Microphthalmia

To the Editor-We read with interest the article "Tyrosinase Expression in Malignant Melanoma, Desmoplastic Melanoma, and Peripheral Nerve Tumors" by Boyle et al.1 In the article, Boyle et al reported that 100% (5/5) of their desmoplastic melanomas stained with antibodies to tyrosinase and HMB-45 using a microwave-based antigen-retrieval immunohistochemical technique. This result surprised us because our immunohistochemical laboratory has been using microwave-based antigen-retrieval techniques for many years and we have never seen consistent staining of desmoplastic melanoma with tyrosinase and HMB-45, except focally in superficially located or in situ epithelioid or nevoid components of the lesions. In fact, our previous study and many others in the literature demonstrated rare if any staining of the spindle cell component of desmoplastic melanoma with tyrosinase and HMB-45.2,3

Most desmoplastic melanomas are positive for S100, but frequently lack the melanocytic differentiation markers such as tyrosinase, gp100 (HMB-45), melan-A, and MITF. In an effort to resolve the difference, we performed tyrosinase immuno-staining on 5 well-characterized desmoplastic melanomas using microwave-based antigen-retrieval techniques and the same tyrosinase antibody (T311) as was used in the mentioned article. In 4 of 5 cases (80%), the tumor cells were completely negative; in 1 case (20%), only superficial more epithelioid tumor cells were positive; however, the deeper more spindled cells were negative for tyrosinase. In all of these cases, internal positive controls stained properly.

We disagree with the conclusion of Boyle et al and would also like to raise 3 questions. First, a conventional microwave oven has no device to control the buffer temperature and can only be estimated at about 100[degrees]C when the buffer is boiling; however, the authors stated that their antigen-retrieval technique was performed at 121[degrees]C, a temperature that likely required a pressure cooker or specially designed microwave oven. In the article, there was no detail about how 121[degrees]C was achieved in a microwave oven. Second, the antigen retrieval condition used in the paper (121[degrees]C, 20 minutes) is very harsh to the tissue. In our experience, heating tissue sections in a pressure cooker more than 5 minutes will often result in dissolution of the tissue section. In addition, we wonder whether the authors used other means to validate their findings so as to exclude the possibility that the staining was due to cross-binding of the antibody to some unrelated epitopes generated by the harsh antigen-retrieval technique? Third, because the photomicrographs of the desmoplastic melanoma showed aggregates of epithelioid to spindled tumor cells, unlike typical desmoplastic melanoma, in which individual tumor cells are embedded in a fibrotic stroma, we question whether these tumors may represent nondesmoplastic spindle cell melanomas, which might account for the discrepancy in the reported findings.

We believe it is misleading to represent desmoplastic melanomas, at least in their predominant spindle cell components, as reacting with HMB-45 and tyrosinase. It is well known that desmoplastic melanoma can mimic scar and benign neural lesions because of its bland spindle cell morphology; and it is dangerous to assume that negative HMB-45 and tyrosinase stains are sufficient to rule out desmoplastic melanoma.

XIAOWEI XU, MD, PhD

PAUL J. ZHANG, MD

DAVID E. ELDER, MB,ChB

Department of Pathology and Laboratory Medicine

Hospital of University of Pennsylvania

Philadelphia, PA 19104

1. Boyle JL, Haupt HM, Stern JB, Multhaupt HA. Tyrosinase expression in malignant melanoma, desmoplastic melanoma, and peripheral nerve tumors. Arch Pathol Lab Med 2002;126:816-822

2. Xu X, Chu AY, Pasha TL, Elder DE, Zhang PJ. Immunoprofile of MITF, tyrosinase, melan-A, and MAGE-1 in HMB45-negative melanomas. Am J Surg Pathol. 2002;26:82-87.

3. Busam KJ, Iversen K, Coplan KC, Jungbluth AA. Analysis of microphthalmia transcription factor expression in normal tissues and tumors, and comparison of its expression with S-100 protein, gp100, and tyrosinase in desmoplastic malignant melanoma. Am J Surg Pathol. 2001;25:197-204.

In Reply.-We wish to thank the authors for their interest in our paper.1 It is not the first time a laboratory claiming to use the same techniques does not reproduce results of another laboratory. Although our finding of positive staining for tyrosinase in desmoplastic melanomas clearly differs from the authors' prior conclusions, our results are similar to previously published studies of Jungbluth et al,2 Orchard,3 and Busam et al,4 who found a proportion of desmoplastic melanomas to stain positively for tyrosinase. In particular, the study by Busam et al,4 cited by these authors as evidence against tyrosinase staining, demonstrated 11 of 20 desmoplastic melanomas to be positive for tyrosinase.

The antigen retrieval and immunohistochemical staining in our study was performed using a Ventana Discovery (Ventana Medical Systems, Tucson, Ariz), which allows the precise setting of 121[degrees]C for 20 minutes. The immunohistochemical technologist from the laboratory that submitted this letter contacted our laboratory and we explained in detail our method of performing the immunohistochemistry. She indicated that the laboratory did not have access to a Ventana Discovery at the time these studies were performed.

Heat does not necessarily destroy tissues. Frozen sections and even EM 130 nm sections have been reported to have been treated by boiling for 10 minutes without affecting tissue integrity.5,6

All tissues in our study were treated the same way. If this resulted in nonspecific staining (cross-binding of the antibody to some unrelated epitope generated by treatment, as the letter suggests), the tissues would stain more homogeneously rather than focally in the tumor cells. The specificity of the antibodies is further supported by the negative areas, such as stroma, as well as by the appropriate staining of known positive controls.

The authors' statement that the photomicrographs of the desmoplastic melanoma showed aggregates of epithelioid tumor cells, and therefore may not represent a desmoplastic melanoma, appears incongruous with the previously published histologic description of desmoplastic melanoma by one of the groups listed in the letter's first reference.7 In this authoritative source, "fasciculated spindle cell areas" and "areas where epithelioid cells are arranged in sheets and nests" are described as characteristic of desmoplastic melanomas. We carefully selected for our study unequivocal desmoplastic melanomas with atypical spindle cells infiltrating a dense desmoplastic stroma, shown in Figure 5, a in our publication.1 Aggregates of epithelioid tumor cells are simply not seen in this photomicrograph.

We agree with the authors' concluding comment that "it is dangerous to assume that negative HMB-45 and tyrosine stains are sufficient to rule out desmoplastic melanoma." And that is precisely why we did not make or imply such a statement in our article.

HELEN M. HAUPT, MD

Department of Pathology

Pennsylvania Hospital

Philadelphia, PA 19107

JERE B. STERN, MD

Dermatopathology Consultation Services

Damascus, MD, and Quest Diagnostics

Baltimore, MD

HINKE A. B. MULTHAUPT, PhD

Imperial College School of Medicine

London, United Kingdom

1. Boyle JL, Haupt HM, Stern JB, Multhaupt HAB. Tyrosinase expression in malignant melanoma, desmoplastic melanoma, and peripheral nerve tumors. Arch Pathol Lab Med. 2002;126:816-822.

2. Jungbluth A, Iversen K, Coplan K, et al. T311: an anti-tyrosinase monoclonal antibody for the detection of melanocytic lesions in paraffin embedded tissues. Pathol Res Pract. 2000;196:235-242.

3. Orchard CE. Comparison of immunohisto-chemical labeling of melanocyte differentiation antibodies melan-A, tyrosinase and HM8-45 with NKIC3 and S100 protein in the evaluation of benign naevi and malignant melanoma. Histochem J. 2000;32:475-481.

4. Busam K, Iversen K, Coplan K, Jungbluth A. Analysis of microphthalmia transcription factor expression in normal tissues and tumors and comparison of its expression with S100 protein, gp100 and tyrosinase in desmoplastic malignant melanoma. Am J Surg Pathol. 2001;255:197-204.

5. Harm CR, Springen MJ, Johnson DH. Antigen retrieval of basement membrane proteins from archival eye tissues. J Histochem Cytochem. 2001;49:475-482.

6. Stirling JW, Graff PS. Antigen unmasking for immunoelectron microscopy: labeling is improved by treating with sodium ethoxide or sodium metaperiodate, then heating on retrieval medium. J Histochem Cytochem. 1995;43:115-123.

7. Elder DE, Murphy CF. Melanocytic Tumors of the Skin. Washington, DC: Armed Forces Institute of Pathology; 1991:167. Atlas of Tumor Pathology, 3rd series, fascicle 2.

Copyright College of American Pathologists Sep 2003
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