Wilms’ Tumour (WT) is a tumour of the kidneys that typically occurs in children. WT1 protein expression in mesothelial cells has become a reliable marker for the diagnosis of mesotheliomas. The NCL-L-WT1-562 (WT49) was found to be very specific and best suited to this facility’s fully automated Immunohistochemistry (IHC) laboratory, when compared with the WT1 6F-H2 clone. The positive reaction of WT49 was restricted to nuclear staining without cytoplasmic staining which has been described in other WT1 antibodies.
Wilms’ tumour (WT) is typically a paediatric malignancy with about 20% of solid tumours arising due to a loss of function mutation of the WT gene on chromosome 11p13. The gene which encodes a number of protein isoforms which share four zinc fingers, recognizes the early growth response (EGR-1) consensus sequence found in promoters of growth factor genes.1, 2 There is evidence to support that the protein encoded by WT1 regulates the transcription of other genes; therefore functioning as both a transcriptional activator and repressor.1, 2 WT1 protein has been localised in WT, malignant mesothelioma and serous ovarian adenocarcinoma.2, 3 Mesotheliomas form part of a spectrum of tumours of which those with epitheloid features, are most common. They usually arise in the chest cavity or abdomen. Those arising from the pleura are almost always caused by heavy occupational exposure to asbestos fibres and dust2, with a latency period for symptoms of disease ranging from 25-45 years4 and the lifetime risk of developing mesothelioma after heavy exposure ranging from 7-10%.4
WT1 protein is conventionally used as a positive marker in differentiating mesothelioma from other neoplastic mimics.1, 2 The importance in making the correct and accurate diagnosis is not only a medico legal issue due to the association of asbestos exposure, but also for therapeutic intervention.
Materials and Methods
An optimal immunostaining protocol was determined using known mesothelioma tumours from archival material from this site. The BOND-MAX instruments were used to perform the staining.
Tissues were fixed in 10% neutral-buffered formalin and processed to paraffin wax by conventional laboratory methods. Sections were cut at 3μm, mounted on commercially available positively charged glass slides and incubated at 60º C for 30 minutes. The appropriate positive and negative controls, which included normal fallopian tube, ovarian adenocarcinoma and mesothelioma, were optimised to test specificity of the antibody and to exclude non-specific staining.
Both clones required antigen retrieval procedures, which were not identical. As per the specification sheets, the WT1 clone 6F-H2 (Dako Copenhagen), required pepsin enzyme retrieval and the NCL-L- WT1-562 clone WT49 (Leica Biosystems, Newcastle) required Heat induced epitope retrieval (HIER) ER2 (AR9640) pH 9.0 for a duration of 30 minutes.
Specifically bound antibodies were visualised using Bond Polymer Refine Detection System (DS9800) with the addition of a DAB enhancer (AR9432).
The optimal protocol and dilution for NCL-L-WT1-562 (WT49) was 1:50 with an incubation time of 15 minutes, with HIER using ER 2 solution (pH 9.0) for 30 minutes. To facilitate optimisation of the WT1 6F-H2 clone, two different retrieval solutions and protocols were employed.
For the first optimisation, as a means of mimicking the suppliers recommended retrieval, antigen retrieval was performed using Bond Enzyme 1 (AR9551) for 10 minutes. The second optimisation used HIER ER2 for 30 minutes. For both these retrieval methods, the primary antibody was standardised at 1:200 with an incubation of 15 minutes at ambient temperature (Table 1).
Results and Discussion
Review of the literature reveals that some studies have shown clear distinctions in WT1 protein expression of mesotheliomas and adenocarcinomas. Mesotheliomas exhibit a strong differential nuclear pattern of staining while adenocarcinomas either exhibit a negative result or a cytoplasmic pattern of staining.3, 5 Using a polyclonal antibody to WT1 (Santa-Cruz, California), Foster et al, 2001 demonstrated strong nuclear staining in 75% of mesotheliomas, with absent nuclear staining in primary pulmonary adenocarcinomas.5 This group further showed that 86% of pulmonary adenocarcinomas demonstrated cytoplasmic staining.5 Hence, for the distinction between adenocarcinomas and mesotheliomas, nuclear staining for WT1 has been shown to be highly specific for mesothelioma. A panel of antibodies, including WT1 protein, has also been suggested to distinguish pancreaticobiliary from ovarian serous carcinomas. The study undertaken by Goldstein et al, 2001, demonstrated varying degrees of WT1 immunoreactivity in as many as 93% of ovarian serous carcinomas when using the WT1 6F-H2 clone.6, 7
According to the manufacturer’s specification sheet, the WT1 6F-H2 requires pepsin as the enzyme of choice for retrieval. Pepsin is a crude enzyme that may destroy some epitopes and alter tissue morphology. For optimal staining, the length of pepsin digestion or any proteolytic enzyme should be proportional to the length of fixation time.8 As a referral centre, our laboratory receives paraffin blocks from a variety of centres across international borders. This material is often limited, fixed and processed sub optimally, making enzyme retrieval difficult to optimise. The purpose of using different retrieval protocols when staining with the 6F-H2 clone was employed as a means of demonstrating the presence or absence of staining of EC’s and therefore definitive, accurate staining of mesotheliomas.
As per the WT1 6F-H2 antibody data sheet, endothelial cells (ECs) are inherently positively demonstrated and the expected staining pattern is nuclear and / or cytoplasmic. The EC’s therefore serves as an internal control, which confirms that the protocol and antibody is performing optimally as per the recommendations of this supplier. The protocol that employed Bond Enzyme 1 produced appropriate positive staining of granulosa cells of the ovary (Figure 1), however false negative staining was observed on a known WT1-positive case of mesothelioma and EC staining was also negative (Figure 2). Sections of the same case which were run in parallel employing HIER were immunoreactive in the tumour and the EC’s. For use of the WT1 6F-H2 antibody on the Bond Instrument, to obtain true positive staining on the mesotheliomas, HIER is required. This in turn produced not only positive demonstration of the expected EC’s, but an increase in background staining, which can make interpretation of the result extremely difficult (Figure 3). As Foster et al, 2001 report, WT1 positivity in only 75% of mesotheliomas, selection of appropriate material for optimisation purposes is critical and should include a number of mesothelioma cases to eliminate the possibility of negative staining results being interpreted as a run error.
The NCL WT49 has a nuclear staining pattern and the staining of ECs is absent. This results in a clean, clear background that makes interpretation easy. No cytoplasmic staining was noted with WT49 (Figure 4). Only one retrieval protocol was used with the NCL-L-WT1-562 (WT49), which produced the expected results (Table 1).
While nuclear staining is sometimes associated with cytoplasmic staining, in the absence of nuclear staining, cytoplasmic staining should be considered as spurious and may even represent cross-reactivity with an epitope unrelated to WT1. Until the width of immunoreactivity of Dako WT1 6F-H2 is completely understood, it may not be a reliable marker for the accurate identification of mesotheliomas.4
Tabulation summary of the protocols used for optimization and staining results.
Antibody and Clone
Dako WT1 6F-H2
Bond Enzyme 1 for 10 minutes
EC and granulosa cells positive.
False negative staining of both EC and mesothelioma.
Dako WT1 6F-H2
Bond ER2 for 30 minutes
EC strongly immunoreactive with mesothelioma positive
NCL-WT1-562 WT 49
ER2 for 30 minutes
EC negative and mesothelioma positive nuclear staining
The NCL-L-WT1-562 (WT49) has for our purposes, produced staining far superior to the Dako WT1 6F-H2 clone. It has produced a clean crisp stain that is well localised and easy to interpret. The retrieval solution for the NCL-L-WT1-562 (WT49) has proven to be less harsh on epitopes and cellular morphology of the tissue structures; with the variables of fixation time and resultant negative impact on tissue integrity easier to overcome. With the impact of automation addressing global technical skill shortages, coupled with clinical demand for high throughput, automation decreases laboratory variation and provides a platform for consistent reproducible results. This translates into fewer repeats, improved diagnostic quality and impacts positively on laboratory cost constraints.
However, cognisance of manufacturer specifications when using primary antibodies from various sources needs to be carefully factored in to the optimisation process; especially on an open automated system. Responsibility lies with the laboratory to determine an initial base line of accurate staining for validation as per the manufacturer recommendations.
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Amin KM, et al. Wilms’ tumor 1 susceptibility (WT1) gene products are selectively expressed in malignant mesothelioma. American Journal of Pathology 1995; Vol 146(2):344-356.
Lee SB, Haber DA. Wilms’ tumor and the WT1 gene. Experimental Cell Research. 2001; 264:74–99.
Kumar-Singh S, et al. WT1 mutation in malignant mesothelioma and WT1 immunoreactivity in relation to p53 and growth factor receptor expression, cell-type transition, and prognosis. J Pathol 1997; 181:67-74.
Foster MR, Johnson JE, Olson SJ, Allred DC. Immunohistochemical analysis of nuclear versus cytoplasmic staining of WT1 in malignant mesotheliomas and primary pulmonary adenocarcinomas. Archives of Pathology and Laboratory Medicine 2001; 125:1316-1320.
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