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• W2000707699 abstract "To the Editor: I would like to address several issues pertaining to WT1 immunohistochemical (IHC) staining of endometrial serous carcinomas (ESCs) raised by the recent studies by Acs et al. and Egan et al. and the accompanying editorial by Dr. McCluggage (1–3). In contrast to our earlier study in which we found no WT1 immunoreactivity in 18 ESCs (4), Acs et al. reported WT1 positivity in 10 of 16 (63%) ESCs and Egan et al. reported WT1 positivity in 2 of 31 (6.3%) ESCs. I completely agree with the suggestion by Egan et al. and Dr. McCluggage that these disparate results were possibly due to variability in antibodies, reaction conditions, or scoring systems. Furthermore, I suggest that differences in methodologies between the studies prohibit any meaningful comparisons of their results with those reported in our study. One difference was the size of the tissue section that was stained and evaluated. We and Acs et al. utilized standard tissue block sections, whereas Egan et al. used a tissue section compilation of three 2-mm diameter needle cores. Using the original WT1 stained slides from our study, an Olympus digital microscope, and the Photoshop CS program (Adobe Systems, Inc, San Jose, CA), the mean tissue section area per slide was 3.88 cm2 (range, 2.6–4.8 cm2). All of the sections in our study were from the luminal component of large-volume ESCs. In contrast, the calculated mean tissue section area per slide in the study by Egan et al was 0.094 cm2, assuming each core was a perfect column ([area = πr2, radius = 0.1 cm] × 3 needle cores). Nuclear regulatory proteins such as WT1, p53, and CDX2 often have variable expression and immunoreactivity. Additionally, it is my experience that antibodies to these molecules usually produce false-positive (rather than false-negative) immunoreactivity in under-fixed or over-antigen retrieved tissue. Microarray tissue sections may be a valid methodology for IHC screening a large number of neoplasms or comparing staining protocols, but in my opinion were an inappropriate choice for validating or refuting the findings of a prior study that used standard-area tissue sections. I am confident the results reported by Egan et al. are accurate in the context of the sectional area that was stained and examined. However, in my opinion, the 41.3-fold mean tissue section area difference between the studies invalidates their comparison of WT1 IHC results. A second important issue were the differences in the IHC staining procedures used in each study. Acs et al. used 5 μm-thick tissue sections and we used 3 μm-thick sections. Acs et al. used an antigen retrieval protocol of steam heat for 20 minutes and pH 6 citrate buffer, we used steam heat at 95°C for 25 minutes and pH7 EDTA buffer, and Egan et al. used a pepsin predigestion at 37° for 30 minutes followed by an unspecified method of antigen retrieval using pH8 EDTA buffer solution. Although the antibody clone was identical (clone 6F-H2, DakoCytomation Co, Carpenteria, CA), we used a 1:200 dilution and a 20-minute incubation, Acs et al. used a 1:400 dilution for 30 minutes, and Egan used a 1:50 dilution for an unspecified incubation time. Slides were developed in diaminobenzidine for 10 minutes in the Acs et al. study, 4 minutes in our study, and an unspecified time in the Egan et al. study. I cut new tissue sections from the ESCs tissue blocks used in our study and stained each of them with WT1 using the IHC procedure described in each study (Egan et al. protocol: 20 minutes antibody incubation). Endothelial cells that stained strongly with WT1 reactive with our IHC protocol were only weakly reactive with the Acs et al. protocol. There was also weak diffuse background staining of the entire section in 10 of 18 (55%) cases, including nuclear and cytoplasmic reactivity of ESC cells, lymphocytes, and endometrial stromal cells. The Egan et al. IHC protocol produced completely different results: tissue sections on 13 of the 18 slides (72%) fell off or were folded, torn, or shredded following antigen retrieval. The five cases with intact sections had diffuse, strong, background staining of the entire tissue section and nuclear chromatin fine detail was destroyed. Many benign and ESC cell nuclei had multiple nucleolar-like dots. The poor WT1 IHC results we obtained using these two IHC protocols are not surprising. Directly importing IHC staining protocols without modification almost always produces suboptimal results. I do not wish to construe that the IHC staining procedures used by either group of authors were inappropriate. Both were undoubtedly optimized and validated at each respective institution. The components of each author’s IHC staining protocol reflects the unique tissue fixation and processing factors of the institution. Importantly, these factors and the resultant IHC staining protocol also induce unique alterations of many target antigens and shifts antibody avidity in my opinion. These issues have not been extensively investigated by immunohistochemical manufacturers to my knowledge. My limited experiments lead me to believe that different methodologies can cause large variation in the proportion and intensity of staining. Altered antigen conformation was not an overly important issue when target antigens were cytoplasmic intermediate filaments, large cell membrane bound glycoproteins, or organelle confined molecules. However, it has become a vitally relevant issue because the sensitivity of IHC staining has increased and target antigens have moved to small and short-lived molecules. This observation may explain the WT1 staining results of Egan et al. in which the authors describe the immunoreactivity pattern as granular in the legend of their Figure 2. WT1 typically produces a homogeneous, uniform, “painted-on” pattern of nuclear staining that gradually fades to negative toward the periphery of the nucleus. In my laboratory, I can produce a granular nuclear staining pattern by using an overly concentrated WT1 antibody solution and by over-incubation in diaminobenzidene. Comparing results of studies that used markedly different immunohistochemical staining protocols is invalid. A third important issue that invalidates the comparison of results was the use of different scoring systems by the authors. In the Acs et al. study, ESCs with any nuclear WT1 staining, regardless of the intensity or the proportion of stained nuclei, were classified as WT1 positive. In the Egan et al. study, neoplasms with moderate or strong nuclear staining were classified as WT1 positive. None of the carcinomas in our study had moderate or strong WT1 nuclear staining. Three cases (17%) had extremely weak nuclear staining in <5% of the neoplastic cells. We classified these three carcinomas as WT1 negative from the benchmark perspective of diffuse strong nuclear WT1 staining of ovarian serous carcinomas. WT1 is a transcription factor type nuclear regulatory molecule that controls expression of differentiation and proliferation genes (5–7). Low intensity WT1 staining is therefore an expected finding in nuclei of normal cells, similar to other nuclear regulatory molecules such as p53. In my opinion, classifying weak WT1 nuclear staining as a positive result is incorrect. The misclassification of ESCs with normal low-intensity nuclear WT1 staining as WT1 positive possibly explains how Acs et al. reported that 10 of the 16 (62%) ESCs were WT1 positive, yet the mean and median staining scores in these cases were 7.5 and 23.2, respectively (staining scoring system range: 0–300). Additionally, it is difficult to reconcile their Figure 1D, which shows “moderate-to-strong” WT1 nuclear staining, with their reported results. Given their published mean and mean staining scores, the photographed field appears to have been extremely small. In my opinion, the markedly lower cut point for classifying a carcinoma as WT1-positive used by Egan et al. invalidates the conclusions they drew from comparing their results with ours. The problems that ensue from the use of different and inappropriately low staining cut-points are not novel. The results reported in three early studies on cytokeratin 7 and cytokeratin 20 coordinate staining, each of which used a different positive result cut-point ranging from any (>1%) to 25%, were so different that no reliable conclusions could be drawn from them as a group (8–10). Varying the threshold of stained cells cut-point can alter the distribution of positive results by up to 35% (11). I am disappointed to see these problematic issues persist. Delving into these issues in a gynecologic pathology journal may seem picayune and out of place. I believe it is important for all pathologists, especially those who believe that immunohistochemistry is a straightforward and standardized procedure, to be cognizant of the potential impact of these factors. Problems resulting from these issues seem to be increasing and may be partly due to the amplification of effects from a popular web site (www.immunoquery.com). Using these three studies as an example, the meta-analysis program used by this web site would report that 18% (12 of 65 cases) of ESCs are WT1 positive. The clinical utility of this result is dubious. Attention to procedural and methodological factors is central to generating accurate results that allow valid comparisons with the results reported in previous studies. Neal S. Goldstein, M.D. Director, Immunohistochemistry and Molecular Pathology, William Beaumont Hospital, Royal Oak, Michigan" @default.
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• W2000707699 title "WT-Staining in Endometrial Serous Carcinomas" @default.
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