Neu Oncoprotein Research Articles

Concordance Between Immunohistochemistry (IHC) and Silver Situ Hybridization (SISH) in Endometrial Carcinoma Diagnosis: Using HER-2/neu.

Human epidermal growth factor receptor 2 (Her-2/neu) has been demonstrated as biomarker in Endometrial Carcinoma (EC). This study was conducted to assess the concordance between Sudanese women between immunohistochemistry (IHC) and Silver Situ Hybridization (SISH) for EC diagnosis using HER-2/neu. This was a retrospective cross sectional study performed in the state of Khartoum, Sudan. This research used eighty-eight samples of embedded blocks of formalin fixed paraffin referred to the histopathology lab from 2007-2013. The technique of tissue microarray (TMA) was used in which paraffin blocks were prepared before IHC and SISH were subjected. In this analysis, 88 specimens were previously diagnosed as endometrial adenocarcinoma. The number of Her-2/new oncogene positive cases among Sudanese women by using IHC were (15.9%), however Her-2/neu amplification in EC were 11.5% by using SISH analysis and strong correlation between Her-2/neu IHC and Her-2/neu SISH is existed, since p-value is 0.,000. SISH is a reliable technique that can be used for detecting Her-2/neu oncoprotein and it has many advantages over other methods, also SISH can be used as an alternative to FISH technique.

Role and significance of HER-2/neu as a biomarker in the premalignant and malignant lesions of uterine cervix

Cancer of uterine cervix is the most common cancer among Indian females. Surgery is the main modality of treatment along with radiation and chemotherapy depending upon clinical stage of the tumor and surgical findings. However, patients with advanced or recurrent disease have a poor survival despite the use of cisplatin-based combination chemotherapy. Hence, there is an increasing need for developing novel therapeutic target that can replace or be added to the current therapy for these patients. The present study was conducted to evaluate the presence of Her-2/neu expression. Two hundred cervical specimens were included in this study. These comprised cases with diagnosis of cervical intraepithelial neoplasia, squamous cell carcinoma, adenocarcinoma. HER-2/neu immunostaining was performed by using BioGenex monoclonal mouse anti-human HER-2/neu Receptor IgG1 antibody. Higher expression of HER-2/neu was noted in malignant lesions as compared to premalignant lesions. Intensity of staining also correlated with grade of malignant tumor, clinical stage and lymph node metastasis. The over-expression of HER-2/neu oncoprotein is found to be associated with poor prognosis, metastatic potential and aggressive biological behaviour.

Abstract 2831: Identification of cell specific expression signatures using NGS global gene expression profiles from LCM procured breast carcinoma and adjacent stromal cells

Abstract Our goal is to determine relationships of gene expression profiles to patient-associated characteristics, breast carcinoma pathology and biomarker status with clinical outcome to improve assessment of prognosis and therapy selection. Procedures: De-identified frozen tissue biopsies of human breast carcinomas which were collected and stored under stringently controlled conditions were employed. Patient-related features (e.g., age, race, menopausal status, nodal status, clinical treatment and response) and results from biochemical analyses of established tumor markers (e.g., estrogen and progestin receptors, EGF receptor, HER-2/neu oncoprotein) guided tissue selection. Structural integrity of a tissue was first evaluated by light microscopy of H&E stained sections. To decipher uniqueness and clinical utility of gene expression profiles from specific cell types, Laser Capture Microdissection (LCM) with a PixCell IIeTM (Arcturus®, Thermo Fisher Scientific) was used to non-destructively collect either carcinoma cells or adjacent stromal cells on CapSureTM LCM caps. A second intact serial tissue section was processed identically to that of the section used for LCM as a control. Each of the two independent cell captures from the same tissue section and the second intact tissue section were independently extracted for RNA. RNA was assessed for integrity, purified and subjected to novel analyses using the Ion AmpliSeqTM RNA Breast Cancer Research Panel containing 1174 genes. Sequencing workflow included templating on the Ion Chef TM System and multiplexed sequencing on the Ion S5TM XL sequencing system and Ion 540TM chip. Results: Each sequencing library yielded approximately 4-5 million reads with a mapping rate of >98% that was achieved after alignment to the human transcriptome. Each sample was sequenced in triplicate with technical reproducibility near 99% with a mean of 1035 genes classified as detected. Cluster analyses of gene expression profiles revealed clear distinctions between stromal and carcinoma cells procured by LCM compared to intact tissue driven by strongly differentially expressed genes such as vimentin (VIM), MPRIP, stanniocalcin-2 (STC2) and estrogen receptor beta (ESR2). Conclusions: Collectively, the results indicate that molecular signatures of breast carcinoma cells procured by LCM reveal unique gene expression profiles compared to those of the intact tissue section or the adjacent stromal cells. These novel findings are likely to discern molecular features unique to a breast carcinoma that may be related to clinical outcome to improve assessment of patient prognosis and therapy selection. Supported in part by a grant from the Phi Beta Psi Charity Trust (JLW & SAA) and a CTSP Award from the Commonwealth of Kentucky (JLW). For research use only. Citation Format: Jeoffrey J. Schageman, Kristi Lea, Sarah A. Andres, Kelli S. Bramlett, James L. Wittliff. Identification of cell specific expression signatures using NGS global gene expression profiles from LCM procured breast carcinoma and adjacent stromal cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2831. doi:10.1158/1538-7445.AM2017-2831

Epithelial tumours of the lacrimal gland: a clinical, histopathological, surgical and oncological survey

Editor, Sarah Line′a von Holstein and associates present a review of the primary epithelial tumours of the lacrimal gland describing in the first part the frequency, demographics, clinical presentation and diagnostic features (von Holstein et al. 2013). In the second part, primarily tumour-specific histological characteristics are given. Finally, treatment modalities including surgical procedures and medical oncology as well as prognosis are discussed. Authors are to be commended for their effort to have performed such wide review on epithelial tumours of the lacrimal gland. Those orbital lesions, in fact, still represent a challenge for both clinicians and researchers. However, some relevant information regarding the pathological features and the prognosis was missed. In the section on pathology and molecular characteristics of those tumours, the authors reported that the immunohistological staining of biomarkers, including the androgen receptor, p53 and the HER-2/neu oncoprotein, is strong and diffuses in the malignant area. In this context, it would be important to report even the relation between the expression of apoptosis-related markers and the malignant epithelial tumours of the lachrymal gland (Strianese et al. 2007). Particularly, it was found in a re-examination of the 21 specimens of malignant epithelial tumours of the lacrimal gland that the expression of p53 was directly correlated with the apoptotic index, while expression of Bcl-2 was inversely correlated (Strianese et al. 2007). This expression pattern has been already described in other tumours (Greenblatt et al. 1994). Presumably, the correlation between apoptotic index, p53 and Bcl-2 indicates that tumour cells have lost or down-regulated their survival gene Bcl-2 and up-regulated the expression of p53, to attempt damage repair. Studies on apoptosis and apoptosis-related factors in salivary gland tumours have shown similar correlation as that found in lacrimal gland tumours (Soini et al. 1998). In addition, a statistically significant positive relationship for number of apoptotic cells (TUNEL) and p53, and an inverse correlation for Bcl-2 staining, was demonstrated with overall survival (Strianese et al. 2007). The correlation with survival of apoptotic index, p53 and Bcl-2 expression suggests that the more tumour cells go in apoptosis, up-regulating p53 and down-regulating Bcl-2, the better the survival of patients. As a role of apoptosis regulatory proteins in the pathogenesis of malignant epithelial lacrimal gland tumours was established, the hypothesis that evaluation of the expression of apoptosis-related markers in these tumours may provide a prognostic tool was reported (Strianese et al. 2007). This latter information should be mentioned in a review on epithelial tumours of the lacrimal gland, to provide adequate information for the clinician on prognostic factors of those tumours. As these tumours are rare (Bonavolontà et al. 2013), very little information about the molecular changes leading to their development and progression has so far been reported. Studies on targeting of the apoptosis pathway in epithelial tumours of the lacrimal gland represent an interesting new insight and, indeed, are considered by most researchers the new frontier for the understanding and management of the malignancies (Chaabane et al. 2013). Eventually, this letter may complete the wide review of van Holstein and associates.

Role of Nuclear Factor-κB in female Breast Cancer: A Study in Indian Patients

The nuclear factor κB (NF-κB) is a super family of transcription factors which plays important roles in development and progression of cancer. The present investigation concerns NF-κB /p65 activity in human breast cancers with overexpression of ER, PR, HER-2/neu, as well as the significance of p65 expression with regard to menopausal status, stage, grade, tumor size, nodal status, and NPI of invasive ductal carcinomas in Eastern India. In this hospital based study 57 breast cancer patients attending a Breast Clinic of a reputed institute of Eastern India were assessed for p65 protein expression in breast tumor tissue samples by Western blotting. ER, PR and HER-2/neu expression was determined by immunohistochemistry. NF-κB/p65 was significantly associated with advanced stage, large tumor size (≥5 cm), high grade, negative ER, negative PR, and positive HER-2/neu. High NF-κB/p65 expression was more frequent in patients with a high NPI (NPI≥5.4, 84.6%) compared with low NPI (<5.4, 44.4%) and this association was statistically significant (p=0.002). NF-κB/p65 overexpression was associated with advanced stage, large tumor size, high grade, and high NPI which are poor prognostic factors linked to enhanced aggressiveness of the disease. NF-κB/p65 expression implies aggressive biological behavior of breast cancer and this study validates significant association of NF-κB /p65 overexpression with negative estrogen and progesterone receptor status and overexpression of HER-2/neu oncoprotein. In our good clinical practice, patients with NF-κB positive tumors need to be treated aggressively.

Circulating levels of HER-2/neu oncoprotein in breast cancer

HER-2/neu, also known as c-erbB-2/neu, is an oncogene located in chromosome 17 which encodes HER-2/neu, a transmembrane protein belonging to the EGFR family. The external domain of this protein is released by the cell and can be studied in serum by immunoassay. HER-2/neu in serum is a specific tumor marker and only slight elevations may be found in the absence of malignancy, mainly in association with liver diseases. Likewise, the highest concentrations of this oncoprotein are found in patients with breast cancer, but lower concentrations may be found in other malignancies, particularly ovarian, prostate and lung cancer (mainly adenocarcinomas). HER-2/neu assay sensitivity in patients with untreated primary loco-regional breast cancer is <10% and seems to be related to overexpression in tissue as well as to the most important prognostic factors: tumor size and nodal involvement. Serial HER-2/neu determinations after surgery seem to be useful in the early diagnosis of recurrence, mainly in patients with HER-2/neu overexpression in tissue, but additional studies are necessary to confirm these results. HER-2/neu sensitivity (proportion of patients with abnormal values) in patients with metastasis is around 40%-45%, with a clear relationship to tissue overexpression and to site (higher in visceral metastases) and number of metastases. The clinical utility of HER-2/neu in patients with advanced disease is mainly for therapeutic monitoring. Likewise, in most of the studies published, a relationship has been found between serum HER-2/neu levels (either pretreatment or at follow-up) with tumor response.

Atomic force microscopy in oncocytology

A new atomic force microscopy (AFM) method was used to examine 51 cytology specimens from breast and thyroid tumors and pretumors and 17 uterine cervix scrapes. The authors formulated the main tasks of using AFM in oncocytology: to search for objective criteria for differential diagnosis between intact and cancer cells; to identify viral changes in the squamatous epithelial cells; and to objectify the immunocytochemical reaction of Her-2/neu oncoprotein hyperexpression. The studies have revealed as follows: glandular cancers are characterized by an increased nucleus/cytoplasm height ratio (from 1.8 to 2.5 for breast cancer and from 1.9 to 2.5 for thyroid cancer); the squamatous epithelial cells show an inverse trend (a decrease from 2.96 to 2.08 for keratinizing carcinoma of the cervix uteri); viral squamatous cell lesion appears as a perinuclear groove 200 nm in depth; Her-2/neu oncoprotein hyperexpression corresponds to the increase in cytoplasmic height from 248 to 850 nm.

Low-Grade Cribriform Cystadenocarcinoma of the Parotid Gland: A Neoplasm With Favorable Prognosis, Distinct From Salivary Duct Carcinoma

Low-grade cribriform cystadenocarcinoma of salivary glands is a recently described rare tumor with favorable prognosis. This study reports the case of 50-year-old woman with swelling lasting for 9 months in the right parotideomasseteric area. Grossly, the tumor was well circumscribed and dominated by cystic space. Microscopically, the neoplasm consisted of well-demarcated islets, some of them cystically dilated. The architecture of islets varied from solid to cribriform and micropapillary without comedo-type necroses. The tumor cells featured no significant cytologic atypia. Immunohistochemically, luminal cells showed expression of cytokeratins (CK), CK7, CK18, and S100 protein. In addition, immunostains for CK5/6, CK14, p63 protein, and smooth muscle actin displayed a continuous rim of myoepithelial cells around all tumor nests. In contrast, detection of CK20, hormonal receptors (androgen, estrogen, and progesterone), epidermal growth factor receptor and Her-2/neu oncoprotein was negative. The patient is free of disease for 2 years. The relationship between low-grade cribriform cystadenocarcinoma and salivary duct carcinoma is discussed.

Immunoprevention and Immunotherapy of Mammary Carcinoma

Cancer immunoprevention posits that the enhancement of immune defenses in healthy individuals could control tumor onset. Immunoprevention of viral tumors is already implemented at the population level for human hepatocellular and cervical carcinomas. Altogether, viral vaccines could prevent more than 10% of all human tumors. The big question is whether immunoprevention can be applied to nonviral tumors, including breast cancer. Promising results were obtained in preclinical models, in particular in HER-2/neu transgenic mice, which are prone to mammary carcinoma development, using vaccines against HER-2/neu oncoprotein p185. The life expectancy of vaccinated mice was more than doubled. Protective immune mechanisms elicited by effective vaccines were mainly based on helper T cell cytokines, in particular γ-interferon, and anti-p185 antibodies. The term "oncoantigens" was coined to define those antigenic molecules that, like HER-2, are indispensable for tumor growth, thus representing the best class of targets for cancer immunoprevention. The study of immunopreventive vaccines against subsequent phases of neoplastic progression showed a dramatic loss of efficacy against established mammary carcinomas, whereas the prevention of micrometastasis growth was successful. Preclinical results provide useful indications for the translation of cancer immunoprevention to humans, and useful hints for cancer immunotherapy.

Abstract 873: Prevalence and correlates of HER2/neu overexpression among invasive breast cancer cases in two hospitals in Puerto Rico

Abstract Introduction Breast cancer (BC) is the most common female malignancy in Puerto Rico. Cases with human epidermal growth factor receptor 2 (HER-2)/neu oncoprotein overamplification are consistently associated with poor prognosis, poor response to endocrine treatment, and an increased likelihood of recurrence. The objective is to evaluate the prevalence of HER2 /neu gene overexpression among a hospital-based female population of BC cases in Puerto Rico, and to determine its association with other clinical characteristics. Methods This cross-sectional study analyzed data from female patients with invasive BC diagnosed between 2000 and 2005, at the I. Gonzalez Martinez Hospital and the Auxilio Mutuo Hospital (n=1,082) in San Juan, Puerto Rico. Information on Her-2 status and other clinical characteristics was retrieved form the hospital's cancer registries and from medical record review. This study was approved by the Institutional Review Boards of the participating hospitals. Logistic regression models were used to evaluate the associations between relevant clinical characteristics and Her-2 status. Results: The prevalence of Her-2/neu overexpression in our study aas 20.9%, whereas 72.3% of the cases were ER positive and 59.3% were PR positive. In multivariate analysis, women with an age at diagnosis ≤ 50 years were 1.74 (95% CI=1.26-2.41) times more likely to be Her-2 positive as compared with those with an age at diagnosis ≥ 50 years. Regarding ER/PR status, women with ER+/PR- and those with ER+/PR+ were 1.97 (95% CI=1.30-2.98) and 2.15 (95% CI=1.49-3.10) times more likely to be Her-2 positive as compared to those with ER-/PR- status. Finally, women with invasive ductal histology were 1.79 (95% CI=1.08-2.95) more likely to be Her-2 positive as compared to those with invasive lobular histology. Women with tumor grade III/IV, tumor size ≥ 2 cm and lymph node metastasis were also more likely to be Her-2 positive (p&amp;lt;0.05). Conclusions: The prevalence of Her-2/neu overexpression in this population of Puerto Rican patients (20.9) and its association with earlier age at diagnosis, tumor size, lymph node metastasis and other clinical characteristics shows that BC patients with Her-2 positive tumors followed similar profiles as Her-2 positive BC patients in the US population. Results from this study will be useful for the development of future BC prevention and control strategies in Puerto Rico. Sponsors: This work was supported by an unrestricted grant from Glaxo SmithKline with additional support from the National Institutes of Health and National Center for Research Resources (NCRR) [grant number G12RR03051 and U54CA96297]. 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 873.

Evaluation of tumor markers (HER-2/neu oncoprotein, CEA, and CA 15.3) in patients with locoregional breast cancer: prognostic value

Tumor markers were studied in the sera of 883 untreated patients with primary breast cancer diagnosed between 1989 and 2007. Abnormal human epidermal growth factor receptor 2 (HER-2)/neu levels (>15 ng/mL) were found in 9.5%, carcinoembryonic antigen (CEA) in 15.9%, and cancer antigen (CA) 15.3 in 19.7% of the patients. One or more tumor markers were abnormal in 305 (34.5%) of the 883 studied patients. Significantly higher serum HER-2/neu levels were found in patients with tissue overexpression of this oncoprotein (p < 0.0001). CEA, CA 15.3, and HER-2/neu (only in those patients with tissue overexpression) serum levels were related with tumor stage (tumor size and nodal involvement) and steroid receptors (higher values in estrogen receptor-negative (ER-) tumors). Univariate analysis showed that HER-2/neu serum levels were prognostic factors in disease-free survival (DFS) and overall survival (OS) only in patients with tissue overexpression. Multivariate analysis in 834 patients show that nodal involvement, tumor size, ER, CEA, and adjuvant treatment were independent prognostic factors in DFS and OS. When only patients with HER-2/neu overexpression in tissue were studied, tumor size, nodal involvement, and tumor markers (one or another positive) were independent prognostic factors for both DFS and OS. HER-2/neu serum levels were also an independent prognostic factor, with CEA, ER, and nodes in 106 patients treated with neoadjuvant treatment. In summary, serum HER-2/neu, CEA, and CA 15.3 are useful tools in the prognostic evaluation of patients with primary breast cancer.

Interleukin-15 and Its Receptor Augment Dendritic Cell Vaccination against theneuOncogene through the Induction of Antibodies Partially Independent of CD4 Help

Interleukin-15 (IL-15) stimulates the diffrentiation and proliferation of T, B, and natural killer cells; enhances CD8(+) cytolytic T-ceII activity; helps maintain CD44(hi)CD8(+) memory T cells; and stimulates immunoglobulin synthesis by B cells. IL-15 is trans-presented to effector cells by its receptor, IL-15Ralpha, expressed on dendritic cells (DC) and monocytes. We examined the antitumor effect of adenoviral-mediated gene transfer of IL-15 and IL-15Ralpha to augment a DC vaccine directed against the NEU (ErbB2) oncoprotein. Transgenic BALB-neuT mice vaccinated in late-stage tumor development with a DC vaccine expressing a truncated NEU antigen, IL-I5, and its receptor (DC(Ad.Neu+Ad_mIL-15+Ad.mlL-15Ralpha)) were protected from mammary carcinomas, with 70% of animals tumor-free at 30 weeks compared with none of the animals vaccinated with NEU alone (DC(Ad.Neu)). The combination of neu, IL-15, and IL-15Ralpha gene transfer leads to a significaintly greater anti-NEU antibody response compared with mice treated with DC(Ad.Neu) or DC(Ad.Neu) combined with either IL-15 (DC(Ad.Neu+Ad.mlL-15)) or lL-15Ralpha (DC(Ad.Neu+Ad.mlL-15Ralpha)). The antitumor effect was antibody mediated and involved modulation of NEU expression and signaIing. Depletion of CD4(+) cells did not abrogate the antitumor effect of the vaccine, nor did it inhibit the induction of anti-NEU aritibodies. Coexpression of IL-15 and IL-15Ralpha in an anticancer vaccine enhanced immune responses against the NEU antigen and may overcome impaired CD4(+) T-helper function.

Telomerase and HER-2/ neu as targets of genetic cancer vaccines in dogs

Pet dogs represent a valuable pre-clinical model to assess the efficacy of oncology drugs. Additionally, canine cancers occur with an incidence similar to that of humans and share many features with human malignancies including histological appearance, tumor genetics, biological behavior and response to conventional therapies. The telomerase reverse transcriptase (TERT) is reactivated in most of human and dog tumors. Similarly, HER-2/ neu oncoprotein is overexpressed in a proportion of canine breast cancers. Therefore, TERT and HER-2/ neu can constitute valid tumor associated antigens (TAA), suitable targets for translational cancer immunotherapy in dogs. In this study, we have evaluated the ability of DNA electroporation (DNA-EP) and Adenovirus serotype 6 (Ad6) to induce immune responses against dog TERT (dTERT) and HER-2/ neu in healthy dogs. Vaccination was effective in all treated animals and the adaptive immune response remained detectable and long-lasting in the absence of autoimmunity or other side-effects. Our results show that DNA-EP/Ad6-based cancer vaccine induces adaptive immune responses against TAA in canine subjects and support further evaluation of this approach in cancer dog patients.

HER-2/neu (c-erbB-2) oncoprotein in hyperplastic endometrial polyps detected in two cats.

The presence of HER-2/neu (c-erbB-2) oncoprotein, oestrogen-alpha receptor (ER), and progesterone receptor (PR) in hyperplastic endometrial polyps (EPs) of two cats with cystic endometrial hyperplasia-pyometra (CEH-P) complex was investigated. Immunohistochemistry assay for ER, PR and c-erbB-2 oncoprotein in the glandular and stromal tissue of the EPs was performed. ER and c-erbB-2 immunoreactivity was observed in the glandular epithelium of the EPs whereas PR immunoreactivity was detected only in the stromal fibroblasts. The c-erbB-2 oncoprotein may play a role with the ER in the pathogenesis of the hyperplastic EPs, although the role of this oncoprotein in the pathogenesis of EPs has yet to be determined.

HER-2/ neu (657-665) represents an immunogenic epitope of HER-2/ neu oncoprotein with potent antitumor properties

The HER-2/ neu oncoprotein is a promising cancer vaccine target. We describe herein a novel HLA-A2.1-restricted epitope, encompassing amino acids 657-665 (AVVGILLVV), which is naturally presented by human breast and ovarian cell lines. HER-2/ neu(657-665), [HER-2(9 657)], binds with high affinity to HLA-A2.1 molecules as revealed by a prediction algorithm (SYFPEITHI) and in functional assays. This peptide was found to be immunogenic in HLA-A2.1 transgenic (HHD) mice inducing peptide-specific CTL, which responded with increased IFNγ production, degranulation, and in vitro as well as in vivo cytotoxicity. Most important, HER-2(9 657) functioned as a therapeutic vaccine by enabling HHD mice to reject established transplantable tumors. Cured mice resisted tumor growth when re-challenged with the same tumor, demonstrating the capacity of HER-2(9 657) to generate tumor-specific memory immune response. Finally, this peptide was also found to be immunogenic in PBMCs from HLA-A2.1 + patients with HER-2/ neu + breast cancer. Our data encourage further exploitation of HER-2(9 657) as a promising candidate for peptide-based cancer vaccines.

Results from the first phase I clinical study of the novel Ii-Key/HER2/neu(776–790) hybrid peptide vaccine in patients with prostate cancer

3011 Background: HER-2/neu(776–790) represents an immunogenic epitope from the HER-2/neu oncoprotein whose immunogenicity is highly potentiated upon linkage with the Ii-Key moiety (LRMK) from the MHC class II invariant chain. Herein, we present the results of the first phase I clinical trial of the Ii-Key/HER-2/neu(776–790) (AE37) vaccine in patients (pts) with prostate cancer. Methods: Androgen-dependent (AD) and androgen-independent (AI) pts with HER-2/neu+ (IHC: 1–3) prostate adenocarcinomas were eligible. Concomitant medication with bicatulamide and LHRH or docitaxel was allowed. All pts received 6 monthly vaccinations with the AE37 vaccine (500 μg of AE37 plus 125 μg of GM-CSF) administered in two doses intradermally 5cm apart in the same extremity for each vaccination cycle. Immunologic responses were measured monthly in vitro by the IFNγ-based ELISPOT assay using pts’ PBMC and in vivo at the beginning and end of immunizations using DTH. Local dermal reactions were also measured after each vaccination. Results: Eligible pts [AD (n=18), AI (n=10)] were at stages T1–3N0M0, GS: 3–7 (n=18); T1–3N+M0, GS: 6–7 (n=2); T1–3N0M+, GS: 6–9 (n=4) and T1–3N+M+, GS: 6–9 (n=4). All pts had standard treatment prior to vaccinations, including surgery (S) (n=9); hormonal treatment (HT) (n=4); S+HT (n=6); S+HT+radiotherapy (RT); (n=2); S+chemotherapy (CH) (n=2); HT+RT (n=2); CH (n=3) and S+HT+CH (n=2). During vaccinations, 11 pts were free of any treatment, while 5 pts who had progressive disease received additional chemotherapy; the remainder received HT alone or combined with RT. 25 pts completed all vaccinations. Toxicity and side effects beyond grade-2 were not observed. GM-CSF was reduced by 50% for subsequent vaccinations when a local reaction of 100mm or greater was observed. DTH reactions to the parent HER-2/neu(776–790) peptide were increased (compared to pre-vaccination) for all pts, while approximately half the pts responded with significantly increased IFNγ responses to AE37 and/or parent HER-2/neu(776–790) peptide in at least 2 sequential vaccination cycles. Conclusions: The AE37 vaccine is safe and well tolerated. AE37 is also capable of eliciting potent and specific immunologic responses in prostate cancer pts. [Table: see text]

Comprehensive immunohistochemical analysis of Her-2/neu oncoprotein overexpression in breast cancer: HercepTest™ (Dako) for manual testing and Her-2/neuTest 4B5 (Ventana) for Ventana BenchMark automatic staining system with correlation to results of fluorescence in situ hybridization (FISH)

Overexpression of Her-2/neu-oncoprotein is used as marker for Herceptin therapy. To investigate the sensitivity and specificity of automatic immunohistochemistry (Benchmark, Ventana), we compared the results to the manual testing (Dako) in 130 breast carcinomas and validated the results by fluorescence in situ hybridization (FISH). Manual and automatic immunohistochemistry of Her-2/neu-oncoprotein using two different antibodies (HercepTest, Her-2/neuTest 4B5) was analyzed. FISH was performed in all cases with uncertain or strong overexpression in either immunohistochemical stainings or with different immunohistochemical results. Same immunohistochemical results were seen in 73.8%. Two cases with overexpression, detected with Her-2/neuTest 4B5 and confirmed by FISH, showed no overexpression using HercepTest. From 21 cases with 2+ by Her-2/neuTest 4B5, 15 cases had no gene amplification (two of them with 3+ HercepTest); three cases showed a gene amplification (one of them with failing overexpression by HercepTest); two other cases were polysomic; one could not be analyzed. Ventana immunohistochemistry seems to be of same reliability like Dako with a little better concordance to FISH in our study.

Identification of a Novel Immunogenic HLA-A*0201-Binding Epitope of HER-2/neu with Potent Antitumor Properties

HER-2/neu oncoprotein is overexpressed in a variety of human tumors and is associated with aggressive disease. Immunogenic HER-2/neu CTL epitopes have been used as vaccines for the treatment of HER-2/neu positive malignancies with limited success. By applying prediction algorithms for MHC class I ligands and proteosomal cleavages, in this study, we describe the identification of HER-2/neu decamer LIAHNQVRQV spanning residues 85-94 (HER-2(10(85))). HER-2(10(85)) proved to bind with high affinity to HLA-A2.1 and was stable for 4 h in an off-kinetics assay. This peptide was immunogenic in HLA-A2.1 transgenic (HHD) mice inducing peptide-specific CTL, which responded to tumor cell lines of various origin coexpressing human HER-2/neu and HLA-A2.1. This demonstrates that HER-2(10(85)) is naturally processed from endogenous HER-2/neu. Five of sixteen HER-2/neu+ HLA-A2.1+ breast cancer patients analyzed had HER-2(10(85))-reactive T cells ranging from 0.35-0.70% of CD8+ T cells. Depletion of T regulatory cells from PBMC enabled the rapid expansion of HLA-A2.1/HER-2(10(85))pentamer+/CD8+ cells (PENT+/CD8+), whereas significantly lower numbers of CTL could be generated from unfractionated PBMC. HER-2(10(85))-specific human CTL recognized the HER-2/neu+ HLA-A2.1+ tumor cell line SKBR3.A2, as determined by IFN-gamma intracellular staining and in the high sensitivity CD107alpha degranulation assay. Finally, HER-2(10(85)) significantly prolonged the survival of HHD mice inoculated with the transplantable ALC.A2.1.HER tumor both in prophylactic and therapeutic settings. These data demonstrate that HER-2(10(85)) is an immunogenic peptide, capable of eliciting CD8-mediated responses in vitro and in vivo, providing the platform for further exploitation of HER-2(10(85)) as a possible target for anticancer immunotherapy.

The Correlation of Serum HER-2/neu and CA15-3 in Patients with Metastatic Breast Cancer

Purpose: The extracellular domain (ECD) of the HER-2/ neu oncoprotein, whose molecular weight is the range from the 95 kD to 105 kD, is shed into the blood from the cell surface via, proteolysis by a metalloprotease. A monoclonal antibody immunoassay has been developed for measuring the circulating concentrations of serum HER-2/neu ECD (following serum HER-2/neu). Serum HER-2/neu has been reported to be correlated with an increased tumor volume in those patients suffering with breast cancer. We measured the serum CA15-3 level, which is a surrogate marker of the tumor burden, we analyzed the correlation of the serum CA15-3 with the serum HER-2/neu and we analyzed the association of both markers with the clinical outcomes. Methods: The sera for the analysis of both HER-2/neu and CA15-3 were obtained from 99 healthy Korean women, 66 primary breast cancer patients and 43 metastatic breast caner patients. The serum HER-2/neu level was measured quantitatively with using an ADVIA Centaur � automated immunoassay analyzer (Bayer Health Care LLC, Diagnostics Division, Tarrytown, New York, USA) and the CA 15-3 level was measured via radioimmunoassay. Results: The serum HER-2/neu level was increased 23 metastatic cancer patients (53%). On the analysis of the correlation of serum HER-2/neu and CA15-3, the correlation coefficient (r) was 0.8072. Thus a positive serum HER-2/neu test in breast cancer patients was highly associated with the CA15-3 level for assessing whether metastasis was present or not. For the relationship between primary breast cancer and metastatic breast cancer, the former was classified as the control group and the latter as the patient group. The results of the Receiver Operation Characteristic (ROC) curve for serum HER-2/neu and CA15-3 showed no statistically significant differences (p=0.176) but the diagnostic efficacy of the serum HER-2/neu test was measured more exactly than that of CA15-3 and CA15-3 a tool for measuring a tumor marker for the diagnosis of whether metastasis was present or not. Conclusion: Serum HER-2/neu is a significant independent predictive prognostic factor for metastatic breast cancer patients. In view of the results we have achieved so far the serum HER-2/neu level in metastatic breast cancer patients may play an important roll as an independent tumor marker.

Alphaviral vector-transduced dendritic cells are successful therapeutic vaccines against neu-overexpressing tumors in wild-type mice.

While dendritic cell (DC) vaccines can protect hosts from tumor challenge, their ability to effectively inhibit the growth of established tumors remains indeterminate. Previously, we have shown that human DCs transduced with Venezuelan equine encephalitis virus replicon particles (VRPs) were potent stimulators of antigen-specific T cells in vitro. Therefore, we investigated the ability of VRP-transduced DCs (VRP-DCs) to induce therapeutic immunity in vivo against tumors overexpressing the neu oncoprotein. Transduction of murine DCs with VRPs resulted in high-level transgene expression, DC maturation and secretion of proinflammatory cytokines. Vaccination with VRP-DCs expressing a truncated neu oncoprotein induced robust neu-specific CD8(+) T cell and anti-neu IgG responses. Furthermore, a single vaccination with VRP-DCs induced the regression of large established tumors in wild-type mice. Interestingly, depletion of CD4(+), but not CD8(+), T cells completely abrogated inhibition of tumor growth following vaccination. Taken together, our results demonstrate that VRP-DC vaccines induce potent immunity against established tumors, and emphasize the importance of the generation of both CD4(+) T cell and B cell responses for efficient tumor inhibition. These findings provide the rationale for future evaluation of VRP-DC vaccines in the clinical setting.