Discrimination between uterine serous papillary carcinomas and ovarian serous papillary tumours by gene expression profiling

High-grade ovarian serous papillary cancer (OSPC) and uterine serous papillary carcinoma (USPC) represent two histologically similar malignancies characterised by markedly different biological behavior and response to chemotherapy. Understanding the molecular basis of these differences may significantly refine differential diagnosis and management, and may lead to the development of novel, more specific and more effective treatment modalities for OSPC and USPC. We used an oligonucleotide microarray with probe sets complementary to >10 000 human genes to determine whether patterns of gene expression may differentiate OSPC from USPC. Hierarchical cluster analysis of gene expression in OSPC and USPC identified 116 genes that exhibited >two-fold differences (P<0.05) and that readily distinguished OSPC from USPC. Plasminogen activator inhibitor (PAI-2) was the most highly overexpressed gene in OSPC when compared to USPC, while c-erbB2 was the most strikingly overexpressed gene in USPC when compared to OSPC. Overexpression of the c-erbB2 gene and its expression product (i.e., HER-2/neu receptor) was validated by quantitative RT-PCR as well as by flow cytometry on primary USPC and OSPC, respectively. Immunohistochemical staining of serous tumour samples from which primary OSPC and USPC cultures were derived as well as from an independent set of 20 clinical tissue samples (i.e., 10 OSPC and 10 USPC) further confirmed HER-2/neu as a novel molecular diagnostic and therapeutic marker for USPC. Gene expression fingerprints have the potential to predict the anatomical site of tumour origin and readily identify the biologically more aggressive USPC from OSPC. A therapeutic strategy targeting HER-2/neu may be beneficial in patients harbouring chemotherapy-resistant USPC.     

Validation of a Tissue Microarray to Study Differential Protein Expression in Inflammatory and Non-Inflammatory Breast Cancer

AIMS: Inflammatory breast cancer (IBC) is an aggressive subtype of breast cancer with poor prognosis. The mechanisms responsible for the aggressive clinical evolution are incompletely understood. We constructed a tissue microarray (TMA) and validated its use in translational IBC research. Differential expression of proteins that might play a role in causing the IBC phenotype was studied. METHODS AND RESULTS: A TMA containing 34 IBC and 41 non-stage matched non-IBC tumours was constructed. Five core biopsies were taken for each IBC and three cores for each non-IBC tumour. The TMA was validated using three approaches: (1) the excellent concordance between immunohistochemical results of the initial pathological examination and the results obtained with the TMA for ER, PR and HER2/neu (kappa > 0.74); (2) the known differential expression between IBC and non-IBC for four bio-markers in IBC (ER, PR, p53 and HER2/neu) was confirmed ( p < 0.01); (3) the HER2/neu status using three different antibodies (CB11, TAB250 and HercepTest) was highly concordant (kappa > 0.75). Furthermore, the overexpression of E-Cadherin and RhoC GTPase in IBC ( p < 0.05) was confirmed. We did not find a differential expression pattern for carbonic anhydrase IX (CA IX) and EGFR. CONCLUSIONS: Using different approaches, we have validated the use of our TMA for studying differential protein expression in IBC and non-IBC. We confirm the overexpression of E-Cadherin and RhoC GTPase in IBC. The lack of differential expression for CA IX and EGFR might suggest the pathways are equally utilised in both types of breast cancer.     

乳腺癌患者血清her-2/neu水平及相关性研究

目的　研究乳腺癌患者血清her 2 /neu的水平、影响因素及临床意义。方法　采用酶联免疫吸附 (ELISA)方法检测 10例正常人、3 1例乳腺良性病变患者、53例可手术乳腺癌及 2 6例晚期转移性乳腺癌患者血清her 2 /neu的水平。结果　正常人和乳腺良性病变患者血清her 2 /neu阳性率均为 0。 53例可手术乳腺癌患者中 ,10例血清her 2 /neu水平高于正常 ,血清her 2 /neu水平与肿瘤大小有相关性 (P <0 .0 5) ,与淋巴结、受体状况无关。 2 6例晚期转移性乳腺癌患者中 ,16例血清her 2 /neu阳性 ,但阳性率 (61.5% )与转移部位无明显相关。结论　血清her 2 /neu的水平与肿瘤良恶性、肿瘤分期及瘤负荷有关 ,并有可能用于指导复发转移性乳腺癌的治疗     

Mapping the HLA-A24-restricted T-cell epitope peptide from a tumour-associated antigen HER2 / neu: possible immunotherapy for colorectal carcinomas

HER2 / neu is a potential antigen candidate for immunotherapy because of its correlation to a poor prognosis and high expressions in many kinds of epithelial tumours. Especially in the colorectal carcinomas, the higher expression of HER2 / neu is recognized in metastatic regions as well as in primary sites. Several CTL epitopes restricted by HLA-A2.1 and -A3 were identified so far, however epitopes restricted by HLA-A24, that is one of the most common allele in Japanese and Caucasians, have not been identified. In this paper, we showed identification of a CTL epitope peptide of HER2 / neu restricted by HLA-A24. HLA-A24 binding peptides selected by an analysis based on HLA-A24 binding motifs were determined for their binding affinities to HLA-A24 molecules. The peptide with a sequence of RWGLLLALL (position 8-16) named HE1 showed the highest affinity. We induced CTLs from CD8(+)cells of HLA-A24 healthy donors by stimulation with HE1-pulsed autologous dendritic cells. The CTLs showed cytotoxic activity against not only the peptide-pulsed target cells but also HLA-A24 colorectal tumour cell lines that endogenously overexpressed HER2 / neu. The antigen-specificity was confirmed by cold target inhibition assay using HE1-pulsed target cells. In summary, HER2 / neu peptide, RWGLLLALL, may contribute to the induction of antitumour immunity with the peptide-based immunotherapy for the colorectal carcinomas.     

Effect of α-difluoromethyl-ornithine on the expression and function of the epidermal growth factor receptor in human breast epithelial cells in culture

We have previously shown that ornithine decarboxylase (ODC) overexpression enhances the transforming effects of HER-2neu and epidermal growth factor (EGF) in normal MCF-10A human breast epithelial cells. Our data suggest that such potentiation may be mediated by activation of the mitogen-activated protein kinase (MAPK) pathway and, possibly, STAT signalling. To further explore the interaction between the polyamine pathway and EGF/HER-2neu signalling in this system, we inhibited endogenous ODC activity with -difluoromethylornithine (DFMO) and assessed the effects of this blockade on the expression of EGF receptors (EGFR) and HER-2neu as well as activation of downstream EGF target genes. We found that DFMO administration to MCF-10A cells increased EGF-R mRNA and protein levels in a dose-response fashion, while HER-2neu expression was not affected. The effect of DFMO was mediated through polyamine depletion since it could be reversed by exogenous putrescine administration. Our results also indicated that the increase in EGFR induced by DFMO was not a non-specific consequence of inhibition of cell proliferation. The upregulated EGFRs were functional since they could be phosphorylated by EGF and they were able to promote phosphorylation of downstream signalling molecules including ERK, STAT-3, and STAT-5. We propose that physiologic levels of ODC activity may be critical for regulation of a yet undefined signalling pathway, whose blockade by DFMO leads to a compensatory increase in functional EGFR.     

Antibody dependent cellular phagocytosis (ADCP) and antibody dependent cellular cytotoxicity (ADCC) of breast cancer cells mediated by bispecific antibody, MDX-210

Background: MDX-210 is a bispecific antibody (BsAb) with specificity for both the proto-oncogene product of HER-2/neu (c-erbB-2) and FcRI (CD64). HER-2/neu is overexpressed in malignant tissue of approximately 30% of patients with breast cancer, and FcRI is expressed on human monocytes, macrophages, and IFN- activated granulocytes. We investigated phagocytosis and cytolysis of cultured human breast cancer cells by human monocyte-derived macrophages (MDM) mediated by BsAb MDX-210, its partially humanized derivative (MDX-H210), and its parent MoAb 520C9 (anti-HER-2/neu) under various conditions.Materials and Methods: Purified monocytes were cultured with GM-CSF, M-CSF, or no cytokine for five or six days. Antibody dependent cellular phagocytosis (ADCP) and cytolysis (ADCC) assays were performed with the MDM and HER-2/neu positive target cells (SK-BR-3). ADCP was measured by two-color fluorescence flow cytometry using PKH2 (green fluorescent dye) and phycoerythrin-conjugated (red) monoclonal antibodies (MoAb) against human CD14 and CD11b. ADCC was measured with a non-radioactive LDH detection kit.Results: Both BsAb MDX-210 (via FcRI) and MoAb 520C9 (mouse IgG1, via FcRII) mediated similar levels of ADCP and ADCC. ADCP mediated by BsAb MDX-H210 was identical to that mediated by BsAb MDX-210. Confocal microscopy demonstrated that dual-labeled cells represented true phagocytosis. Both ADCP and ADCC were higher when MDM were pre-incubated with GM-CSF than when incubated with M-CSF.Conclusions: BsAb MDX-210 is as active in vitro as the parent MoAb 520C9 in inducing both phagocytosis and cytolysis of MDM. MDX-210 and its partially humanized derivative, MDX-H210, mediated similar levels of ADCP. GM-CSF appears to superior to M-CSF in inducing MDM-mediated ADCC and ADCP. These studies support the ongoing clinical investigations of BsAb MDX-210 and its partially humanized derivative.     

Evaluation of NMU-Induced Breast Cancer Treated with Sirolimus and Sunitinib on Breast Cancer Growth

Objective: To analyze the effect of sirolimus and sunitinib in blocking the tumor growth and to evaluate the expressions of estrogen receptor (ER), progesterone receptor (PgR), and human epidermal growth factor receptor-2 (HER2/neu) after treated with sirolimus and sunitinib. Methods: Thirty-two female Sprague Dawley rats at age 21-days old were administered intraperitoneally with N-Methyl-N-Nitroso Urea (NMU), dosed at 70mg/kg body weight. The rats were divided into 4 groups; Group 1 (Control, n=8), Group 2 (Sirolimus, n=8), Group 3 (Sunitinib, n=8) and Group 4 (Sirolimus+Sunitinib, n=8), being treated twice when the tumor reached the size of 14.5±0.5 mm and subsequently sacrificed after 5 days. The protein expressions of ER, PgR and HER2/neu of the tumor tissues were evaluated by using immunohistochemistry analysis. Results: Treatment with sirolimus alone lowered expressions of ER and PgR of breast cancer and reduced tumor size. There was no significant difference of ER and PgR expressions between control and sunitinib treated tumor. Sunitinib treated tumors reduce in diameter after the first treatment, however the diameter increases after the second treatment. Histologically, sunitinib treated tumor did not show any aggressive invasive carcinoma of no special type (NST) histological subtypes. In addition, all NMU-induced tumors are HER2/neu-negative scoring. Conclusion: Sirolimus is neither synergistic nor additive with sunitinib for breast cancer treatment.     

变性高效液相色谱快速诊断儿童型脊肌萎缩症

目的建立变性高效液相色谱(denaturing high performance liquid chromatography,DHPLC)快速诊断儿童型脊肌萎缩症(spinal muscular atrophy,SMA)的新方法.方法 (1) 用二重聚合酶链反应(PCR)扩增运动神经元成活基因(survival motor neuron gene,SMN)外显子7、8及周围部分内含子序列;(2)纯化PCR产物以除去其中的引物、dNTP及缓冲液中的盐粒子等成分;(3)采用多重引物延伸反应特异性检测能将SMN1基因与SMN2基因区分开的3个特异性位点;(4)将延伸反应的产物用DHPLC在完全变性的条件下分析.结果将建立的新方法与传统的PCR-酶切法进行盲法对比试验,检测了30例标本(包含20例SMA患儿和10例正常人群外周血基因组标本)以验证新方法的特异性和可靠性,其诊断结果与PCR-酶切法结果完全一致.结论多重引物延伸反应结合DHPLC分析技术是一种可用于临床SMA基因诊断、产前诊断及胚胎种植前遗传学诊断的高效、灵敏、可靠、快速、简便的新方法.     

乳腺癌中HER-2蛋白表达及基因扩增检测的比较研究

目的 比较免疫组化（IHC）法与荧光原位杂交（FISH）法检测乳腺癌组织HER-2状态,分析二者相关性.方法 应用IHC法与FISH法对56例女性新发乳腺癌行HER-2蛋白表达及基因扩增的检测.结果 56例受试者中HER-2蛋白表达（-）、（＋）、（＋＋）、（＋＋＋）者分别为9例（16.1%）、29例（51.8%）、11例（19.6%）、7例（12.5%）;有26例（46.4%）HER-2基因扩增,30例（53.6%）无扩增.HER-2基因扩增主要表现在HER-2（＋＋）和（＋＋＋）中,基因扩增率分别为72 7%和100%.HER-2（＋）中有11例（37.9%）出现基因扩增,HER-2（-）中未见基因扩增.两种方法检测结果的阳性率差异有统计学意义（χ2=19.778,P＜0.01）.HER-2（-）及HER-2（＋＋＋）与FISH结果一致性很好（Kappa=0.969）.HER-2（＋）及HER-2（＋＋）与FISH结果一致性较差（Kappa=0.271）.结论 IHC是HER-2表达初步的筛查方法,HER-2（-）和（＋＋＋）者与基因扩增情况有很好的一致性,可直接指导临床治疗.HER-2（＋）和（＋＋）者中有部分HER-2基因扩增阳性,如行靶向治疗需进一步行FISH检测.