Neu Extracellular Domain Research Articles

Antitumor immunization of mothers delays tumor development in cancer-prone offspring

Maternal immunization is successfully applied against some life-threatening infectious diseases as it can protect the mother and her offspring through the passive transfer of maternal antibodies. Here, we sought to evaluate whether the concept of maternal immunization could also be applied to cancer immune-prevention. We have previously shown that antibodies induced by DNA vaccination against rat Her2 (neu) protect heterozygous neu-transgenic female (BALB-neuT) mice from autochthonous mammary tumor development. We, herein, seek to evaluate whether a similar maternal immunization can confer antitumor protection to BALB-neuT offspring. Significantly extended tumor-free survival was observed in BALB-neuT offspring born and fed by mothers vaccinated against neu, as compared to controls. Maternally derived anti-neu immunoglobulin G (IgG) was successfully transferred from mothers to newborns and was responsible for the protective effect. Vaccinated mothers and offspring also developed active immunity against neu as revealed by the presence of T–cell-mediated cytotoxicity against the neu immunodominant peptide. This active response was due to the milk transfer of immune complexes that were formed between the neu extracellular domain, shed from vaccine-transfected muscle cells, and the anti-neu IgG induced by the vaccine. These findings show that maternal immunization has the potential to hamper mammary cancer in genetically predestinated offspring and to develop into applications against lethal neonatal cancer diseases for which therapeutic options are currently unavailable.

Circulating Her-2/neu extracellular domain in breast cancer patients-correlation with prognosis and clinicopathological parameters including steroid receptor, Her-2/neu receptor coexpression.

HER-2/neu extracellular domain (ECD) can be detected in blood as a soluble circulating protein. The aim of this study was to analyze the relationship between HER-2/neu extracellular domain in the serum and the prognosis in breast cancer patients. We also correlated HER-2/neu ECD with various clinicopathological factors including steroid receptor, HER-2/neu receptor coexpression. The serum from seventy nine patients with invasive breast cancer and twenty individuals without malignancy was analyzed using the enzyme-linked immune adsorbent assay method. The cut-off value was estimated by the ROC curve analysis (15.86 μg/L). HER-2/neu ECD values in the serum of patients with breast cancer were significantly higher than in control subjects. Circulating HER-2/neu ECD was significantly associated with the histological grade of tumors and the status of axillary lymph nodes. Negative correlation was observed between HER-2/neu ECD in the serum and estrogen receptor positivity. When we analyzed HER-2/neu ECD in relation with coexpression of steroid receptor and HER-2/neu receptor in tissue, statistically higher values were found in the subgroup of patients with steroid receptor negative, HER-2/neu negative tumors than in the other subgroups. HER-2/neu ECD was not an independent factor in the univariate and multivariate analysis. However, elevated HER-2/neu ECD levels were found in patients with breast cancer possessing more aggressive phenotype.

Targeting of antigens to B lymphocytes via CD19 as a means for tumor vaccine development.

Ab therapy against surface Ags on tumor cells has demonstrated significant efficacy for some cancers. However, it is costly and patients frequently develop acquired resistance over time. In cases of Ab therapy resistance, T cell responses have been shown to be essential in controlling disease progression. Thus, vaccination that generates a sustained Ab response as well as a T cell response may be more effective and economical. In this article, we have developed a vaccination strategy by targeting protein Ags to B cells via a CD19 single-chain variable fragment miniAb. Using the tumor-associated Ag her-2/neu extracellular domain, we showed that the coengagement of CD19 and BCR induced full B cell activation to produce a high titer of Abs and enhanced CD4 Th2 response and CD8 T cell activation and differentiation. These Abs competitively inhibited humanized her-2/neu Ab binding and were capable of activating the complement and inhibiting human breast cancer growth in vitro. Therapeutic efficacy was demonstrated in vivo using murine mammary carcinoma models. Furthermore, four different extracellular domains of her-2/neu could be targeted to B cells to generate Abs against particular domains with different antitumor properties. This approach may offer a new avenue for vaccine development with significantly lower cost, which may be of use not only for cancer therapy but also for infectious agents.

Open-label, dose-escalation, safety, pharmacokinetic, and pharmacodynamic study of intravenously administered CNF1010 (17-(allylamino)-17-demethoxygeldanamycin [17-AAG]) in patients with solid tumors

17-(Allylamino)-17-demethoxygeldanamycin (17-AAG) is a benzoquinone ansamycin that binds to and inhibits the Hsp90 family of molecular chaperones leading to the proteasomal degradation of client proteins critical in malignant cell proliferation and survival. We have undertaken a Phase 1 trial of CNF1010, an oil-in-water nanoemulsion of 17-AAG. Patients with advanced solid tumors and adequate organ functions received CNF1010 by 1-h intravenous (IV) infusion, twice a week, 3 out of 4 weeks. Doses were escalated sequentially in single-patient (6 and 12 mg/m(2)/day) and three-to-six-patient (≥25 mg/m(2)/day) cohorts according to a modified Fibonacci's schema. Plasma pharmacokinetic (PK) profiles and biomarkers, including Hsp70 in PBMCs, HER-2 extracellular domain, and IGFBP2 in plasma, were performed. Thirty-five patients were treated at doses ranging from 6 to 225 mg/m(2). A total of 10 DLTs in nine patients (2 events of fatigue, 83 and 175 mg/m(2); shock, abdominal pain, ALT increased, increased transaminases, and pain in extremity at 175 mg/m(2); extremity pain, atrial fibrillation, and metabolic encephalopathy at 225 mg/m(2)) were noted. The PK profile of 17-AAG after the first dose appeared to be linear up to 175 mg/m(2), with a dose-proportional increase in C max and AUC0-inf. Hsp70 induction in PBMCs and inhibition of serum HER-2 neu extracellular domain indicated biological effects of CNF1010 at doses >83 mg/m(2). The maximum tolerated dose was not formally established. Hsp70 induction in PBMCs and inhibition of serum HER-2 neu extracellular domain indicated biological effects. The CNF1010 clinical program is no longer being pursued due to the toxicity profile of the drug and the development of second-generation Hsp90 molecules.

A vaccine approach for breast cancer immunotherapy by targeting HER2/neu antigen to B cells via CD19 molecule.

287 Background: Trastuzumab has been proven to be effective in the immunotherapy for Her-2/neu-positive breast cancer. However, this adoptively transferred therapeutic Ab must be continuously given to the patient with huge financial cost. Thus, it would be desirable if tumor vaccines could elicit long-lasting trastuzumab-like Ab. Methods: Constructing fusion protein of anti-mouse CD19 single chain variable fragment (anti-CD19-scFv) with human Her-2/neu extracellular domain P3-4 (P3-4) by extracting total RNA from Rat anti-mouse CD19 hybridoma (1D3) and first strand cDNA was synthesized accordingly. VH and VL were amplified using designed primers. Single chain Fv (VL-VH) was then synthesized by overlapping PCR. Then constructs were sequenced and cloned into pET-20b(+) vectors. Fragments P3-4 of extracellular domains of human Her-2/neu was cloned from pcDNA3.1-Her2 and then cloned into pET-20b(+)-scFv. Series of constructs were expressed and purified according to standard protocol and verified by Western Blot. Results: Both in vitro and in vivo studies demonstrated that fusion proteins anti-CD19-scFv and anti-CD19-scFv-P3-4 but not P3-4 could specifically bind to B cells with high affinity. Mice immunized with anti-CD19-scFv-P3-4 secreted higher titers of IgG and IgM than those from controls (p<0.05). Studies also demonstrated that sera from anti-CD19-scFv-P3-4 but not anti-CD19-scFv or P3-4 immunized mice stained with Her-2/neu expressing SKOV-3 tumor cells. These Abs also competitively inhibited trastuzumab-mediated Ag binding, suggesting that trastuzumab-like Ab responses were elicited. WT mice immunized with anti-CD19-scFv-P3-4 fusion protein then challenged with D2/F2-Her-2 mammary tumor cells showed significantly reduced tumor burden compared to those immunized with control fusion proteins (p<0.05) and had enhanced median overall survival to 45 days versus 34 days in WT mice immunized with either anti-CD19-scFv or P3-4. Conclusions: Targeting of Her-2/neu antigens to B cells stimulates Th-dependent humoral immune responses with anti tumor effect in mouse model. These findings provide a novel avenue for successful development of breast cancer vaccination strategy and help to fight for cancer.

Targeting HER2/neu antigen to B cells via CD19 molecule: A vaccine approach for breast cancer immunotherapy.

e13074 Background: Herceptin has been proven to be effective in the immunotherapy for Her-2/neu+ breast cancer. However, this adoptively transferred therapeutic Ab must be continuously given to the patient with huge financial cost. Thus, it would be desirable if tumor vaccines could elicit long-lasting Herceptin-like Ab. Methods: Constructing fusion protein of anti-mouse CD19 single chain variable fragment (anti-CD19-scFv) with human Her-2/neu extracellular domain P3-4 (P3-4) by extracting total RNA from Rat anti-mouse CD19 hybridoma (1D3) and first strand cDNA was synthesized accordingly. VH and VL were amplified using designed primers. Single chain Fv (VL-VH) was then synthesized by overlapping PCR. Then constructs were sequenced and cloned into pET-20b(+) vectors. Fragments P3-4 of extracellular domains of human Her-2/neu was cloned from pcDNA3.1-Her2 and then cloned into pET-20b(+)-scFv. Series of constructs were expressed and purified according to standard protocol and verified by Western Blot. Results: Both in vitro and in vivo studies demonstrated that fusion proteins anti-CD19-scFv and anti-CD19-scFv-P3-4 but not P3-4 could specifically bind to B cells with high affinity. Mice immunized with anti-CD19-scFv-P3-4 secreted higher titers of IgG and IgM than those from controls (p<0.05). Studies also demonstrated that sera from anti-CD19-scFv-P3-4 but not anti-CD19-scFv or P3-4 immunized mice stained with Her-2/neu expressing SKOV-3 tumor cells. These Abs also competitively inhibited Herceptin-mediated Ag binding, suggesting that Herceptin-like Ab responses were elicited. WT mice immunized with anti-CD19-scFv-P3-4 fusion protein then challenged with D2/F2-Her-2 mammary tumor cells showed significantly reduced tumor burden compared to those immunized with control fusion proteins (p<0.05) and had enhanced median overall survival to 45 days versus 34 days in WT mice immunized with either anti-CD19-scFv or P3-4. Conclusions: Targeting of Her-2/neu antigens to B cells stimulates Th-dependent humoral immune responses with anti tumor effect in mouse model. These findings provide a novel avenue for successful development of breast cancer vaccination strategy and help to fight for cancer.

A Better Immune Reaction to Erbb-2 Tumors Is Elicited in Mice by DNA Vaccines Encoding Rat/Human Chimeric Proteins

The Erbb-2 (neu in rat and Her-2 in humans) tyrosine kinase receptor is an oncoantigen (i.e., a tumor-associated molecule directly involved in cancer progression). Because oncoantigens are self-tolerated molecules, to trigger a response circumventing tolerance, we generated two plasmids (RHuT and HuRT) coding for chimeric neu-Her-2 extracellular and transmembrane proteins that are expressed on the cell membrane of the transfected cells and recognized by monoclonal antibodies reacting against neu and Her-2. RHuT encodes a protein in which the 410 NH(2)-terminal residues are from the neu extracellular domain and the remaining residues from Her-2. Almost symmetrically, HuRT encodes for a protein in which the 390 NH(2)-terminal residues are from Her-2 and the remainder from neu. The ability of RHuT and HuRT to elicit a protective response to neu and Her-2 in wild-type mice and in transgenic mice tolerant to neu and Her-2 proteins was compared with that of plasmids coding for the fully rat or fully human extracellular and transmembrane domains of the Erbb-2 receptor. In most cases, RHuT and HuRT elicited a stronger response, although this chimeric benefit is markedly modulated by the location of the heterologous moiety in the protein coded by the plasmid, the immune tolerance of the responding mouse, and the kind of Erbb-2 orthologue on the targeted tumor.

Serological and immunohistochemical HER-2/neu statuses do not correlate and lack prognostic value for ovarian cancer patients

The serodiagnostics of extracellular domain (ECD) HER-2/neu has turned into an evidenced-based tumour marker for HER-2/neu-positive breast cancer patients. This study investigated the clinical relevance of immunohistochemical and serum HER-2/neu in 44 patients with advanced ovarian cancer. The Hercept-Test® from DAKO Diagnostics was used to analyse immunohistochemical HER-2/neu expression. The HER-2/neu ECD in serum was determined quantitatively by Bayer Immuno 1™ Immunoanalyser. The HER-2/neu serum values were correlated to the clinical course of disease and to established prognostic factors, i.e. progression-free and overall survival. Some 23% of patients (n = 11) expressed HER-2/neu serum levels higher than 15 ng/mL, whereas only 7.7% (n = 2) of the patients examined by immunohistochemistry showed a HER-2/neu overexpression of the tissue. None of them revealed an overexpression of HER-2/neu ECD by serodiagnostics. HER-2/neu overexpression did not correlate significantly to any of the analysed prognostic factors. According to progression-free and overall survival, there was no significant difference between serologically HER-2/neu-positive or negative patients. For ovarian cancer patients, neither high HER-2/neu serum levels, nor immunohistochemically determined HER-2/neu positivity, appear to predict the course of disease. This study shows a lack of association between the immunohistochemical HER-2/neu status and the serum level of solute extracelluar HER-2/neu domain.

Anti-p53 antibodies in serum: relationship to tumor biology and prognosis of breast cancer patients

The aim of this study was to analyze the concentration of anti-p53 antibodies in the serum of breast cancer patients and to correlate these results with various clinical, pathological and biochemical parameters. We also wanted to assess the prognostic significance of these antibodies in our patients. Sera from 61 patients with breast cancer and 20 individuals without malignancies were analyzed using enzyme-linked immunoadsorbent assay. High levels of anti-p53 antibodies were detected in twenty-one (35%) breast cancer patients and one control (5%). The difference was statistically significant. We observed an inverse relationship between the anti-p53 antibodies and the age of the patients. We found significant association of anti-p53 antibodies with tumor size, histological grade of the tumors and the number of axillary lymph nodes involved. The levels of anti-p53 antibodies were higher in patients with negative estrogen and progesterone receptors in comparison with patients with positive steroid receptors, but the difference was not statistically significant. No relation was observed between anti-p53 antibodies neither with the Cathepsin D levels in the cytosol nor with the HER-2/neu extracellular domain in the serum. Patients with primary tumors and higher levels of anti-p53 antibodies had shorter 5-year survival than patients with lower levels of anti-p53 antibodies. Our results support the role of anti-p53 antibodies as a biomarker of less favorable phenotype as well as a prognostic factor for patients with breast cancer.

The clinic significance of the HER-2/neu ECD in patients with recurrent and metastatic breast cancer receiving salvage chemotherapy

Objective To investigate the clinical significance of the extracellular domain (ECD) of the HER-2/neu receptor in serum in patients with recurrent and metastatic breast cancer receiving salvage chemotherapy, nethods HER-2/neu ECD in sera obtained from 27 healthy volunteers,27 patients with be-nign breast disease, and 27 patients with recurrent and metastatic breast cancer around chemotherapy was measured by ELISA,and statistically analyzed in combination with clinical data. Results There was no sig-nificant difference in HER-2/neu ECD levels (below 15 μg/L) between healthy volunteers and patients with benign breast disease (P>0.05). The mean concentration of HER-2/neu ECD in patients with recurrent and metastatic breast cancer before salvage chemotherapy was (47.30±109.06) μg/L. As compared with healthy volunteers and patients with benign breast disease, patients with recurrent and metastatic breast canc-er had significantly higher levels of HER-2/neu ECD (P 0.05). The HER-2/neu ECD levels before chemotherapy had a negative correlation with overall survival (r=-0.601, P 0.05). As compared with patients with baseline below 15 μg/L,patients with initially elevated HER-2/neu ECD levels (>15 μg/L) had lower overall survival rate (P 0.05). Conclusion The monitoring of HER-2/neu ECD levels is helpful to detect recurrence and metastasis of breast cancer,select regimen of salvage chemotherapy as well as evaluate the efficacy and prognosis. Key words: Breast neoplasms; Serum; Therapy

The prognostic and predictive values of circulating HER-2/neu extracellular domain in patients with metastatic breast cancer

The prognostic and predictive values of circulating HER-2/neu extracellular domain in patients with metastatic breast cancer

Protective Immunity Againstneu-Positive Carcinomas Elicited by Electroporation of Plasmids Encoding Decreasing Fragments of Rat Neu Extracellular Domain

We have shown that electroporation of plasmid carrying extracellular and transmembrane domains (EC-TM plasmid) encoded by the rat neu oncogene triggers a protective immune response toward rat p185(neu)-positive tumors in both wild-type BALB/c mice and cancer-prone rat neu-transgenic BALB-neuT mice. To identify the critical fragments that confer this protective immunity, mice were electroporated with plasmids encoding the TM domain associated with decreasing fragments of the EC domain and the antitumor protection afforded, the titer of antibody, and cytotoxic T lymphocyte (CTL) activity elicited to Neu protein were evaluated. Plasmids encoding EC fragments shortened by 70 (EC1-TM plasmid), 150 (EC2-TM), 230 (EC3-TM), 310 (EC4-TM), and 390 (EC5-TM) NH(2)-terminal residues afforded effective protection. Plasmids encoding shorter truncated proteins were ineffective. When the immunogenic protein was retained in the cytoplasm (EC1-TM, EC2-TM, and EC5-TM), only a CTL response was elicited, whereas when it was also expressed on the membrane (EC4-TM) both CTLs and antibodies were induced. EC4-TM encoding a truncated protein with an EC portion of only 344 amino acids conferred protection on both BALB/c and BALB-neuT mice comparable to that of EC-TM.

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.

Gene-specific inhibition of breast carcinoma in BALB-neuT mice by active immunization with rat Neu or human ErbB receptors.

Employing the transgenic BALB-neuT mouse tumor model, we explored the in vivo biologic relevance of immunocompetent epitopes shared among the four ErbB receptors. The outcome of neu-mediated tumorigenesis was compared following vaccination with isogeneic normal rat ErbB2/Neu (LTR-Neu) or xenogeneic human ErbB receptors (LTR-EGFR, LTR-ErbB2, LTR-ErbB3 and LTR-ErbB4), each recombinantly expressed in an NIH3T3 murine cell background. Vaccination using rat LTR-Neu at the stage of atypical hyperplasia potently inhibited neu-mediated mammary tumorigenesis. Moreover, all human ErbB receptors specifically interfered with tumor development in BALB-neuT mice. Relative increase in tumor-free survival and reduction in tumor incidence corresponded to structural similarity shared with the etiologic neu oncogene, as rat orthologue LTR-Neu proved most effective followed by the human homologue LTR-ErbB2 and the other three human ErbB receptors. Vaccination resulted in high titer specific serum antibodies, whose tumor-inhibitory effect correlated with cross-reactivity to purified rat Neu extracellular domain in vitro. Furthermore, a T cell response specific for peptide epitopes of rat Neu was elicited in spleen cells of mice immunized with LTR-Neu and was remotely detectable for discrete peptides upon vaccination with LTR-ErbB2 and LTR-EGFR. The most pronounced tumor inhibition by LTR-Neu vaccination was associated with leukocyte infiltrate and tumor necrosis in vivo, while immune sera specifically induced cytotoxicity and apoptosis of BALB-neuT tumor cells in vitro. Our findings indicated that targeted inhibition of neu oncogene-mediated mammary carcinogenesis is conditional upon the immunization schedule and discrete immunogenic epitopes shared to a variable extent by different ErbB receptors.

Kinetics of serum HER‐2/neu changes in patients with HER‐2‐positive primary breast cancer after initiation of primary chemotherapy

The purpose of the study was to determine the utility of quantitation of the extracellular domain (ECD) of the HER-2/neu receptor in the serum for predicting response to treatment in patients with primary breast cancer receiving neoadjuvant therapy. HER-2/neu ECD was measured in sera obtained from 39 patients with HER-2-amplified stage II-III primary breast cancer undergoing neoadjuvant chemotherapy. Patients were randomly assigned to either 4 cycles of paclitaxel followed by 4 cycles of fluorouracil, epirubicin, and cyclophosphamide (FEC) (n = 10) or to the same chemotherapy with simultaneous weekly trastuzumab for 24 weeks (n = 29). Changes in HER-2 ECD were monitored with the Bayer HER-2/neu assay over 6 months and correlated with pathological response to treatment. Before initiation of chemotherapy, 28.2% of patients had elevated concentration of the HER-2 ECD (>15 ng/mL). The median baseline serum HER-2 ECD concentration was 13.6 ng/mL (mean +/- SD, 20.3 +/- 35.5 ng/mL). A decrease in the median HER-2 ECD levels from baseline to Week 3 and from baseline to Week 6 of chemotherapy was seen regardless of treatment regimen. No significant difference in baseline HER-2 ECD levels was observed between the groups who achieved pathological complete response (pCR) and the group with residual disease (P = .41). However, a 9% drop from Week 3 to Week 6 after initial chemotherapy was predictive of pCR (P = .04). A decrease in serum HER-2 ECD levels early during treatment was associated with pathological response in patients receiving primary chemotherapy, particularly trastuzumab-based regimens. Serum HER-2 ECD levels may serve to monitor neoadjuvant therapy in HER-2-positive primary breast cancer.

Serum HER-2/neu change predicts clinical outcome to trastuzumab-based therapy

500 Background: Trastuzumab monotherapy has a 34% objective response rate (ORR) in patients with HER-2/neu IHC 3+ or FISH-positive first-line metastatic breast cancer (C. Vogel et al, JCO 20:719–726, 2002). Predicting response and survival to trastuzumab-based therapy is an unsolved problem. The HER-2/neu extracellular domain (ECD) is released after cleavage by the ADAM metalloproteinases, and the remaining membrane-bound internal domain is constitutively activated. Trastuzumab inhibits cleavage of the HER-2/neu ECD. Methods: A pooled analysis of 7 trials of first-line trastuzumab therapy (with or without chemotherapy) with serial serum HER-2/neu levels were included. The FDA-approved HER-2/neu ELISA (Oncogene Science/Bayer HealthCare) was used to determine serum HER-2/neu levels. A pretreatment and post-treatment serum (16–120 days) from 307 patients was available. 236 patients had data on overall survival. Kaplan Meier Life table analysis was performed to compare duration of response (DRP), time to progression (TTP), and overall survival (OS). Results: The median decrease in serum HER-2/neu levels for all patients was 31.0% (Range: 98% decrease to 239% increase). Patients with &gt; 20% decrease in HER-2/neu levels had a significantly higher objective response rate (ORR, complete + partial response) and longer DRP, TTP and OS. The results were similar regardless of the timing of the second serum draw (≤ 30 days vs. &gt; 30 days) after the start of trastazumab. Conclusion: Patients with &lt; 20% decrease in serum HER-2/neu levels have decreased benefit from trastuzumab therapy. Patients who do not have a significant decrease in serum HER-2/neu levels should be considered for additional HER-2/neu-targeted therapies. [Table: see text] [Table: see text]

Establishment of ELISA to detect HER-2/neu ECD levels in human sera

目的 建立乳腺癌患者血清HER-2/neu 胞外域蛋白(ECD)水平检测方法,为乳腺癌治疗及预后判断提供参考.方法以识别HER-2/neu 不同抗原表位的2种抗HER-2/neu单克隆抗体(McAb)为基础,建立可定量检测血清HER-2/neu ECD水平的双抗夹心酶联免疫吸附试验法(ELISA).结果本研究建立的HER-2/neu ECD水平检测ELISA所用试剂[包括抗HER-2/neu McAb 5A12包被的酶标板、辣根过氧化物酶(HRP)标记的HER-2/neu McAb 1-2及HRP 底物四甲基联苯胺(TMB)溶液]在1年内保持稳定;批内、批间变异系数分别为3.6%±4.2%、6.9%±3.3%;与HER-2 ECD同源性很高的人表皮生长因子受体(EGFR/HER-1)ECD不发生交叉反应.自制ELISA试剂盒与Bender Medsystems试剂盒检测人血清HER-2 ECD水平的相关性较好(r＞0.75).健康人群(20名)与乳腺癌患者(140例)血清HER-2 ECD阳性率之间的差异无统计学意义(P＞0.05),但与转移乳腺癌患者(10例)血清HER-2 ECD阳性率之间差异有统计学意义(P＜0.01).结论本研究建立的HER-2/neu ECD 双抗夹心ELISA检测方法特异、稳定、可靠,可定量检测乳腺癌患者血清HER-2/neu ECD水平。

Insights into the mechanism of anti-tumor immunity in mice vaccinated with the human HER2/ neu extracellular domain plus anti-HER2/ neu IgG3-(IL-2) or anti-HER2/ neu IgG3-(GM-CSF) fusion protein

In the present study, we demonstrate that a physical association between the extracellular domain of human HER2/ neu receptor (ECD HER2) plus anti-HER2/ neu IgG3-(IL-2) or anti-HER2/ neu IgG3-(GM-CSF) was required to elicit the most effective anti-tumor immune response against a syngeneic tumor expressing rat HER2/ neu. Immune effectors including CD4 +, CD8 +, and NK cells contributed to protection against tumor growth. Vaccinated B-cell deficient mice did not elicit tumor protection, suggesting a critical role for B-cells in a protective immune response. These results provide insights into the mechanisms responsible for the protective tumor immunity elicited when antibody-(IL-2 or GM-CSF) are used as enhancers of vaccines targeting tumor antigens.

HER-2 sérique, cancer du sein et trastuzumab (Herceptine ®)

The HER-2/neu oncogene and its p185 receptor protein are indicators of a more aggressive form of breast cancer. The HER-2/neu status of the tumour guides trastuzumab (Herceptin ®) therapy, a monoclonal antibody directed against the extracellular domain (ECD) of the HER-2/neu oncoprotein. The HER-2/neu ECD is shed from cancer cells into the circulation and is measurable by immunoassay (HER-2/neu Manual Elisa, Oncogene Science, Cambridge, MA, USA). Currently, the HER-2 status of the tumour is evaluated either by morphological techniques, immunocytochemistry or fluorescent in situ hybridization. We studied serum HER-2 ECD before and after primary treatment in a series of 701 primary breast cancers at diagnosis and before first metastases ( N =128). At diagnosis, elevated HER-2 ECD (>12 ng/ml, cut-off level) was found in only 47 (6.7%) patients. Before first metastases, 45% of the samples were over 12 μg/l of HER-2 ECD (cut-off) and were significantly related with pre -and post treatment values ( P =0.0350 and P <0.0001 respectively). Concordance with CA 15.3 was 56.25% (weak correlation, rho =0.263). HER-2 ECD levels differed according to the sites of metastases ( P =0.0199, highest levels found in liver and lung). A median HER-2 ECD lead time of 254 days was found in 18/28 (64.3%) of recurring patients. Premetastatic HER-2 ECD levels showed a strong univariate (Kaplan-Meier method, P =0.0014) and multivariate prognostic values (Cox model, P <0.0001) for survival. We monitored the longitudinal changes in HER-2 ECD values of 33 women with metastatic breast cancer treated with trastuzumab and chemotherapy. Changes in HER-2 ECD serial concentrations reflected the clinical course of disease, increasing HER-2 ECD indicated breast cancer progression while decreasing serum values were indicative of response to therapy. We constructed a model (GEE, generalised estimated equation) that can be used to emulate patients’ response and we found pre-treatment HER-2 ECD to be predictive of the response to trastuzumab : when over 30ng/ml, pretreatment HER-2 ECD is predictive of treatment failure. In recurrent breast cancer, elevated levels of HER-2 ECD allow an early detection of occult metastases and the identification of patients with a high probability of shortened survival. Monitoring HER-2/ neu ECD levels during metastatic breast cancer can provide a real time assessment of a woman’s HER-2/ neu status and can provide important information for therapeutic decisions.

Active Her-2/neu signal transduction in breast cancer –relevance of a ligand-independent activation mechanism and impact on the efficacy of trastuzumab-based treatment

649 Background: Proteolytic shedding of the Her-2/neu extracellular domain (ECD) has been demonstrated to initiate receptor phosphorylation and thereby Her-2/neu activation in vitro. The aim of the present work was to investigate the clinical relevance of ECD cleavage for Her-2/neu activation and the clinical relevance of active intracellular Her-2/neu signalling reflected by tyrosine kinase phosphorylation in patients treated with trastuzumab-based therapy. Methods: Sera from 62 patients receiving trastuzumab-based treatment for Her-2/neu over-expressing metastatic breast cancer were analyzed for pre-treatment ECD levels by enzyme-linked immunosorbent assay (ELISA). The degree of Her-2/neu phosphorylation/activation of tumor specimens was assessed by immunohistochemistry using a Her-2/neu phosphorylation state specific antibody (PN2A). The Her-2/neu phosphorylation status was then correlated with the patients' ECD levels and clinical course of disease. Results: Serum ECD levels were significantly higher in 15 (24%) patients with tumours exhibiting activated Her-2/neu as compared to those without detectable Her-2/neu phosphorylation (median 148.2 ng/ml vs. 28.5 ng/ml, p=0.01). Whereas response rate only tended to be higher in patients with Her-2/neu-phosphorylated breast cancer (47% vs. 34%, p=0.197), both uni- an multivariate analyses revealed that the median progression-free survival under trastuzumab-based treatment was significantly longer in patients with Her-2/neu-phosphorylated breast cancer 11.7 (95% CI 5.2 to 18.3) as compared to the progression-free survival of 4.5 (95% CI 3.4 to 5.6, p=0.001) months observed in patients with tumours without phosphorylated Her-2/neu. Conclusions: These data strongly suggest that proteolytic cleavage of the ECD represents a biologically relevant ligand-independent mechanism of Her-2/neu activation in vivo. The influence of Her-2/neu activation status upon the efficacy of trastuzumab merits further investigation in larger prospective trials. No significant financial relationships to disclose.