c-Kit expression in desmoid fibromatosis. Comparative immunohistochemical evaluation of two commercial antibodies

To determine the frequency of c-Kit staining in desmoids and optimize an assay for clinical use, we stained 19 desmoids from various sites at various dilutions with 2 commonly used rabbit polyclonal, anti-c-Kit antibodies (A4502, DAKO, Carpinteria, CA; C-19, Santa Cruz Biotechnology, Santa Cruz, CA), with and without heat-induced epitope retrieval (HIER) in citrate buffer. Approdpriate external and internal control samples were evaluated for each test condition. At dilutions of 1:50 both antibodies stained substantial numbers of desmoids: with/without HIER, A4502, 89%/63%; C-19, 37%/74%. The staining was cytoplasmic without cell membrane accentuation. However, background stromal staining and nonspecific staining of endothelium and smooth and striated muscle were problematic with both antibodies at 1:50. At higher dilutions, C-19 stained no desmoid; however, diminished staining of external and internal control samples made it unreliable. A4502 similarly stained many fewer desmoids at higher dilutions. However, it retained strong staining of both external and internal control samples and showed much less nonspecific staining. Best results were achieved at 1:250 without HIER; only weak focal staining was present in 1 desmoid. With a simple immunohistochemical method optimized for clinical use, desmoid can be regarded as a c-Kit-negative tumor.     

Comparison of monoclonal versus polyclonal calretinin antibodies for immunohistochemical diagnosis of malignant mesothelioma.

Of putative specific markers for diffuse malignant mesothelioma, nuclear staining with Zymed polyclonal calretinin antibody has shown the best specificity to date for epithelial diffuse malignant mesothelioma versus adenocarcinoma. We compared specificity and sensitivity of this polyclonal antibody for diagnosis of diffuse malignant mesothelioma with a new monoclonal antibody from DAKO. One hundred eighteen adenocarcinomas and 111 diffuse malignant mesotheliomas-70 epithelial, 22 sarcomatous, and 19 biphasic-were immunostained with calretinin antibodies from Zymed (polyclonal rabbit, prediluted, PAD:DC8) and DAKO(monoclonal mouse, 1:100, clone DAK Calret 1) using manufacturer-recommended procedures. Cases were blinded and assessed for nuclear versus cytoplasmic staining, percent positive cells, and background. Both antibodies showed similar positive predictive values for diffuse malignant mesothelioma by nuclear staining (Zymed=95%; DAKO=97%). False positives in 4 (3.4%) and 2 (1.7%) adenocarcinomas, respectively, stained greater than 10% of cells. Sensitivity for epithelial malignant mesothelioma was slightly less for DAKO antibody (Zymed=80%; DAKO=73%). Neither antibody performed well on sarcomatous malignant mesothelioma (Zymed=2/22; DAKO=1/22). Both antibodies are useful in the diagnosis of epithelial malignant mesothelioma, although monoclonal antibody is slightly less sensitive.     

The effects of antibody clone and pretreatment method on the results of HER2 immunostaining in cytologic samples of metastatic breast cancer: A query and a review of the literature

The standardization and use of heat-induced epitope retrieval (HIER) is particularly important with immunohistochemical markers that direct the course of cancer treatment, such as Herceptin therapy. Increasingly, many laboratories are performing immunohistochemical analysis using various antibodies and methodologies for HER2/neu. We attempted to determine the effects of antibody clone and pretreatment methods on the interpretation of HER-2/neu staining in cytologic samples. Cell block sections from 54 cases of metastatic breast cancer (24 FNAs, 30 effusions) were analyzed for HER2 expression using antibodies to CB-11, TAB250, and A0485. Antibodies were analyzed with and without HIER. One pathologist using the FDA-approved scoring system for the HercepTest reviewed all slides in a blinded fashion. Five of fifty-four cases (9%) using CB-11 showed a significant increase in HER2 immunoreactivity using HIER (i.e. from 0/1+ to 2-3+). However, in twenty-nine of fifty-four cases (54%), the cytoplasmic background was significantly higher after HIER. With the A0485 antibody, two of fifty four cases (4%) showed a significant increase in immunoreactivity using HIER, while seventeen of fifty-four cases (31%) exhibited only more pronounced cytoplasmic staining. HIER pretreatment did not increase HER2 staining in any TAB250 stained sample, rather four of fifty-four cases (7%) showed a significant decrease in staining with HIER. We conclude that HIER may enhance membrane staining with the CB-11 and A0485 antibodies, but also increases cytoplasmic background. Loss of antigenicity is seen when HIER is used with TAB250.     

Selecting antibodies to detect HER2 overexpression by immunohistochemistry in invasive mammary carcinomas.

There is an increasing clinical demand for HER2 analysis in breast cancer, especially since the release of trastuzumab. The authors assessed the ability of immunohistochemistry to detect HER2 overexpression in invasive mammary carcinomas (IMC) using five antibodies. Paraffin-embedded samples of 86 IMCs (T2N0) were used to compare the immunohistochemical overexpression of HER2 using two polyclonal antibodies (HercepTest [DAKO] and A0485 [DAKO]) and three monoclonal antibodies (CB11 from two different laboratories, Biogenex and Novocastra, and 4D5 [Genentech]). All immunostainings were scored according to the FDA-approved HercepTest recommendations. The HercepTest-positive cases were compared with gene amplification by FISH (Oncor Inform, Ventana). The HercepTest was positive in 31 of the 86 cases (36.1%). The DAKO antibody A0485 was positive in 25 of the 66 (37.8%). Monoclonal antibody 4D5 was positive in only 15 of the 86 cases (17.4%). There was almost total agreement in results between the two CB11 antibodies: 25 of the 86 positive cases (29.1%). All cases positive for CB11 or 4D5 were HercepTest positive. Most of the HercepTest 2+ cases were negative when using either monoclonal antibody. FISH was positive in 19 of the 20 HercepTest 3+ cases and negative in 5 HercepTest 2+ cases. Three CB11-2+ cases showed no amplification by FISH. In three FISH-positive cases the immunohistochemistry showed no overexpression by all antibodies used. These findings suggest that immunohistochemistry may be used reliably as a primary methodology for evaluating HER2; however, the use of polyclonal antibodies may not be adequate to assess HER2 overexpression. CB11, regardless of the manufacturer (Biogenex or Novocastra), showed better concordance with FISH (kappa=0.83) than did the polyclonal antibodies.     

1D5 and 6F11: An immunohistochemical comparison of two monoclonal antibodies for the evaluation of estrogen receptor status in primary breast carcinoma

The optimal monoclonal antibody to examine steroid hormone receptor status of primary breast carcinoma has yet to be defined. Estrogen receptor status was evaluated in 592 cases using routinely prepared paraffin-embedded tissue samples from primary breast carcinomas with the 1D5 (DAKO, Carpinteria, CA) and 6F11 (Novocastra, Newcastle upon Tyne, England) monoclonal antibodies. The stains were compared, assessing the percentage of positive cells stained and their intensity. They also were examined for nonspecific cytoplasmic staining and fixation artifact. In addition, a cost analysis for their production was performed. Overall, 1D5 and 6F11 showed a 97.5% concordance rate. 6F11 stained a significantly higher percentage of cells (P < .0001), more intensely (P < .0001), with less nonspecific cytoplasmic staining (P < .0001). There was no significant difference in fixation artifact between the 2 clones. The cost of antibody used for preparing a 1D5-stained slide was 86% more than for preparing a 6F11-stained slide (dollars 14.27 vs dollars 7.67).     

Determination of proliferation index in neoplasms using different Ki-67 equivalent antibodies

Paraffin sections from 23 tumours were immunohistochemically stained with the following four Ki-67 equivalent antibodies: monoclonal MIB-1 (DAKO), monoclonal MM1 (Novocastra), polyclonal NCL-Ki-67p (Novocastra), and polyclonal Rah Ki-67 (DAKO). Ki-67 labelling indices were determined by counting in exactly the same area in each case. MIB-1 showed the highest labelling index in 21 of the 23 cases, and the mean MIB-1 index was approximately 30% higher than that of the other antibodies. The differences between MM1, NCL-Ki-67p and Rah Ki-67 were small and non-significant. There was a positive correlation between each of the four antibodies. As these findings may be of importance when the Ki-67 labelling index is used as a criterion for tumour grading or for clinical prognostication, this necessitate identification of the antibody used in every case.     

Accuracy of the determination of S100 protein expression in malignant melanoma using a polyclonal antibody directed against S100 and monoclonal antibodies specific for S100 alpha and beta

Abstract

S100 proteins are present in a variety of tissues and perform regulatory functions in numerous metabolic processes. They have an important role in many human cancers, including malignant melanoma. Both polyclonal and monoclonal antibodies have been used to investigate S100 expression in melanoma tissue sections. This study aimed to determine the accuracy and sensitivity of these two types of antibodies in detecting S100 proteins in paraffin processed tissue cases of malignant melanoma. The study compared routinely used rabbit polyclonal anti-S100 antibody raised against both anti-S100A and B isoforms (Dako, Glostrup, Denmark), as per studies by Timar, and compared and contrasted findings with mouse monoclonal anti-S100A and anti-S100B antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). The study involved the assessment of formalin-fixed paraffin-embedded tissue blocks from 56 cases of malignant melanoma, consisting of 23 superficial spreading, nine nodular, eight lentigo maligna, five acral lentigenous forms, five metastatic melanomas (two sentinel lymph node positive cases and three cases of nodal involvement from cases of elective nodal groin dissections), and six cases of desmoplastic malignant melanoma (DMM). The slides were stained by immunohistochemical methods on an automated platform (BenchMark XT; Roche, USA) and employing the iView detection system. All slides were examined by routine light microscopy by two independent assessors. The best results for both intensity of staining and percentage of positive tumor cells were achieved with polyclonal anti-S100 antibody and monoclonal anti-S100B antibody. Anti-S100A antibody yielded weaker staining intensity (with mean intensity of 1·8, compared to 2·8 for both anti-S100B antibody and polyclonal anti-S100 antibody), and a lower percentage of positive melanoma cells (an average of 74% for anti-S100A, compared to 95% for both anti-S100B antibody and polyclonal anti-S100 antibody). This result was statistically significant (P<0·01). Staining in cases of DMM gave the same results (P<0·01). The conclusion from this study is that polyclonal anti-S100 antibody and monoclonal anti-S100B antibody are more suitable than monoclonal anti-S100A antibody for diagnostic investigations of malignant melanoma, irrespective of the histological type of melanoma.     

Immunohistochemical analysis of nuclear versus cytoplasmic staining of WT1 in malignant mesotheliomas and primary pulmonary adenocarcinomas

CONTEXT: Previous studies have indicated certain immunohistochemical markers, including WT1, may be helpful in distinguishing adenocarcinomas from mesotheliomas, but to date there are no reliable, widely accepted, commercially available antibodies positive in mesotheliomas and negative in adenocarcinomas. We compared the nuclear and cytoplasmic staining patterns of WT1 in these 2 malignancies using a commercially available antibody and examined the expression of 2 other previously reported positive markers, calretinin and thrombomodulin. METHODS: Sixty-seven mesotheliomas and 51 adenocarcinomas, all paraffin embedded, were retrieved from recent case files. The diagnosis of mesothelioma was based on typical clinical and morphologic features, as well as immunohistochemistry; electron microscopy had been performed on 16 cases. The diagnosis of adenocarcinoma was based on typical light microscopic findings and a positive stain for mucin. Commercially available antibodies to WT1, thrombomodulin, and calretinin were applied. Because of the conflict surrounding calretinin, 2 anticalretinin antibodies (from Chemicon Inc and Zymed Laboratories) were utilized. RESULTS: Fifty of 67 mesotheliomas showed strong nuclear staining with WT1. No adenocarcinomas (0/51) showed nuclear staining. Twenty-three of 67 mesotheliomas were positive for thrombomodulin, and 35 of 67 mesotheliomas were positive for calretinin with the Chemicon antibody. Nine of 15 mesotheliomas were positive for calretinin with the Zymed antibody. CONCLUSIONS: Thrombomodulin and calretinin did not prove useful in discriminating between mesotheliomas and adenocarcinomas. The degree of positivity with calretinin may be dependent on the specific antibody utilized. Nuclear staining for WT1 is highly specific for mesothelioma and, in the appropriate clinical setting, can be a helpful adjunct in the distinction between adenocarcinomas and mesotheliomas.     

两种商业化二抗在免疫组织化学染色中一致性的比较

目的比较两种商业化二抗在免疫组织化学染色中的一致性。方法以常见肿瘤组织样本为待检组织,选取18种常用免疫组织化学染色一抗,按国际专家特设委员会的建议设立阳性对照及阴性对照。使用DAKO自动免疫组织化学染色平台,其他实验条件一致,以两种不同二抗进行免疫组织化学染色,标准组二抗为DAKO聚合物系统(DAKO EnVision FLEX,High pH),实验组二抗为Power-StainTM试剂盒(Power-StainTM 1.0 Poly HRP DAB Kit for Mouse+Rabbit)。染色后,采集图像,医师单盲法进行定位、定性以及半定量评价;两组选取同一区域,均以数字病理图像测量方法定量测量吸光度校正值、测量区域面积值以及灰度图阳性积分吸光度值,计算得到平均吸光度均值。结果两组所有样本染色的定位均准确,定性一致,两组半定量测量数据对两组强度比较、两组阳性百分率比较、两组平均吸光度均值比较,组间差异均无统计学意义。结论两种商业化二抗在免疫组织化学染色中具有良好的一致性。     

CD79a is heterogeneously expressed in neoplastic and normal myeloid precursors and megakaryocytes in an antibody clone-dependent manner

CD79a, a component of the B-cell antigen receptor complex, can also be expressed in certain non-B-cell malignancies. The reported frequency of CD79a expression in acute myeloid leukemias (AML) ranges from 0% to 90%. We evaluated 39 bone marrow biopsy specimens (29 AML and 10 normal cases) using 5 different commercially available anti-CD79a monoclonal antibody (MoAb) clones. Of 7 acute promyelocytic leukemia (APL) cases, 6 (86%) stained for CD79a with clones HM47/A9 (Novocastra, Newcastle Upon Tyne, England) and HM57 (DAKO, Carpinteria, CA) but were negative with clones 11E3 (Novocastra), and JCB117 (DAKO). Half of 6 acute megakaryoblastic leukemia (AMKL) cases and normal megakaryocytes in 14 (67%) of 21 cases were immunoreactive using clone 11D10 (Novocastra). Approximately one third of non-APL/non-AMKL AML and myeloid precursors in normal marrow specimens stained with clones HM57 and 11D10. This heterogeneity of CD79a expression in AML, megakaryocytes, and myeloid precursors is MoAb clone-dependent, likely owing to different epitope detection, and may be of diagnostic usefulness.     

The best immunohistochemical panel for differentiating hepatocellular carcinoma from metastatic adenocarcinoma

It can be difficult to differentiate hepatocellular carcinoma (HCC) from metastatic adenocarcinoma (MA). An appropriate immunohistochemical panel is required for the differential diagnosis. This study aimed at finding the best panel, including hepatocyte-specific antigen (Hepatocyte), pCEA, CD10, Villin, CD34, TTF-1, MOC-31, CK7, and CK20 antibodies. Sixty-eight cases of HCC and 107 cases of MA were investigated. Hepatocyte positivity was seen in 95.6% of HCCs and in 1.9% of MAs. pCEA was expressed in 47.8% of HCCs and in 86.8% of MAs. CD10 stained 73.13% of HCCs and 36.9% of MAs. Villin was positive in 23.5% of HCCs and in 81.0% of MAs. Canalicular staining with pCEA, CD10, and Villin was seen only in HCCs. Sinusoidal CD34 staining was seen only in 42.6% of HCCs. A small subset of HCCs demonstrated cytoplasmic TTF-1 and MOC-31. CK7 was expressed in 29.4% of HCCs and in 29.9% of MAs, whereas CK20 stained 14.7% of HCCs and 62.6% of MAs. In conclusion, Hepatocyte should be combined with pCEA, MOC-31, CD10, and CD34. Canalicular staining with pCEA, CD10, and Villin is specific for HCC. CK7 and CK20 expression may be seen in some HCCs. We suggest that the best panel for discriminating HCC from MA should contain Hepatocyte, MOC-31, pCEA, CD10, and CD34.     

Antigen retrieval and primary antibody type affect sensitivity but not specificity of CD117 immunohistochemistry

Aims:  To investigate the effects of antigen retrieval and primary antibody selection on specificity and sensitivity of CD117 immunohistochemistry.
Methods and results:  A survey and literature review were performed to determine the most commonly used CD117 antibodies. Of six such antibodies, three (Neomarkers polyclonal RB-1518, Novocastra monoclonal T595 and Santa Cruz polyclonal C19) were rejected as only suboptimal immunoreactivity was produced despite the use of various immunohistochemical protocols. Immunohistochemistry using the three remaining antibodies (Cell Marque polyclonal CMC766, Dako polyclonal A4502 and Epitomics monoclonal YR145) was performed, with and without (for Dako and Epitomics antibodies) antigen retrieval, on 32 gastrointestinal stromal tumours (GISTs) and on 139 neoplasms (comprising 24 neoplasm types) that are differential diagnoses for GIST and/or have been reported to express CD117. Antigen retrieval generally increased the sensitivity but did not alter the specificity of immunoreactivity with the three antibodies. The different antibodies showed variations in sensitivity, but did not stain different spectrums of neoplasm type. A small number of neoplasms showed scattered nuclear immunopositivity (particularly seen without antigen retrieval), which was regarded as representing cross-reactivity.
Conclusions:  Antigen retrieval and changing between the three antibodies tested affect sensitivity but not specificity of CD117 immunohistochemistry. Antigen retrieval does not produce false-positive CD117 immunostaining.     

Comparison of Ki-67 equivalent antibodies

AIMS: To compare commercially available Ki-67 equivalent antibodies with regard to qualitative and quantitative immunohistochemical staining characteristics. METHODS: The following antibodies were used: monoclonal MIB-1 (Immunotech), monoclonal MM1 (Novocastra), polyclonal NCL-Ki-67p (Novocastra), and polyclonal Rah Ki-67 (Dako). All immunostainings were evaluated in squamous epithelium from formalin fixed and paraffin wax embedded pharyngeal tonsils. Labelling indices (LIs) were recorded twice to test their reproducibility. RESULTS: By application of all four antibodies the nuclear staining could be either diffuse, granular, or a combination of both (classified as granular in this study). The diffuse pattern generally showed a strong or moderate staining intensity, whereas the granular pattern displayed a continuum from strong to very weak, making it difficult to discriminate between positive and negative nuclei. The diffuse staining pattern was seen in approximately 59% of the nuclei with the MIB-1 antibody and in 35-45% when the other antibodies were used. The following mean LIs were recorded: MIB-1, 31%; NCL-Ki-67p, 21%; Rah Ki-67, 17%; and MM1, 14%. The reproducibility was excellent for all four antibodies, with the mean of differences between the two runs of counts ranging from 1.1% to 1.5%. CONCLUSIONS: The four tested Ki-67 equivalent antibodies revealed differences in qualitative and quantitative staining characteristics, which resulted in considerable variations in registered LIs. The MIB-1 antibody appears to have a higher sensitivity for detecting the Ki-67 antigen than the other three tested antibodies. These differences are important to consider when proliferative activity is determined by the Ki-67 LI.     

Comparison of the Dako EGFR pharmDx kit and Zymed EGFR antibody for assessment of EGFR status in colorectal adenocarcinoma.

Immunohistochemistry is widely used to assess epidermal growth factor receptor (EGFR) expression on colorectal carcinomas to select patients for treatment with cetuximab, an anti-EGFR antibody. The data comparing different commercial EGFR antibodies is limited, and no cost comparisons have been made. We analyzed 65 advanced colorectal cancers from 36 patients using the EGFR pharmDx kit (DakoCytomation) and Clone 31G7 (Zymed Laboratories, Inc). EGFR expression was seen in 35 (53%) tumors (21 primary, 14 metastatic) with the Dako pharmDx kit. The Zymed antibody showed positive results in 41 (63%) tumors (25 primary, 16 metastatic). The cost per test was $40.00 with the pharmDx kit and $3.52 with the Zymed antibody. The Zymed antibody detects 10% more cases of colorectal cancer as EGFR positive, and is 10 times cheaper than the Dako pharmDx kit. There is little justification for the use of expensive kits for testing EGFR expression, when other available antibodies without the kit can give comparable or superior results.