抗体膜片法检测乳腺癌患者血清C-erbB-2蛋白及其方法学评价

目的:建立血清C-erbB-2蛋白的抗体膜片检测方法,并进行方法学评价.方法:抗体膜片法检测血清C-erbB-2蛋白,免疫组化方法检测乳腺组织C-erbB-2蛋白.结果:30例乳腺癌、26例乳腺增生、30例正常对照组血清中C-erbB-2蛋白阳性率分别为40.0%、7.7%和6.7%,三组相比较有显著差异(P＜0.05);乳腺癌组织C-erbB-2蛋白阳性和阴性者其血清C-erbB-2蛋白阳性率分别为91.67%和11.11%,血清与乳腺组织C-erbB-2蛋白表达具有相关性(P＜0.01);抗体膜片法检测血清C-erbB-2蛋白的灵敏度、特异度和准确度分别为40.0%、92.8%和74.4%.结论:抗体膜片法检测血清C-erbB-2蛋白与乳腺癌病理学诊断符合率高,且方法简便易行,临床具有实用价值.     

Immunohistochemical study of human breast tumors using monoclonal antibodies to intermediate filament proteins (nonproliferating epithelial structures in breast dysplasia)

An immunohistochemical analysis of nonproliferating epithelial structures was carried out in 10 samples of human breast dysplasia and in 4 samples of tissue surrounding mammary gland carcinoma. Monoclonal mouse antibodies against individual prekeratins of rat monolayer epithelial antibodies of clone C12 against rat prekeratin with the molecular mass 49 kilodalton and antibodies of clone E3 against rat prekeratin with the molecular mass 40 kilodalton-monoclonal antibodies against vimentin (clone 30), as well as polyclonal antibodies against smooth muscle myosin and against the basement membrane glycoprotein laminin were used. The lining epithelium of all glandular structures reacted only with C12 antibodies. Two variants of myoepithelial cells containing myosin were detected. Variant I contains myosin and vimentin and is localized in intralobular ducts. Variant 2 contains myosin and prekeratin, recognized by E3 antibodies and is found in extralobular ducts.     

Immunohistochemical reactivity of a monoclonal antibody prepared against human breast carcinoma

Summary The reactivity profile of an IgM monoclonal antibody, MBR1, raised against the human breast cancer cell line MCF7, was studied in a variety of human tumours and non-neoplastic tissues by light microscopic immunohistochemistry. The range of reactivity included specific types of non-neoplastic epithelial cells and a number of epithelial tumours. Most mammary carcinomas reacted with MBR1, but adenocarcinomas and squamous carcinomas from different sites were also strongly positive. Different patterns of immunoreactivity were apparent in microscopically normal tissues, in tissues with inflammatory changes and in carcinomas. Heterogeneous staining, despite morphological similarities, was documented in neoplastic and non-neoplastic epithelial cells. The reactivity of MBR1 was different from that reported for other monoclonal antibodies, but revealed similarities to that of monoclonal antibodies and polyclonal sera against human milk fat globule membrane.     

Immunohistochemical analysis of rat liver using a monoclonal antibody (HAM8) against gap junction

Four monoclonal antibodies were raised against crude gap junction fractions of rat liver to clarify the distribution of gap junctions during animal development and to analyze gap junction expression in vivo and the polarity of hepatocytes in vitro. Among the monoclonal antibodies obtained, HAM8 antibody recognized the 27-kDa rat liver gap junction protein connexin 32. This antibody recognized gap junctions at the contiguous faces of hepatocytes, and the antigen was also observed in exocrine pancreas and salivary gland but not in kidney, heart, esophagus, or thymus. HAM8 did not react with amphibian or fish liver, heart, esophagus, stomach, or intestine as assessed via the immunofluorescence method on frozen sections. A few hepatocytes and many hemopoietic cells were seen in rat fetal liver at 15 days of gestation. HAM8 antigen was expressed on some hepatocytes but not on any hemopoietic cells. As the fetus grew, the number of hepatocytes in the liver increased gradually, together with the amount of HAM8 antigen. The distribution of HAM8 antigen at 25 days after birth was similar to that in adult liver. When the expression of HAM8 antigen was examined in primary cultured hepatocytes using the immunofluorescence method, the antigen was observed clearly between the hepatocytes. However, most of the HAM8 antigen on the free surface of hepatocytes disappeared within 4 hr. HAM8 antigen was not expressed on AH7974 rat hepatoma cells when they formed small islets in the rat peritoneal cavity or within the liver. When HAM8 IgG antibody was injected intravenously, the HAM8 signal was expressed in the liver. © 1993 Wiley-Liss, Inc.     

A new rabbit monoclonal antibody (4B5) for the immunohistochemical (IHC) determination of the HER2 status in breast cancer: comparison with CB11, fluorescence in situ hybridization (FISH), and interlaboratory reproducibility.

The 2 methodologies in current clinical use to assess HER2 status in breast cancer are: fluorescence in situ hybridization (FISH) (gene amplification) and immunohistochemistry (protein over-expression). A consistent finding has been that 3% to 15% of breast cancers over-express HER2 protein without evidence for gene amplification. Accurate determination of the HER2 status has implications for selecting patients most likely to respond to trastuzumab. We report here our preliminary experience with a new anti-HER2 rabbit monoclonal antibody, 4B5. The evaluation of HER2 status in 2 different cohorts of breast cancer cases (Single Institution (SI) and Multinational (MN)) with a total of 322 breast cancer cases was performed on an automated staining system (Ventana Medical Systems, Inc, Tucson, AZ) and scored by 3 pathologists (0-3+), for comparison with CB11 staining results (PATHWAY) and FISH (PathVysion). Interlaboratory reproducibility of automated staining results and interpretation was determined on a subset of the SI cohort at 3 separate laboratories. Rabbit monoclonal 4B5 demonstrated sharper membrane staining with less cytoplasmic and stromal background staining than CB11. In the SI cohort, the staining results for 4B5 were highly comparable with those obtained for CB11 with an overall concordance of 93.3%. In the multinational cohort, the overall concordance with CB11 was 84.7%. This lower level of concordance was associated with a much higher overall agreement of 4B5 with FISH (89.5%), compared with agreement of CB11 with FISH (81.2%). The difference in the performance of CB11 in the MN cohort versus the SI cohort may be due to differences in tissue fixation and processing in a centralized, high volume laboratory in an academic medical center versus multiple sites in the international community with potentially nonstandardized techniques. The staining results with 4B5 indicate that it has a more robust performance than CB11 because the correlation of 4B5 with FISH was nearly equivalent (88.2% MN; 89.3% SI) in both cohorts. Interlaboratory reproducibility was also excellent (kappa 1.0). RMoAb 4B5 provides excellent sensitivity, specificity, and interlaboratory reproducibility for the detection of HER2 status in breast cancer.     

Immunohistochemical characterization of an anti-epithelial monoclonal antibody (mAB lu-5)

Summary A mouse monoclonal antibody (mAB lu-5) was prepared using a lung cancer cell line as an antigen. The selected clone produces an IgG with a gamma-1 heavy chain and a kappa-light-chain. Immunohistochemical testing of mAB lu-5 on 117 normal tissue biopsies and 474 tumours revealed reactivity with an intracytoplasmic, formaldehyderesistant antigen present in most epithelial and mesothelial cells, but absent in mesenchymal cells. The antibody can therefore be used as a first order, pan-epithelial marker. It proved also useful for fast tumour diagnosis on frozen sections.     

Long-term survival in breast cancer related to overexpression of the c-erbB-2 oncoprotein: an immunohistochemical study using monoclonal antibody NCL-CB11.

In a previous series we have shown poor short-term (3-5 years) survival for patients with tumours overexpressing the c-erbB-2 oncoprotein. In this study we employed archival paraffin-embedded tissue from patients who underwent mastectomy 10-12 years prior to assessment (n = 187). Immunohistochemical staining was carried out by an indirect immunoperoxidase technique using the novel monoclonal antibody NCL-CB11. Tumours were scored according to intensity of membrane staining. Patient and tumour information was obtained by scrutiny of clinical records. Survival analysis was carried out for both time to relapse and time to death, using the log rank test. Patients with tumours demonstrating intense membrane staining had a poor prognosis compared with the rest, with a steeply sloped survival curve over the first 4 years; the survival difference was still evident at 12 years follow-up (P less than 0.001). The survival advantage for c-erbB-2 negative patients was maintained in lymph node negative patients (P less than 0.001). However, c-erbB-2 status did not influence survival in the node positive group, where all patients had a uniformly poor outlook. These results applied to both time to relapse and time to death. In conclusion, c-erbB-2 status, determined using NCL-CB11, is a powerful prognostic indicator, defining in particular node negative patients with a particularly poor prognosis, and for whom alternative therapeutic strategies may be appropriate.     

抗人乳腺癌单克隆抗体BG6的免疫组织化学研究

Paraffin sections from a series of human tissues were examined by an indirect immunoperoxidase method with BG6 antibody. Data showed that primary breast cancer (96%) or lymph nodes with metastatic tumors (94%) gave positive result with BG6 antibody. Weak staining was observed in some sections of nonbreast tumors and no evidence of positive immunoperoxidase staining was obtained in benign breast hyperplasia, normal tissues and embryonic tissues examined. It is considered that McAb BG6 might be used as a specific marker for breast cancer.     

Immunofluorescent localization of C-erbB-2 oncoprotein in breast cancer: a preliminary study

Monoclonal antibody CIBCgp185 of IgG2a isotype has been generated against C-erbB-2 oncoprotein using BT474 breast carcinoma cell line as immunogen. Earlier studies have revealed that this MAb has potential application as diagnostic tool in the detection of breast cancers overexpressing C-erbB-2. In the present study, the reactivity pattern of this MAb was studied on frozen sections of 28 malignant and 15 normal breast tissues and on cultured mammary tumor cell lines by indirect immunofluorescent staining. Results indicated that gp185C-erbB-2 was overexpressed in 6 malignant specimens, indicating correlation with immunohistochemical analysis studied previously. These results indicate that immunofluorescence might also be used to study C-erbB-2 overexpression in breast cancers and could serve as a confirmatory test for IHC. However, further studies with large number of cases are needed to confirm these results.     

Immunohistochemical study of monoclonal antibody MGD-1 in gastric carcinoma

This paper reports a pathological and immunohistochemical study of gastric carcinoma for immunoreactivity with a monoclonal antibody. MGD-1, raised against cells from an adenocarcinoma of stomach. Fifty-four of 61 gastric carcinomas (89%) were positive for MGD-1. Metastatic gastric carcinoma in local nodes was positive in all 11 such cases. Out of 40 examples of chronic atrophic gastritis, only three, with mild dysplasia, were positive (7.5%). Forty cases with normal gastric mucosa were negative. The MGD-1 detection-rate of well- and poorly-differentiated gastric carcinoma was 85% and 93% respectively. The metastatic cells and cells infiltrating the submucosa and muscular layer were more frequently positive and showed stronger staining with MGD-1 than those in mucosa. These results show that MGD-1 possesses a high degree of specificity for gastric carcinoma and could be used diagnostically.     

Immunohistochemical study of c-erbB-2 expression in primary breast cancer

An immunohistochemical (IHC) study of the c-erbB-2 protein was performed in paraffin-embedded tissues from 506 primary breast carcinomas. An overexpression of c-erbB-2 was detected in 32% of the tumors and was correlated with a negative estrogen receptor status, increasing tumor size as well as axillary lymph node involvement. The five-year disease free survival was analyzed in 183 patients who have been followed for at least five years. No statistically significant association of c-erbB-2 status with survival was shown. However, longer survival in women over 50 years compared to under 50 years of age was detected among the c-erbB-2 positive patients. In the multivariate Cox's regression analysis, lymph node and vascular invasions were independent prognostic indicators among these patients. But c-erbB-2 status and other factors did not predict the relapse of breast cancer. However, these data may not negate the benefit of c-erbB-2 detected by IHC for identification of patients who have a poor prognosis and require more aggressive adjuvant therapy. Further studies in a larger group of patients with longer follow-up time may provide more valid information.     

Immunohistochemical identification of cytotoxic lymphocytes using human perforin monoclonal antibody.

Perforin is a potent cytolytic pore-forming protein expressed in cytoplasmic granules of cytotoxic T lymphocytes and natural killer cells. A new monoclonal antibody raised against human perforin was used to detect both in vitro and in vivo perforin expression in cytotoxic cells. Immunohistochemical analysis of human peripheral blood mononuclear cells cultured in recombinant interleukin-2 (rIL-2) showed strong granular cytoplasmic staining of the IL-2 activated cytotoxic cells. Fresh-frozen tissue sections from patients with heart allograft rejection were also stained. Strong granular cytoplasmic staining of the mononuclear inflammatory infiltrate characteristic for perforin in cardiac allograft rejection was observed. The detection and quantitative analysis of perforin-associated cytotoxic cells by the human anti-perforin monoclonal antibody will help to evaluate the significance of these functionally distinct cytotoxic cells in human tissue.     

HER2 assessment on core biopsy specimens using monoclonal antibody CB11 accurately determines HER2 status in breast carcinoma

Purdie C A, Jordan L B, McCullough J B, Edwards S L, Cunningham J, Walsh M, Grant A, Pratt N & Thompson A M
(2010) Histopathology 56, 702–707
HER2 assessment on core biopsy specimens using monoclonal antibody CB11 accurately determines HER2 status in breast carcinoma Aims: HER2 status is a prognostic factor in breast carcinoma and predicts response to trastuzumab therapy. Trastuzumab is effective in the neoadjuvant, adjuvant and advanced disease settings. Knowledge of HER2 status early in the diagnostic and therapeutic process is vital for treatment planning. HER2 analysis is usually carried out by immunohistochemistry or fluorescence in situ hybridization (FISH). The aim of this study was to establish whether HER2 immunohistochemistry using monoclonal antibody CB11 and carried out on the initial diagnostic core biopsy specimen accurately predicts HER2 amplification status. Methods and results:  This is the largest study to date in which HER2 protein expression has been assessed by CB11 immunohistochemistry on the diagnostic core biopsy specimen and correlated with the result of FISH. Using FISH as the definitive HER2 status, we studied 568 invasive breast cancers using CB11 immunohistochemistry on core biopsy. This analysis had a sensitivity of 99.4%, specificity of 93.9%, false‐positive frequency of 3.9% and false‐negative frequency of 1.1%. These data are as good as those obtained from analysing resection specimens alone in UK national reference centres. Conclusions:  CB11 immunohistochemistry accurately predicts HER2 amplification status and can be reliably carried out on core biopsy specimens of breast carcinoma.     

Immunohistochemical study of alpha-macroglobulins in breast cancer

Immunohistochemistry of macroglobulins (alpha2-macroglobulin and alpha2-glycoprotein associated with pregnancy) in parenchyma and stroma of the normal breast, breast fibroadenoma and carcinoma was studied. The tissues of the normal breast had low levels of both proteins. The parenchyma of fibroadenoma had an increased level of alpha2-macroglobulin. The parenchyma of breast carcinoma showed an increase of alpha2-glycoprotein level. An efficient therapeutical radiation was accompanied by an alpha2-glycoprotein fall and alpha2-macroglobulin rise. The data can be used as diagnostic and prognostic criteria of breast fibroadenoma and carcinoma.     

5－bromo－2－deoxyuridine标记法

以５－ｂｒｏｍｏ－２－ｄｅｏｘｙｕｒｉｄｉｎｅ标记法检测了ＳＰ２／０细胞的增殖动力学，并与３Ｈ－ＴｄＲ掺入法作了比较，结果表明本法是可靠的，且有很高的特异性。该法较３Ｈ－ＴｄＲ掺入的传统方法简便、敏感、精确，可广泛应用于细胞增殖动力学、肿瘤生物学、遗传学、分子生物学等多个领域。     

Immunohistochemical demonstration of mitochondria in routinely processed tissue using a monoclonal antibody

We report results obtained using the monoclonal antibody M-II 68, which recognizes inner mitochondrial membrane in routinely processed (formalin-fixed and paraffin-embedded) tissue by light microscopic immunohistochemistry. In ten normal brains, the range of immunoreactivity in various cell types and locations was defined. The most intense staining was observed in Purkinje cells, in neurons of cranial nerve nuclei, pons and substantia nigra, as well as in choroid plexus epithelial cells. By comparison with this control group, one case of primary mitochondrial encephalomyopathy exhibited increased staining of endothelial and vascular smooth muscle cells, choroid plexus epithelial cells, and neurons of various locations. Scattered ragged-red fibres were heavily labelled in one case of mitochondrial myopathy, while ten muscles without mitochondriopathy were left unstained. Our method is able to detect accumulations of mitochondria and increases in mitochondrial cristae density. It could prove useful for differential diagnosis of routine biopsy material and for clarification of cell types involved in mitochondrial cytopathies.     

Immunohistochemical study of nasopharyngeal carcinoma using monoclonal keratin antibodies

Nasopharyngeal carcinoma (NPC) provides a unique opportunity to evaluate distinctive epidemiologic features and a possible etiologic relationship with Epstein-Barr virus (EBV) in human malignancy. The lack of a uniformly accepted pathologic classification for NPC has limited the application of this data, although the World Health Organization (WHO) developed a classification that may solve this problem. Monoclonal keratin antibodies were used for staining of NPC for evaluation of its assistance in diagnosis and classification. In the present immunohistochemical study, monoclonal keratin antibodies, designated AE1, AE2, and AE3, and a polyclonal keratin antibody (RAK) were used for study of the presence of keratin in 121 cases of NPC obtained from China and the United States. AE1 monoclonal antibody, which recognizes keratin protein classes 56.5K, 50K, and 40K, was shown to be the most sensitive and specific for NPC tumor cells among the keratin antibodies studied. In addition, some different keratin expression patterns could be identified between different kinds of epithelium and different tumor groups, with possible relevance to the histogenesis of the histologic subtypes of NPC.     

Immunohistochemical definition of antigenic determinants of pregnancy-associated alpha 2-glycoprotein (alpha 2-PAG) using monoclonal antibodies

Human pregnancy-associated alpha 2-glycoprotein (alpha 2-PAG) is a high molecular weight glycoprotein in normal sera. The protein is present in high concentration in the sera of pregnant females and in abnormally low concentration in association with conditions connected with abnormalities of mucosal immunity. Indirect immunoperoxidase techniques using poly- and monoclonal antibodies were employed to identify pregnancy-associated alpha 2-PAG in different tissues. Four monoclonal antibodies were selected from a battery of antibodies with defined specificities in order to ascertain reactivity with various epitopes of the antigen. The antibodies were applied to paraffin sections of breast, colon, salivary gland, and tonsil, and different fixation regimes were used in the preparation of the tissues. The polyclonal antibodies were found to stain plasma cells and epithelial lumina evenly in all the tissues included whereas the monoclonal antibodies were shown to stain certain components selectively. In breast and salivary glands, all four monoclonal antibodies could identify alpha 2-PAG, but in tonsil and colon, only two were reactive. This difference in epitope expression might reflect the internal processing of alpha 2-PAG, and lack of availability of certain epitopes may be indicative of functional blocking of certain domains.     

Immunohistochemical research on human breast tumors using monoclonal antibodies to intermediate filament proteins. Cancer of the breast

Immunomorphologic study of 29 breast cancer cases using monoclonal antibodies to proteins of intermediate filaments shown to differentiate the lining epithelium from myoepithelium in the non-proliferating epithelial structures of the mamma, has shown the cells in the majority of tumours (according to the International WHO Classification defined as infiltrating ductal, lobular, and tubular cancer forms) to contain prekeratin (PK) C12, specific for normal lining epithelium, but not for the myoepithelium. In cases of cancer with chondroid metaplasia (a malignant variant of the so-called "mixed tumour") the cells contained PK E3, vimentin and structural myosin, normally specific for myoepithelium. The cell heterogenicity in PK C12 content or its absence noted in the infiltrating cancers with predominance of a solid component can indicate a high degree of tumour anaplasia. It is concluded that usage of monoclonal antibodies to PK C12, invariably found in the cells of fibrotic invasion foci, can be a useful indicator for early diagnosis of infiltrative tumour growth.