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The Authors Reply

Acs, Geza M.D., Ph.D.; Zhang, Paul J M.D.

International Journal of Gynecological Pathology: October 2004 - Volume 23 - Issue 4 - p 407-408
doi: 10.1097/01.pgp.0000139664.50629.dc
Letters to the Editor

To the Editor:

We completely agree with Dr. Goldstein that the differences in methodologies hamper meaningful comparison between our studies. Regarding the immunohistochemical staining protocol, as implied by Dr. Goldstein, our protocol was optimized and validated under the conditions of our laboratory. Dr. Goldstein has proven his point by an attempt to simply duplicate our protocol in his laboratory and observed less sensitive staining with higher background. Under our conditions, we observed no background staining at all, and endothelial cells reacted strongly with the antibody.

Dr. Goldstein suggested the possibility of false nuclear reactivity due to methodological problems, such as over-antigen retrieval or too high an antibody concentration. More importantly, we did not observe any immunoreactivity in benign endometrium in any of our cases. Therefore, it is very unlikely that the WT-1 nuclear staining in tumor cells of our cases were simply due to nonspecific reactivity or false staining artifact.

We believe that the major difference between the results of the studies arises from the interpretation of the results, in particular the different cut-off values used for classifying a case as positive versus negative. Dr. Goldstein suggests that because WT1 is a transcription factor (1,2), low intensity WT1 staining is an expected finding in the nuclei of normal cells. We cannot agree with this statement for several reasons. Previous studies have established that the WT1 gene is normally expressed in only a small number of human organs, such as the developing kidney, gonads, mesothelium, and ovarian surface epithelium (3). In all these cells with normal WT1 expression, strong and diffuse nuclear immunoreactivity can be detected. In contrast, cells not normally expressing WT1, such as benign endometrial glandular cells, show no immunoreactivity at all (as also seen in our study). Second, comparison to problems with cut-off using cytokeratins 7 and 20 is probably not the best approach to this problem. In our opinion it is not valid to compare expression of structural proteins with that of a transcription factor with practically unknown functions in ovarian (or endometrial) carcinogenesis. Because we do not know the role of WT1 in these processes, we believe that, especially in the presence of complete lack of staining in the benign counterpart, any level of expression is potentially significant, and cannot be disregarded. Abnormal, increased WT1 expression has been reported in tumors arising from epithelial cells normally not expressing WT1, such as breast (4–6) and lung (7) cancers. In addition, it is not surprising that WT1 is expressed in endometrial carcinoma cells. In fact WT1 expression has been previously shown in cultured endometrial carcinoma cells (8), and we have also shown WT1 expression by Western blotting and RT-PCR in several endometrial carcinoma cell lines (KLE and RL95, unpublished data). Since the submission of our manuscript, we have encountered two more endometrial serous carcinomas confined to the uterus that showed diffuse moderate to strong WT1 immunoreactivity. Thus, it seems that WT1 is indeed expressed in a subset of endometrial serous carcinomas. Although the reactivity is usually focal and weak, we had three cases in our report that showed relatively diffuse, moderate to strong staining, with scores close to 100 (Fig. 5 in our article). We chose the example for Fig. 1D to simply illustrate the point that strong to moderate WT1 immunoreactivity can occur in endometrial serous carcinomas.

From a practical point of view, we agree with Dr. Goldstein that WT1 expression is usually heterogeneous in the tumors. In fact, we observed heterogenous staining in some ovarian serous carcinomas as well. We agree that there is a statistically highly significant difference between WT1 immunoreactivity in ovarian and endometrial serous carcinomas. However, WT1 expression seems to show variable degrees of overlap between serous carcinomas in the two sites, likely depending, at least in part, on the methodology used. Thus, even with more cases being analyzed in detail, it might still be difficult to establish a cutoff value that is reproducible and usable in practice among all pathology laboratories. Because of these difficulties, investigators should always validate their own test performed in their laboratory and determine their own comfortable cut-off value for any marker used for determination of tumor lineage and primary site. In summary, it is safe to say that together with all pathological and clinical findings, one can favor an ovarian or endometrial origin of a serous carcinoma if there is diffuse strong or no WT1 immunoreactivity, respectively, without providing a specific cutoff value<j> generated in one study from one laboratory. To establish a primary site for a tumor, one should never rely purely on the result of a single immunohistochemical stain,<j> particularly in cases of small biopsies from metastatic lesions.

Geza Acs, M.D., Ph.D.

Paul J. Zhang, M.D.

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