PEG). The surface of PEG–ADV was then further conjugated with anti-HER2/neu monoclonal antibody (MAb), Herceptin (Trastuzumab; HER) to grant HER2/neu over-expressed breast cancer cells specific targeting. The PEG–ADV and Herceptin immobilized PEG–ADV (HER–PEG–ADV) extents of retargeting were evaluated, as compared to those of naked ADV. In summary, HER–PEG–ADV exhibited more enhanced level of GFP expression than PEG–ADV did for MDA-MB-435 and MDA-MB-468 cells (a HER2/neu positive cell line), but not for a HER2/neu deficient U251N cells. PEGylated ADV significantly reduced innate immune response likewise, as judged from the amount of interleukin 6 released from macrophage cells. Consequently, this study suggests that HER–PEG–ADV conjugates enable ADV to become more potential therapeutic tools through overcoming the limitation of ADV against immune system and non-specificity.

Systemic targeting of CpG-ODN to the tumor microenvironment with anti-neu-CpG hybrid molecule and T regulatory cell depletion induces memory responses in BALB-neuT tolerant mice.

We have shown that neu transgenic mice are immunotolerant and that immunizations with dendritic cells (DC) pulsed with neu-derived antigens were not able to control tumor growth in these animals. We tested whether, by modulating the tumor microenvironment with Toll-like receptor ligands, it could be possible to induce the activation of antitumor responses in neu mice. Our results indicate that only intratumoral (i.t.) injections of CpG-ODN induce an antitumor response in neu mice. To target the CpG-ODN to the tumor site anywhere within the body, we chemically conjugated an anti-Her-2/neu monoclonal antibody (mAb) with CpG-ODN. The anti-neu-CpG hybrid molecule retained its ability to bind to Her-2/neu(+) tumors, activate DCs, and induce antitumor responses. Our results indicated that injections of anti-neu-CpG induced the rejection of primary tumors in 100% of BALB/c mice and only in approximately 30% of BALB-neuT mice. After challenging the BALB/c and BALB-neuT mice, we observed that BALB/c mice developed a protective memory response; in contrast, BALB-neuT mice succumbed to the challenge. After injections of anti-neu-CpG, T regulatory cells (T-reg) were drastically reduced at the tumor site, but a large number were still present in the lymphoid organs. When BALB-neuT mice were treated with anti-neu-CpG plus anti-GITR mAb, but not with anti-CD25 mAb, 100% of the BALB-neuT mice rejected the primary tumor and developed a protective memory response indicating the critical role of T-regs in regulating the repertoire against self antigens. Taken together, these results indicate that CpG-ODN-targeted therapy and depletion of T-regs optimally activate a primary response and generate a protective memory response against self-tumor antigens.

Immunoglobulin G Fragment C Receptor Polymorphisms and Clinical Efficacy of Trastuzumab-Based Therapy in Patients With HER-2/neu–Positive Metastatic Breast Cancer

The anti-HER-2/neu monoclonal antibody trastuzumab has been shown to engage both activatory (fragment C receptor [Fc gamma R]IIIa; Fc gamma RIIa) and inhibitory (Fc gamma RIIb) antibody receptors and Fc gamma R polymorphisms have been identified that may affect the antibody-dependent cell-mediated cytotoxicity (ADCC) of natural-killer cells/monocytes. In this study, we tested whether Fc gamma R polymorphisms are associated with clinical outcome of patients with breast cancer who received trastuzumab. Fifty-four consecutive patients with HER-2/neu-amplified breast cancer receiving trastuzumab plus taxane for metastatic disease were evaluated for genotype for the Fc gamma RIIIa-158 valine(V)/phenylalanine(F), Fc gamma RIIa-131 histidine(H)/arginine(R), and Fc gamma RIIb-232 isoleucine(I)/threonine(T) polymorphisms. Trastuzumab-mediated ADCC of patients' peripheral blood mononuclear cells (PBMCs) was measured by chromium-51 release using a HER-2/neu-expressing human breast cancer cell line as a target. Controls comprised thirty-four patients treated with taxane alone. Our population was in Hardy-Weinberg equilibrium except for the Fc gamma RIIb polymorphism. The Fc gamma RIIIa-158 V/V genotype was significantly correlated with objective response rate (ORR) and progression-free survival (PFS). Also, there was trend significance in ORR and PFS for the Fc gamma RIIa-131 H/H genotype. The combination of the two favorable genotypes (VV and/or H/H) was independently associated with better ORR and PFS compared with the other combinations. The ADCC analysis showed that V/V and/or H/H PBMCs had a significantly higher trastuzumab-mediated cytotoxicity than PBMCs harboring different genotypes. These data support for the first time the hypothesis that Fc gamma R-mediated ADCC plays an important role in the clinical effect of trastuzumab. Prospective studies are needed to confirm the role of Fc gamma R polymorphisms in predicting clinical outcome of patients with breast cancer treated with trastuzumab-based therapy.