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Immunophenotypic Differences Between Uveal and Cutaneous Melanomas
Satori Iwamoto, MD, PhD;
Robert C. Burrows, PhD;
Robert E. Kalina, MD;
David George, MD;
Michael Boehm, MD;
Mark A. Bothwell, PhD;
Rodney Schmidt, MD, PhD
Arch Ophthalmol. 2002;120:466-470.
ABSTRACT
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Objective To determine the immunophenotypic differences between uveal and cutaneous
melanomas, employing standard melanoma markers as well as p75 neurotrophin
receptor (p75NTR) and microphthalmia transcription factor (MITF).
Design Fifteen uveal melanomas (5 spindle, 5 epithelioid, and 5 mixed uveal
subtypes) were immunolabeled with a panel of antibodies that included S100,
tyrosinase, melan-A, HMB-45 and HMB-50 combination, MITF, and p75NTR. The
results were tabulated on the basis of intensity and pervasiveness of the
labeling and compared with a prior study on cutaneous spindle and epithelioid
melanomas.
Results In contrast to its strong labeling of cutaneous melanomas, S100 immunolabeling
of uveal melanomas was weak and variable. p75NTR, known to differentiate spindle
from epithelioid melanomas of the skin, did not immunolabel uveal melanomas.
HMB-45, HMB-50, tyrosinase, melan-A, and MITF immunolabeled all uveal melanomas
strongly, irrespective of the histologic subtype, but not cutaneous melanomas.
Microphthalmia transcription factor was especially clear in its labeling of
uveal melanomas.
Conclusions Although cutaneous and uveal melanomas share many molecular markers
in common, there are differences between the 2 types of melanoma. First, the
level of expression of S100 differs between cutaneous and uveal melanomas.
Second, while cutaneous melanomas can be further subdivided into spindle and
epithelioid types based on their immunophenotype, the uveal melanomas cannot.
INTRODUCTION
IN 1931, CALLENDER proposed a classification of uveal melanomas based
on 2 major cell types, spindle and epithelioid. This classification has been
demonstrated to be a valuable indicator of prognosis. Patients with spindle
melanoma cells exhibit a better clinical course than those with epithelioid
cell types. Over the years, numerous modifications of the Callender classification
have been published, as thoroughly reviewed by McLean.1
While tumor size, mitotic activity, tumor-infiltrating lymphocytes, age, and
tumor vascular pattern2 have prognostic value,
the Callender classification by cell type as modified by the Armed Forces
Institute of Pathology remains one of the most reliable prognostic determinants.3 More recently, Moshari and McLean4
reported that the mean length of the longest nucleoli measured on silver-stained
sections has a predictive value as reliable as cell type. A similar tendency
of nucleolar size was detected by electron microscopy in 1972.5
Cutaneous melanomas can also be classified into spindle and epithelioid
cell types. Furthermore, studies have revealed distinct differences in the
immunophenotype between spindle and epithelioid melanomas, especially when
cells are labeled by the antibody to the p75 neurotrophin receptor (p75NTR).6-7 With the exception of S100, standard
melanoma markers, including HMB-45, HMB-50, tyrosinase, and melan-A, are strongly
expressed in epithelioid cutaneous but are poorly expressed in spindle cutaneous
melanomas.8-11
In contrast, certain Schwann cell markers, particularly the p75NTR, are strongly
expressed in spindle cutaneous but not in epithelioid melanomas.6
Microphthalmia transcription factor (MITF) is also preferentially expressed
in epithelioid but not spindle subsets.12
Prior immunohistochemical studies of uveal melanomas have involved S100,13-19
HMB-45,20-21 melan-A,19, 22 and tyrosinase.22
There have been reports suggesting differences between uveal and cutaneous
melanomas based on S100 labeling,15-16
but to our knowledge, there has been no systemic side-by-side comparison using
a large panel of antibodies. We sought to determine whether the differences
in immunophenotype between spindle and epithelioid cells noted in cutaneous
melanomas6-7 were also applicable
to uveal melanomas. Our hope was that such studies could be helpful in the
cellular classification and perhaps have prognostic value in uveal melanomas.
We examined the immunophenotype of 5 spindle, 5 epithelioid, and 5 mixed
uveal melanomas. Detailed analyses of immunolabeling were performed based
on signal intensity and pervasiveness. We compared the results with our recent
report of the immunophenotype of spindle and epithelioid cutaneous melanomas.
A comparison was made for the expression of S100, melan-A, HMB-45, HMB-50,
tyrosinase, MITF, and p75NTR in the 3 categories of uveal melanomas. This
is the first report, to our knowledge, of p75NTR expression in uveal melanomas.
MATERIALS AND METHODS
TISSUES
Choroidal melanoma tissues were retrieved from the archives of the Department
of Pathology at the University of Washington Medical Center (Seattle). Choroidal
melanomas were divided into spindle (n = 5), epithelioid (n = 5), and mixed
epithelioid and spindle melanomas (n = 5) on the basis of the pathology reports.
Their histologic makeup was reconfirmed prior to immunocytochemical labeling.
Six normal eye specimens (with no history of pathology) were obtained from
the pathology department and the Lions Eye Bank (Seattle).
IMMUNOCYTOCHEMISTRY
Five-micrometer formalin-fixed, paraffin-embedded sections were obtained
from the archived blocks, dewaxed, rehydrated, and treated with 3% hydrogen
peroxidase. For S100 labeling, nonspecific binding was blocked by incubating
slides with normal goat serum for 10 minutes. Following heat-induced epitope
retrieval in 10 mM of sodium citrate at a pH of 6.0, slides were immunolabeled
with the following antibodies for 40 minutes at room temperature: p75NTR antibody,
1:200 (mouse monoclonal MS-394-PI; Neomarkers, Fremont Calif); S100, 1:8000
(Z311; Dako, Carpinteria Calif); melan-A, 1:50 (clone A103, M7196; Dako);
tyrosinase, 1:50 (monoclonal clone #T311, NCL-TYROS; Novocastra/Vector Lab,
Burlingame, Calif); HMB-45/-50 cocktail, 1:1600/1:500 (monoclonal antibodies
HMB-45 and HMB-50; University of Washington); MITF, 1:25 (a gift from David
Fisher, MD, PhD, Dana Farber Cancer Institute, Boston, Mass). After several
rinses with phosphate-buffered isotonic sodium chloride solution (PBS), slides
were incubated with biotinylated secondary antibodies for 25 minutes. The
slides were then rinsed in PBS followed by incubation with avidin-biotin complex
(Vector Elite, Burlingame) for 25 minutes at room temperature. Binding was
visualized using 3,3' diaminobenzidine as a chromogen, with nickel chloride
enhancement and a methyl green counterstain. For negative controls, we used
substituted diluted normal rabbit serum for the primary antibody. Some of
the uveal melanoma specimens were immunolabeled concurrently with some of
the cutaneous melanoma specimens, although the results of the cutaneous melanomas
have been reported separately.6
SCORING
The immunolabeled specimens were interpreted according to the intensity
and the pervasiveness of the immunocytochemical staining. The intensity was
ranked on a scale of 0 to 3 as follows: 0 = absence of labeling; 1 = low;
2 = moderate; and 3 = high. The pervasiveness or extent of labeling was ranked
on a scale of 0 to 3 as follows: 0 = absence of labeling; 1 = focal or less
than 10% of cells; 2 = patchy or 10% to 75% of cells; and 3 = diffuse or more
than 75% of cells. Internal positive controls are discussed in the "Results"
section. Scores were independently rated by at least 2 authors. Four of the
authors had been involved in the cutaneous melanoma study with which this
set of data has been compared. Agreement of scores was reached in all cases.
Data are given as mean ± SEM. Results were graphed for comparison purposes
and placed side by side with corresponding results of spindle and epithelioid
cutaneous melanoma as reported previously.5
PHOTOGRAPHY
For Figure 1, images of labeled
slides were directly collected using a CoolSnap digital camera (R. S. Photometrics,
Tucson, Ariz) attached to a Leitz Orthoplan (Leica Microsystems Inc, Bannockburn,
Ill) microscope (magnification to the charge-coupled device chip x27.5).
Images were saved as 1392 x 1040 24 bit RGB TIFF (tagged image file
format) files. The figure was assembled in Adobe Photoshop 5 (Adobe Systems
Inc, San Jose, Calif) with minimal contrast and brightness enhancement.
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Figure 1. Photomicrographs of a spindle
uveal melanoma illustrating immunophenotypic labeling patterns. Note the complete
absence of p75NTR (neurotrophin receptor) immunolabeling and the relatively
low level of S100 immunolabeling. In contrast, HMB-45, tyrosinase, melan-A,
and MITF (microphthalmia transcription factor) all show extensive immunoreactivity
of the tumor (original magnification x27.5). H&E indicates hematoxylin-eosin.
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RESULTS
A total of 15 uveal melanomas, including 5 spindle, 5 epithelioid, and
5 mixed types, were immunolabeled with a panel of antibodies that included
p75NTR, S100, HMB-45, HMB-50, tyrosinase, melan-A, and MITF. The mean ±
SEM values of the intensity and of the pervasiveness are shown with our previous
results from the immunophenotypic differences in expression patterns between
epithelioid and spindle melanomas of the skin6
(Table 1 and Figure 2). The cutaneous melanoma results for all markers except
MITF are presented in Figure 2 and Table 1 for comparison purposes only.
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Relative Immunophenotypic Expression Patterns of Cutaneous and Uveal
Melanomas*
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Figure 2. Immunophenotypic results from
uveal melanomas compared with results of cutaneous melanoma data obtained
from a previous study.6 A shows staining intensity
while B shows pervasiveness of staining.The MITF results reflect the uveal
melanoma data only. Note the complete absence of p75NTR labeling in any of
the melanomas, and the high level of MITF staining for both intensity and
pervasiveness.
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Nontumor ocular tissue from tumor-containing eyes served as internal
controls. We also labeled 6 normal eye specimens (with no ocular pathology)
to establish normal immunolabeling patterns. For MITF, the positive internal
controls included the retinal pigment epithelium and the variable labeling
of stromal cells of the iris, ciliary body, and choroid. For melan-A, variable
but distinct staining of the stromal cells of the ciliary body and iris was
used as an internal positive control. For tyrosinase and the HMB-45 and HMB-50
antibodies, there was immunolabeling only of tumor cells, with minimal immunolabeling
of normal retinal or uveal tissues. For S100, internal controls included the
stromal cells of the iris and ciliary body and the nerve fiber layer of the
retina. For the p75 neurotrophin receptor labeling, the peripheral nerves
of the iris and ciliary body as well as the Müller glial cells of the
retina were used as the internal positive controls.
p75NTR was absent in all of the choroidal melanoma specimens, which
was in striking contrast to its labeling of spindle melanomas in the skin.
S100 labeled 3 of 5 spindle melanomas with an intensity of 0.6 ± 0.24
and a pervasiveness of 0.8 ± 0.37. In the epithelioid melanomas, 4
of 5 specimens were labeled with an intensity of 1.2 ± 0.37 and a pervasiveness
of 1.4 ± 0.4. In the mixed melanomas, 5 of 5 specimens were labeled
with an intensity of 1.4 ± 0.24 and a pervasiveness of 2.1 ±
0.18. HMB-45 AND HMB-50 labeled 5 of 5 specimens in the spindle melanomas
with an intensity of 2.8 ± 0.2 and 2.7 ± 0.2 for pervasiveness.
For the epithelioid melanomas, 5 of 5 specimens were labeled with an intensity
of 2.5 ± 0.22 and a pervasiveness of 2.8 ± 0.2. In the mixed
melanomas, 5 of 5 specimens were labeled with an intensity of 3.0 and a pervasiveness
of 2.6 ± 0.24.
Tyrosinase labeled 5 of 5 spindle melanomas with an intensity of 2.4
± 0.24 and a pervasiveness of 3.0. In the epithelioid melanomas, 5
of 5 specimens were labeled with an intensity of 2.6 ± 0.24 and a pervasiveness
of 2.6 ± 0.24. In the mixed melanomas, 5 of 5 specimens were labeled
with an intensity of 2.3 ± 0.2 and a pervasiveness of 2.2 ±
0.37. In spindle melanomas, melan-A labeled 5 of 5 specimens with an intensity
of 2.8 ± 0.2 and pervasiveness of 3. In the epithelioid melanomas,
5 of 5 specimens were labeled with an intensity of 2.8 ± 0.2 and a
pervasiveness of 3.0. In the mixed melanomas, 5 of 5 specimens were labeled
with an intensity and pervasiveness score of 3.0. Microphthalmia transcription
factor labeled 5 of 5 specimens for each of the 3 types of choroidal melanomas,
spindle (n = 5), epithelioid (n = 5), and mixed (n = 5), with an average value
of 3.0 for both intensity and pervasiveness. Of particular interest was the
clear specificity of this marker owing to the intense nuclear localization
of the signal (Figure 1). Patterns
of immunoreactivity for each antibody are shown in Figure 1 for a spindle uveal melanoma.
COMMENT
This study was undertaken to compare the immunophenotypic expression
patterns between melanomas of the skin and of the uvea. We had initially set
out to determine whether p75NTR, which can distinguish spindle from epithelioid
skin melanomas,6-7 would also
be helpful in distinguishing spindle from epithelioid populations within uveal
melanomas. Such a marker would have potential prognostic value in uveal melanomas.
However, the absence of p75NTR immunolabeling in all of the uveal melanomas
despite the presence of all internal positive controls is a striking feature
that distinguishes cutaneous from uveal melanomas. Thus, the p75NTR, a marker
that is useful in distinguishing spindle from epithelioid melanomas in the
skin, is not helpful in identifying the spindle cells of uveal melanomas.
It is possible that labeling with p75NTR reflects desmoplasia, which is a
common feature in spindle cutaneous melanomas but is rarely, if ever, present
in uveal melanomas.
Another interesting difference between cutaneous and uveal melanomas
was apparent with S100 immunolabeling. S100 immunoreactivity was relatively
low when measured by intensity and pervasiveness in the uveal melanomas; conversely,
in the skin melanomas, the reactivity was high. The difference could be due
to different S100 isoforms. The low immunoreactivity to the S100 antibody
in uveal compared with skin melanomas is in accord with prior detailed studies
by Kan-Mitchell et al15-16 and
others who have reported decreased S100 compared with HMB-45 immunolabeling
in primary18-19 and in metastatic20 uveal melanomas.19-20
The remaining markers, HMB-45, HMB-50, tyrosinase, melan-A, and MITF
also showed differences in expression between uveal and cutaneous melanomas
in that they were expressed at high levels whether the uveal melanoma was
of the spindle, epithelioid, or mixed type. In contrast, in skin melanomas,
these same antibodies are expressed at high levels only in the epithelioid
types and expressed poorly in the spindle subsets.6 Figure 2 and Table 1 compare immunolabeling patterns between skin and uveal melanomas
that were performed in our laboratory; however, it should be noted that the
comparison is a historical rather than a side by side comparison. Although Figure 2 and Table 1 do not present data on MITF-labeling of cutaneous melanomas,
MITF is also poorly expressed in cutaneous spindle melanomas.12
Our MITF expression patterns confirm an earlier preliminary study by Sharara
et al,23 which found that MITF does not distinguish
uveal from cutaneous melanomas.
Due to the differences in immunolabeling patterns between skin and uveal
melanomas, the current panel of antibodies does not have the same diagnostic
power for uveal melanomas as it does for cutaneous melanomas to delineate
spindle from epithelioid cell types. However, this study also demonstrates
that the markers melan-A, tyrosinase, HMB-45, HMB-50, and MITF are all superior
markers to S100 for the identification of uveal melanomas.
AUTHOR INFORMATION
Submitted for publication July 24, 2001; final revision received November
16, 2001; accepted December 11, 2001.
This work was supported by an American Society of Dermatologic Surgery
grant and Dermatologist-Investigator Award from the Dermatology Foundation,
Evanston, Ill (Dr Iwamoto), a grant from the Carl J. Herzog Foundation, Exeter,
NH (Dr Iwamoto), grants RO1 DC02863 and RO1 NS 33200 from the National Institutes
of Health, Bethesda, Md (Dr Bothwell), and Pathology Professional Funds, University
of Washington, Seattle (Dr Schmidt).
We thank Takeo Iwamoto, MD, PhD, Weill Medical College of Cornell University,
New York, NY, for discussion and review of the manuscript. We thank John Olerud,
MD, Szolt Argenyi, MD, and James Orcutt, MD, PhD, for critical review of the
manuscript. We thank Lorraine Gibbs, BA, HT (ASCP), Regina Bowman, BA, HT
(ASCP), and Holly Predd, BA, for technical support and discussion. We also
thank the immunocytochemistry laboratory at the University of Washington for
performing the ICC studies.
Corresponding author and reprints: Satori Iwamoto, MD, PhD, Department
of Medicine, Division of Dermatology, University of Washington Medical Center,
Box 356524, Seattle, WA 98195-6524 (e-mail: siwamoto{at}u.washington.edu).
From the Departments of Medicine (Dr Iwamoto), Physiology and Biophysics
(Drs Iwamoto and Bothwell), Radiology/Imaging Research Laboratory (Dr Burrows),
Ophthalmology (Drs Kalina, George, and Boehm), and Pathology/Immunocytochemistry
(Dr Schmidt), University of Washington Medical Center, Seattle, Wash.
REFERENCES
| |
1. McLean I. Uveal nevi and malignant melanomas. In: Spenser W, ed. Ophthalmic Pathology: An Atlas
and Textbook. 4th ed. Philadelphia, Pa: Saunders Co; 1996:2121-2217.
2. Folberg R, Rummelt V, Parys-Van Ginderdeuren R, et al. The prognostic value of tumor blood vessel morphology in primary uveal
melanoma. Ophthalmology. 1993;100:1389-1398.
ISI
| PUBMED
3. McLean IW. Prognostic features of uveal malignant melanoma. Ophthalmol Clin North Am. 1995;8:143-153.
4. Moshari A, McLean IW. Uveal melanoma: mean of the longest nucleoli measured on silver-stained
sections. Invest Ophthalmol Vis Sci. 2001;42:1160-1163.
FREE FULL TEXT
5. Iwamoto T, Jones IS, Howard GM. Ultrastructural comparison of spindle A, spindle B, and epithelioid-type
cells in uveal malignant melanoma. Invest Ophthalmol. 1972;11:873-889.
FREE FULL TEXT
6. Iwamoto S, Burrows RC, Agoff SN, Piepkorn M, Bothwell M, Schmidt R. The p75 neurotrophin receptor, relative to other Schwann cell and melanoma
markers, is abundantly expressed in spindled melanomas. Am J Dermatopathol. 2001;23:288-294.
FULL TEXT
|
ISI
| PUBMED
7. Iwamoto S, Odland PB, Piepkorn M, Bothwell M. Evidence that the p75 neurotrophin receptor mediates perineural spread
of desmoplastic melanoma. J Am Acad Dermatol. 1996;35:725-731.
FULL TEXT
|
ISI
| PUBMED
8. Wick MR, Swanson PE, Rocamora A. Recognition of malignant melanoma by monoclonal antibody HMB-45: an
immunohistochemical study of 200 paraffin-embedded cutaneous tumors. J Cutan Pathol. 1988;15:201-207.
FULL TEXT
|
ISI
| PUBMED
9. Carlson JA, Egbert BM. Desmoplastic neurotropic malignant melanoma. Pathology (Phila). 1994;2:339-357.
PUBMED
10. Longacre TA, Egbert BM, Rouse RV. Desmoplastic and spindle-cell malignant melanoma: an immunohistochemical
study. Am J Surg Pathol. 1996;20:1489-1500.
FULL TEXT
|
ISI
| PUBMED
11. Blessing K, Sanders D, Grant J. Comparison of immunohistochemical staining of the novel antibody melan-A
with S100 protein and HMB-45 in malignant melanoma and melanoma vaiants. Histopathology. 1998;32:139-146.
FULL TEXT
|
ISI
| PUBMED
12. Granter SR, Weilbaecher KN, Quigley C, Fletcher CD, Fisher DE. Microphthalmia transcription factor: not a sensitive or specific marker
for the diagnosis of desmoplastic melanoma and spindle cell (non-desmoplastic)
melanoma. Am J Dermatopathol. 2001;23:185-189.
FULL TEXT
|
ISI
| PUBMED
13. Cochran AJ, Wen DR. S100 protein as a marker for melanocytic and other tumours. Pathology. 1985;17:340-345.
ISI
| PUBMED
14. Garrido CM, Arra A. Value of the S100 protein in the study of nevus and ocular melanomas. Ophthalmologica. 1987;194:201-203.
ISI
| PUBMED
15. Kan-Mitchell J, Rao N, Albert DM, Van Eldik LJ, Taylor CR. S100 immunophenotypes of uveal melanomas. Invest Ophthalmol Vis Sci. 1990;31:1492-1496.
FREE FULL TEXT
16. Kan-Mitchell J, Liggett PE, Taylor CR, et al. Differential S100 beta expression in choroidal and skin melanomas:
quantitation by the polymerase chain reaction. Invest Ophthalmol Vis Sci. 1993;34:3366-3375.
FREE FULL TEXT
17. Kochavi O, Munichor M, Lichtig C, Meyer E. Immunohistochemical staining techniques of intraocular tumors. Ann Ophthalmol. 1992;24:347-351.
ISI
| PUBMED
18. Burnier MN, McLean IW Jr, Gamel JW. Immunohistochemical evaluation of uveal melanocytic tumors: expression
of HMB-45, S100 protein, and neuron-specific enolase. Cancer. 1991;68:809-814.
FULL TEXT
|
ISI
| PUBMED
19. Heegaard S, Jensen OA, Prause JU. Immunohistochemical diagnosis of malignant melanoma of the conjunctiva
and uvea: comparison of the novel antibody against melan-A with S100 protein
and HMB-45. Melanoma Res. 2000;10:350-354.
FULL TEXT
|
ISI
| PUBMED
20. Luyten GP, Mooy CM, Post J, Jensen OA, Luider TM, de Jong PT. Metastatic uveal melanoma: a morphologic and immunohistochemical analysis. Cancer. 1996;78:1967-1971.
FULL TEXT
|
ISI
| PUBMED
21. Martins M, Scull JJ, Alcocer CE, Deschenes J, Antecka E, Burnier MN. Immunohistochemical expression of S100B in choroidal melanomas. Can J Ophthalmol. 1997;32:378-381.
ISI
| PUBMED
22. de Vries TJ, Trancikova D, Ruiter DJ, van Muijen GN. High expression of immunotherapy candidate proteins gp100, MART-1,
tyrosinase and TRP-1 in uveal melanoma. Br J Cancer. 1998;78:1156-1161.
ISI
| PUBMED
23. Sharara NA, Sippy BD, O'Reilly FM, et al. Microphthalmia protein: new diagnostic tool in the diagnosis and classification
of cutaneous, uveal and central nervous system melanocytic neoplasms [abstract]. Invest Ophthalmol Vis Sci. 2001;42(suppl):S-217.
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