Fine-Needle Aspiration Biopsy of Malignant Melanoma: A Cytologic and lmmunocytochemical Analysis Thomas J. Simmons, M.D., and Sue Ellen Martin, M.D.,
The immunoreactivity of alcohol-fixed cell blocks from 15 f n e needle aspiration (FNA) specimens of malignant melanoma was investigated using monoclonal antibodies to keratin and vimentin intermediate filaments, melanoma cytoplasmic antigen (HMB45), and 9100, as well as polyclonal antibodies to S-100. The results were compared with the immunoprofiles obtained using formalin-fxed surgical specimens from I 0 of the same patients, In all cases, immunostaining for keratin was negative and immunostaining for vimentin was positive. Immunostaining for HMB-45 was positive in 13/15 aspirates and in 9/10 surgical specimens. Immunostainingfor 4100 protein showed the greatest variability in staining, with 5/15 fine needle aspiration biopsies and 9/10 surgical specimens being positive using the polyclonal antibody and only 1/15 FNA specimens and 7/10 surgical specimens being positive using the monoclonal S-I00 reagent. Our findings indicate that immunocytochemical studies can be very useful as an adjunct in the FNA diagnosis of melanoma. Also included in our series is an unusual variant of malignant melanoma, the so-called signet ring melanoma. Given the location of the anal verge, the use of immunocytochemical markers was essential in establishing the correct diagnosis in this case. While S-I00 protein is of limited value as a marker of melanoma in alcohol-fixed FNA specimens, a definitive diagnosis of malignant melanoma can be made using a panel of antibodies including keratin, vimentin, and HMB-45. Diagn Cytopathol 1991; 7:380-386. Key Words: Cytodiagnosis; HMB-45; Immunoperoxidase; S- 100 protein; Tumor markers
Malignant melanoma is a common neoplasm with a propensity for widespread metastasis. Fine-needle aspiration (FNA) cytology has been demonstrated to be useful in documenting recurrent or metastatic malignant mela's2
Received June 30, 1988. Accepted June 10, 1989. From the Department of Pathology, University of Southern California School of Medicine, Los Angeles, CA. Address reprint requests to Sue Ellen Martin, M.D., Ph.D., Department of Pathology, USC School of Medicine, HMR 304, 2011 Zonal Ave, Los Angeles, CA 90033.
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noma in patients with a known primary. 3-6 In amelanotic lesions and in cases without a previous history of melanoma, however, the cytologic diagnosis of melanoma can be very difficult, because amelanotic malignant melanoma can mimic a variety of other neoplasms. Because immunohistochemistry has proved to be a valuable adjunct in the histologic diagnosis of malignant melanoma, we investigated the usefulness of the application of immunocytochemistry to the FNA diagnosis of malignant melanoma. Immunohistochemical studies on formalinfixed, paraffin-embedded tissue have shown that malignant melanoma is characteristically negative for keratin intermediate filaments, 7,8 and positive for vimentin intermediate filaments, 7,8 S- 100 protein, 7-10 and melanoma cytoplasmic antigen (HMB-45). 1 1 , 1 2 In this paper, we report the immunostaining characteristics of alcohol-fixed cell block material from 15 FNA specimens of metastatic malignant melanoma. In 10/15 cases, formalin-fixed, paraffin-embedded surgical tissue was available for comparison of the immunostaining profiles.
Materials and Methods Case Selection Smears and cell blocks from 22 cases of malignant melanoma diagnosed by FNA cytology between the years 1983 and 1987 were retrieved from the files of the LAC-USC Medical Center and the USC Cytology Laboratory. Only those cases were included in this study in which the patient had a confirmed diagnosis of malignant melanoma and there was sufficient material for immunocytochemical evaluation. Criteria for establishing a diagnosis of melanoma included a documented history of melanoma, demonstrable intracellular melanin, or premelanosomes confirmed by electron microscopy. A total of 15 cases were included in this study, 10 of which also had surgical pathology specimens available for immunostaining (see Table I, below). In cases 1, 2, 4, and 15, the surgical 0 1991 WILEY-LISS, INC.
FNA BIOPSY OF MELANOMA
specimen immunostained was from the primary site. In the remaining cases, the surgical specimen immunostained was from the metastasis.
was used at a dilution of 1:100. CLA was used at a dilution of 1:25. The antikeratin reagent used was a cocktail of three mouse monoclonal antibodies and was prepared by mixing cytokeratin (prediluted, Biogenex Laboratories) and AE- 1/AE-3 (1: 100 dilution, Hybritech, San Diego, CA).
Processing of FNA Specimens All FNA specimens were obtained using a 2 1- or 22-gauge needle and a 10-cc syringe. All specimens were processed using the standard procedures employed by the Medical Center. Alcohol-fixed smears were prepared and stained by rapid H&E and Papanicolaou stains, and an air-dried Diff-Quik stained smear was generally prepared. The needle was washed in 80% ethyl alcohol to collect material for cell block preparations. After fixation and centrifugation, the specimens were placed in agar and routinely processed for paraffin embedding. In addition to H&E stains, the cell block sections of the aspirates were immunostained using a battery of commercially available polyclonal and monoclonal antibodies.
Antibodies Polyclonal rabbit antisera to bovine S- 100 protein (PS100, Dako Corporation, Santa Barbara, CA) recognizes both the a and /3 subunits of S-100 protein and was used at a dilution of 1:200. Mouse monoclonal antibodies used included antibodies to S-100 (MoAb 079, Chemicon International, El Segundo, CA), melanoma cytoplasmic antigen (HMB-45, Enzo Biochem, Inc., New York, NY), vimentin intermediate filaments (Biogenics Laboratories, Dublin, CA), keratin intermediate filaments, and common leukocyte antigen (CLA, Dako). MoAb 079 reacts with both the a and /3 subunits of S-100 protein and was used at a dilution of 1:300. HMB-45 reacts with a cytoplasmic antigen that is present in most melanomas but absent in carcinomas, sarcomas, and lymphomas. l 1 This antibody Table I.
Immunocytochemistry Sections were immunostained using the avidin-biotin complex immunoperoxidase (ABC) procedure. l 3 After deparaffinization and hydration of cell block sections, endogenous peroxidase activity was blocked by incubation in 0.6% hydrogen peroxide for 30 min. Slides stained for keratin were placed in 0.05% protease (type XXIII, Sigma Chemical Company, St. Louis, MO) and incubated at 37°C for 5 min. All slides were then incubated sequentially with 5% normal horse serum, primary antibody, biotinylated secondary antibody (Vector Laboratories, Burlingame, CA), and the ABC reagent (Vector Laboratories). All incubations were performed at room temperature for 30 min. Either diaminobenzidine (DAB) or 3-amino-9-methylcarbazole in N,N-dimethylformamide (AEC) was used as a chromogen and the slides counterstained with hematoxylin. Positive and negative control slides were prepared, and all slides were immunostained concurrently for each antibody to control for variation in staining technique.
Results The clinical features of all the cases are summarized in Table I. Twelve of the 15 patients aspirated had a documented history of malignant melanoma. In the remaining three cases, the diagnosis was confirmed by the presence of melanin pigment identified on the H&E sections and
Clinical Features of Patients Studied With Metastatic Melanoma
Case
Age/sex
FNA site
Site of primary
Surgical pathology'
1 2 3 4 5 6 7 8 9
61/F 53/F 72/F 63/F 38/F 40/F 5 1/F 26/F 20/M 32/F 68/M 44/M 72/M 70/M 85/M
Inguinal lymph node (LN) Shoulder nodule Paraspinous mass Inguinal LN Inguinal LN Iliac LN Extradural mass Neck LN Axillary LN Ovarian mass Axillary LN Hepatic nodules Inguinal LN Hepatic nodules Inguinal LN
Anal verge Scalp Unknownb Foot Foot Leg Back Shoulder Unknown' Leg Arm Unknownb Leg Foot Anal verge
Yes Yes No Yes Yes Yes No Yes Yes Yes No No Yes No Yes
10 11
12 13 14 15
aSurgical specimen available for immunohistochemical evaluation. In cases 1, 2, 4, and 15, the surgical specimen was from the primary site. In the remaining cases, the surgical specimen was from the metastasis. bAbundant melanin pigment present in FNA. 'Electron microscopy of biopsy showed premelanosomes. Diagnostic Cytopathology, Vol 7, No 4
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confirmed by special (Fontana) stains (cases 3 and 12) or premelanosomes (case 9) within tumor cells. In 9/15 cases the metastatic tumor was aspirated from a lymph node (LN); in 3 cases, from a soft tissue mass; in 2 cases, from liver; and in 1 case, from ovary. Surgical specimens were available for comparison in 10/15 cases. The cytologic features of the FNA specimens are compared in Table 11. The tumor cell population in the aspirates varied from primarily cohesive groups of cells (5 cases, see Fig. l a ) to predominantly single cells (4 cases, see Fig. lb), with an even mixture of clusters and individual cells in 6 cases. In 12 cases, the cells had an epithelial appearance. In case 15, many of the cells assumed a signet ring appearance, mimicking the cytologic appearance of an adenocarcinoma (see Fig. C-1A and C-1B). In three cases, a combined epithelioid and spindle-cell pattern or a predominantly spindle-cell pattern was seen. Melanin pigment was identified in six cases, with abundant intracellular and extracellular melanin pigment in three cases (See Fig. 2a) and focal scanty cytoplasmic melanin pigment in three cases. Nucleoli were present in 14/15 cases and prominent in 6 cases. Multinucleated tumor cells and intranuclear cytoplasmic inclusions were observed in 6/ 15 cases (see Fig. 2b). Immunocytochemical analysis of all FNA specimens and ten surgical cases is shown in Table 111. As expected, all of the aspirates and surgical specimens were negative for keratin and CLA and positive for vimentin. HMB45 was positive in 13/15 aspirates and in 9/10 surgical specimens. Immunostains for polyclonal anti-S- 100 were positive in 9/10 surgical specimens but only 5/15 FNA specimens. Immunostaining with monoclonal anti-S- 100 showed positive staining in only 1/15 alcohol-fixed FNA specimens, while 7/10 of the corresponding surgical specimens were positive. A typical immunostaining pattern for
MS-100 and HMB-45 is illustrated in Figure C-2B and C-2C. Interestingly, in case 2, while the FNA of the metastatic lesion was negative for HMB-45 and S-100, the primary tumor was positive for both of these antigens. While this may indicate the existence of a second primary, i.e., a sarcoma, the morphologic appearance of the cells in the tumor lesions is identical, and the differential staining most likely represents antigenic variation between the primary and metastatic malignant melanoma.
Discussion Fine-needle cytodiagnosis of recurrent malignant melanoma has been used successfully for a number of years. 3-6 In a patient with a previous history of cutaneous melanoma, recurrent or metastatic melanoma in lymph nodes, subcutaneous nodules, and other sites can be diagnosed by FNA with high accuracy. 3,5 Patients presenting with metastatic disease without a previous history of primary melanoma may be more of a diagnostic challenge. If gross extracellular or focal intracytoplasmic melanin granules are seen in smears, the diagnosis is easily made. Unfortunately, a significant number of melanomas are amelanotic or contain only focal melanotic areas. Melanin pigment was identified in 43% of our cases, a finding comparable to that of Hadju and Savino, who reported melanin pigment to be present in 50% of the FNA specimens of malignant melanoma they studied. In the absence of melanin pigment, the use of cytologic features such as prominent nucleoli, intranuclear cytoplasmic inclusions, cellular discohesion, and the presence of multinucleated cells can suggest the diagnosis of melanoma. 3,5*6 None of these features alone, however, is specific for melanoma. Immunohistochemical studies have been useful in the histologic diagnosis of malignant melanoma. One of the most widely used immunohistochemical markers of ma-
Table 11. Cytologic Features of Melanoma in FNA Case
Cohesion"
Pattern
1 2
Single cells Cohesive Mixed Cohesive Mixed Single cells Mixed Single cells Single cells Cohesive Cohesive Cohesive Mixed Mixed Mixed
Epithelial Spindle EpitheliaVspindle Epithelial Epithelial Epithelial Epithelial Epithelial Epithelial Epithelial Epithelial Epithelial/spindle Epithelial Epithelial Epithelial
3 4 5
6 7 8 9 10 11 12
13 14 15
Pigment
C -
EC -
EC EC -
C
-
C -
Nucleoli
+ + ++ + ++ + + ++ ++ + ++ + ++ +
Multinucleated cells
Nuclear inclusions
-
+ + + + +
-
-
+,
Diagnostic Cytopathology, Vol 7, No 4
-
-
-
-
-
-
-
-
-
-
-
-
-
+
+
C, focal cytoplasmic melanin pigment; EC, abundant cytoplasmic and extracellular melanin; +, present; + prominent; -, absent. aSpecimens could be classified as predominantly single cells, predominantly cohesive clusters of cells, or a combination of both (mixed).
382
+ + + + + -
Fig. C-1A
Fig. C-1B
Fig. C-2A
Fig. C-2B
Fig. C-2C Figs. C-1-C-6 Fig. C-1. Smears of FNA of inguinal mass in patient (case 15) with primary rectal melanoma. (A) Cells demonstrate nucleoli and pale cytoplasm without pigmentation. Note “cell-within-cell” arrangement (Papanicolaou stain, original magnification X 250). (B) Cell demonstrating vacuolated cytoplasm containing condensed material resembling inspissated mucin (Papanicolaou stain, original magnification X 250). Fig. C-2. Sections of alcohol-fixed cell block of same aspirate. (A) Many cells show vacuolated cytoplasm and resemble signet ring cells (H&E, original magnification X 100). (B) Immunostains for S-100 protein using monoclonal antibody MoAb 079 shows no positive staining (original magnification X 100). (C) Immunostaining for melanoma cytoplasmic antigen using monoclonal antibody HMB-45 shows strong positive specific granular staining of cytoplasm of majority of cells (original magnification, x 100).
SIMMONS AND MARTIN
Fig. 1. (a) FNA specimen of malignant melanoma showing cohesive cluster of malignant cells (H&E of cell block section, X 200). (b) FNA specimen of malignant melanoma showing predominantly single cells (H&E of cell block section, X400)
lignant melanoma is S-100 protein. While S-100 protein is not specific for melanoma and is, in fact, present in many normal tissues as well as in a variety of neoplasms, 9,10*14 most series cite close to 100% immunoreactivity to S-100 protein in malignant melanoma using formalin-fixed, paraffin-embedded tissues and polyclonal anti-S- 100 antisera. 9-11,15 Monoclonal antibodies to S-100 protein have shown a lower incidence of positive staining. 16-18 In a recent study of 40 mucosal, cutaneous, and metastatic malignant melanomas, we found that 85% stained positively for polyclonal S-100 and 70% stained positively for the monoclonal anti-S- 100 reagent. l 8 In the present study, both monoclonal and polyclonal anti-S- 100 reagents reacted with a substantially smaller percentage of FNA biopsies of malignant melanoma than of surgical specimens from the same patients. Only 1/15 aspirates (7%) stained with monoclonal S-100 as compared with 7/10 (70%) surgical specimens from these patients. Only 5/15 (33%) aspirates were positive with polyclonal S-100 compared with 9/10 (90%) surgical specimens. While several factors could have contributed to this discrepancy, we believe that alcohol fixation of the FNA specimens is the primary etiologic factor. It has been 384
Diagnostic Cytopathology, Vol % No 4
noted that S-100, an acidic protein soluble in saturated ammonium sulfate, loses immunoreactivity secondary to solubility in alcohol and acetone. l 9 The small size of the tissue fragments in FNA specimens is likely to allow rapid penetration by the alcohol and may facilitate loss of S-100 protein from the cells. While we have not evaluated immunostaining of alcohol-fixed smears of melanoma, such staining may also reflect loss of this antigen. In this regard, it may be advisable to utilize formalin fixation in cases in which there is a suspected diagnosis of malignant melanoma based on either clinical history or preliminary, onsite evaluation of the aspiration. In practice, however, we have found it difficult to implement usage of a panel of different fixatives as opposed to a single fixative for cell block preparations. In contrast to our findings with immunostaining of FNA of melanoma for S-100 protein, immunostaining of these FNA for keratin, vimentin, and HMB-45 did not show significant discrepancies between the aspirates and the surgical specimens. None of the aspirates or surgical specimens stained with keratin, whereas all of the specimens stained with vimentin. The fact that malignant melanoma does not stain with keratin has been noted be-
FNA BIOPSY OF MELANOMA
Fig. 2. (a) F N A of malignant melanoma showing extensive melanin pigment (H&E section of cell block, X 1,000). (b) F N A specimen of malignant melanoma showing intranuclear inclusions, prominent nucleoli, and multinucleated cells (H&E section of cell block, X 1,000).
fore73s,10,11 and can be very useful in distinguishing poorly differentiated carcinoma from melanoma. Positive staining of melanoma for vimentin intermediate filaments is also well documented.8 HMB-45 is a relatively recent marker of malignant melanoma developed by Gown et al. We have found this antibody to be a sensitive and useful marker for melanoma, staining approximately 90% of all melanomas. l 8 The value of immunohistochemistry is well illustrated in case 15. This most unusual variant of malignant melanoma, called signet ring cell melanoma, 2o is at best a diagnostic dilemma, especially given the primary location in this case of the anal verge. Strong positive staining for HMB-45, globular cytoplasmic staining for vimentin, and negative staining for keratin support a diagnosis of melanoma as opposed to adenocarcinoma. This study has demonstrated that immunocytochemistry can be very helpful in the accurate and definitive cytologic diagnosis of malignant melanoma in FNA specimens. The use of a single immunostain, however, is not prudent. Rather, a panel of antibodies, including keratin, vimentin, CLA, and HMB-45, should be used to separate melanoma from other malignancies when melanin pig-
ment is not present. We have found that s-100 is not a useful marker of malignant melanoma in alcohol-fixed material.
Acknowledgment The authors thank Ms. Sandra Teasley for her technical assistance in performing the immunostains and Ms. Gina Madrid for assistance in preparation of the manuscript.
References 1. Kopf AN, Bart RS, Rodriguez-Sains RS. Malignant melanoma: A review. J Dermatol Surg Oncol 1977; 3:43. 2. Das Gupta T, Brasfield R. Metastatic melanoma: A clinicopathologic study. Cancer 1964; 17:1323-9. 3. Perry PD, Seigler HF, Johnston WW. Diagnosis of metastatic malignant melanoma by fine needle aspiration biopsy: A clinical and pathologic correlation of 298 cases. JNCI 1986; 77:1013-9. 4. Kline TS, Kannan V. Aspiration biopsy and melanoma. Am J Clin Pathol 1982; 77597-601. 5. Hafstrom L, Nugander A, Jonsson PE, Lindberg LG. Fine needle aspiration cytodiagnosis of recurrent malignant melanoma. J Surg Oncol 1980; 15:229-34. 6. Hadju SI, Savino A. Cytologic diagnosis of malignant melanoma. Acta Cytol (Baltimore) 1973;17:320-7. 7. Battifora H. Recent progress in the immunohistochemistry of solid tumors. Semin Diagn Pathol 1984; 1:251-71. Diagnostic Cytopathology, Vol 7,No 4
385
SIMMONS AND MARTIN Table 111. Comparison of Immunocytochemical Profiles of FNA and Corresponding Surgical Specimens in Malignant Melanomaa
Cuse
Keratin
1 2 3 4
-/-//NA -/-/-/- /NA -/-
5
6 7 8 9 lo 11 12 13 14 15
-/-/- /NA -/NA -/- /NA -/-
Totals FNA Sureical
0/15 0/10
Vimentin
HMB-45
+ +/+ + + +/+ + + +/NA + +/+ + + +/+ + + +/+ + + +/NA + +/+ +/+ + + +/+ + +/NA +/+ + +/NA + +/+ +
+ +/+ + -/+ + +/NA +/+ + +/+ + + +/+ /NA + +/+ +/++/+ + +/NA ++/NA + +/+ ++/NA + +/+ +
15/15 10/10
13/15 9/10
+/NA
-
MS- I00
PS- I00
-/+ + -/+ - /NA -/+ + -/+ +
Common leu koeyte antigen
-//NA -/-
-//NA
- /NA - /NA
/NA -/NA
-/+ + - /NA +/+
-/-/-/NA -/-/-/-/NA -/-/- /-/NA - /NA -/-/NA -/-
1/15 7/10
5/15 9/10
0/15 0/10
-/-
-/+ /NA -/+ + -/+ + -
+
-/+ -/+
-/+ - /NA -/+
+
-/+ +/+ +/+ +
aResults are expressed as (immunostaining of FNA)/(immunostaining of surgical). NA, surgical specimen not available for immunohistochemical evaluation; HMB-45, monoclonal antibody to melanoma cytoplasmic antigen; MS-100, monoclonal S-100; PS-100, polyclonal S-100; +, weakly positive; + +, strongly positive; -, negative. 8. Taylor CR. Immunomicroscopy: A diagnostic tool for the surgical pathologist. Philadelphia: WB Saunders, 1986:282-6. 9. Kahn HJ, Marks A, Thom H, Baumal R. Role of antibody to S-100 protein in diagnostic pathology. Am J Clin Pathol 1983; 79:341-7. 10. Drier JK, Swanson PE, Cherwitz DL, Wick MR. S-100 protein immunoreactivity in poorly differentiated carcinomas. Arch Pathol Lab Med 1987; 11 1:447-52. 11. Gown AM, Vogen AM, Hoak D, Gough F, McNutt MA. Monoclonal antibodies specific for melanocytic tumors distinguish subpopulation of melanocytes. Am J Pathol 1986; 123:195-203. 12. Walts AE, Said JW, Shintaku P: Cytodiagnosis of malignant melanoma: Immunoperoxidase staining with HMB-45 antibody as an aid to diagnosis. Am J Clin Pathol 1988; 90:77-80. 13. Hsu S, Raine L, Fanger H. Use of avidin-biotin-immunoperoxidase complex (ABC) in immunoperoxidase techniques: A comparison between ABC and unlabelled antibody (PAP) procedures. J Histochem Cytochem 1981; 29577-80. 14. Nakajima T, Watanabe S, Sato Y, Kameja T, Hirota T, Shimasato Y. An immunoperoxidase study of SlOO protein distribution in normal and neoplastic tissues. Am J Surg Pathol 1982; 6:715-27.
386
Diagnostic Cytopathology, Vol 7, No 4
15. Kindblom LG, Lodding P, Rosengren L, Baudier J, Haglid K. S-100
16.
17.
18.
19. 20.
protein in melanocytic tumors. Acta Pathol Microbiol Immunol Scand 1984; 92:219-30. Gatter KC, Ralfiiaen E, Skinner J, Brown D, Heryet A, Pulford KAF, Hor-Jensen K , Mason DV. An immunocytochemical study of malignant melanoma and its differential diagnosis from other malignant tumours. J Clin Pathol 1985; 38:1353-7. Loeffel SC, Gillespie GY, Mirmiran SA, Miller EW, Golden P, Askin FB, Seigal GP. Cellular immunolocalization of S-100 protein within fixed tissue sections by monoclonal antibodies. Arch Pathol Lab Med 1985; 109:117-22. Fitzgibbons PL, Chaurushiya P, Nichols PW, Chandrasoma PT, Martin SE. Primary mucosal malignant melanoma: An immunohistochemical study of 12 cases with comparison to cutaneous and metastatic melanomas. Hum Pathol 1989; 20:269-72. Takahashi K. S-100 immunoreactivity in normal human peripheral blood lymphocytes. Am J Clin Pathol 1988; 89:453. Sheibani K, Battifora H. Signet-ring cell melanoma: A rare morphologic variant of malignant melanoma. Am J Surg Pathol 1988; 12: 28-34.
The immunoreactivity of alcohol-fixed cell blocks from 15 f n e needle aspiration (FNA) specimens of malignant melanoma was investigated using monoclonal antibodies to keratin and vimentin intermediate filaments, melanoma cytoplasmic antigen (HMB45), and 9100, as well as polyclonal antibodies to S-100. The results were compared with the immunoprofiles obtained using formalin-fxed surgical specimens from I 0 of the same patients, In all cases, immunostaining for keratin was negative and immunostaining for vimentin was positive. Immunostaining for HMB-45 was positive in 13/15 aspirates and in 9/10 surgical specimens. Immunostainingfor 4100 protein showed the greatest variability in staining, with 5/15 fine needle aspiration biopsies and 9/10 surgical specimens being positive using the polyclonal antibody and only 1/15 FNA specimens and 7/10 surgical specimens being positive using the monoclonal S-I00 reagent. Our findings indicate that immunocytochemical studies can be very useful as an adjunct in the FNA diagnosis of melanoma. Also included in our series is an unusual variant of malignant melanoma, the so-called signet ring melanoma. Given the location of the anal verge, the use of immunocytochemical markers was essential in establishing the correct diagnosis in this case. While S-I00 protein is of limited value as a marker of melanoma in alcohol-fixed FNA specimens, a definitive diagnosis of malignant melanoma can be made using a panel of antibodies including keratin, vimentin, and HMB-45. Diagn Cytopathol 1991; 7:380-386. Key Words: Cytodiagnosis; HMB-45; Immunoperoxidase; S- 100 protein; Tumor markers
Malignant melanoma is a common neoplasm with a propensity for widespread metastasis. Fine-needle aspiration (FNA) cytology has been demonstrated to be useful in documenting recurrent or metastatic malignant mela's2
Received June 30, 1988. Accepted June 10, 1989. From the Department of Pathology, University of Southern California School of Medicine, Los Angeles, CA. Address reprint requests to Sue Ellen Martin, M.D., Ph.D., Department of Pathology, USC School of Medicine, HMR 304, 2011 Zonal Ave, Los Angeles, CA 90033.
380
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PhD.
noma in patients with a known primary. 3-6 In amelanotic lesions and in cases without a previous history of melanoma, however, the cytologic diagnosis of melanoma can be very difficult, because amelanotic malignant melanoma can mimic a variety of other neoplasms. Because immunohistochemistry has proved to be a valuable adjunct in the histologic diagnosis of malignant melanoma, we investigated the usefulness of the application of immunocytochemistry to the FNA diagnosis of malignant melanoma. Immunohistochemical studies on formalinfixed, paraffin-embedded tissue have shown that malignant melanoma is characteristically negative for keratin intermediate filaments, 7,8 and positive for vimentin intermediate filaments, 7,8 S- 100 protein, 7-10 and melanoma cytoplasmic antigen (HMB-45). 1 1 , 1 2 In this paper, we report the immunostaining characteristics of alcohol-fixed cell block material from 15 FNA specimens of metastatic malignant melanoma. In 10/15 cases, formalin-fixed, paraffin-embedded surgical tissue was available for comparison of the immunostaining profiles.
Materials and Methods Case Selection Smears and cell blocks from 22 cases of malignant melanoma diagnosed by FNA cytology between the years 1983 and 1987 were retrieved from the files of the LAC-USC Medical Center and the USC Cytology Laboratory. Only those cases were included in this study in which the patient had a confirmed diagnosis of malignant melanoma and there was sufficient material for immunocytochemical evaluation. Criteria for establishing a diagnosis of melanoma included a documented history of melanoma, demonstrable intracellular melanin, or premelanosomes confirmed by electron microscopy. A total of 15 cases were included in this study, 10 of which also had surgical pathology specimens available for immunostaining (see Table I, below). In cases 1, 2, 4, and 15, the surgical 0 1991 WILEY-LISS, INC.
FNA BIOPSY OF MELANOMA
specimen immunostained was from the primary site. In the remaining cases, the surgical specimen immunostained was from the metastasis.
was used at a dilution of 1:100. CLA was used at a dilution of 1:25. The antikeratin reagent used was a cocktail of three mouse monoclonal antibodies and was prepared by mixing cytokeratin (prediluted, Biogenex Laboratories) and AE- 1/AE-3 (1: 100 dilution, Hybritech, San Diego, CA).
Processing of FNA Specimens All FNA specimens were obtained using a 2 1- or 22-gauge needle and a 10-cc syringe. All specimens were processed using the standard procedures employed by the Medical Center. Alcohol-fixed smears were prepared and stained by rapid H&E and Papanicolaou stains, and an air-dried Diff-Quik stained smear was generally prepared. The needle was washed in 80% ethyl alcohol to collect material for cell block preparations. After fixation and centrifugation, the specimens were placed in agar and routinely processed for paraffin embedding. In addition to H&E stains, the cell block sections of the aspirates were immunostained using a battery of commercially available polyclonal and monoclonal antibodies.
Antibodies Polyclonal rabbit antisera to bovine S- 100 protein (PS100, Dako Corporation, Santa Barbara, CA) recognizes both the a and /3 subunits of S-100 protein and was used at a dilution of 1:200. Mouse monoclonal antibodies used included antibodies to S-100 (MoAb 079, Chemicon International, El Segundo, CA), melanoma cytoplasmic antigen (HMB-45, Enzo Biochem, Inc., New York, NY), vimentin intermediate filaments (Biogenics Laboratories, Dublin, CA), keratin intermediate filaments, and common leukocyte antigen (CLA, Dako). MoAb 079 reacts with both the a and /3 subunits of S-100 protein and was used at a dilution of 1:300. HMB-45 reacts with a cytoplasmic antigen that is present in most melanomas but absent in carcinomas, sarcomas, and lymphomas. l 1 This antibody Table I.
Immunocytochemistry Sections were immunostained using the avidin-biotin complex immunoperoxidase (ABC) procedure. l 3 After deparaffinization and hydration of cell block sections, endogenous peroxidase activity was blocked by incubation in 0.6% hydrogen peroxide for 30 min. Slides stained for keratin were placed in 0.05% protease (type XXIII, Sigma Chemical Company, St. Louis, MO) and incubated at 37°C for 5 min. All slides were then incubated sequentially with 5% normal horse serum, primary antibody, biotinylated secondary antibody (Vector Laboratories, Burlingame, CA), and the ABC reagent (Vector Laboratories). All incubations were performed at room temperature for 30 min. Either diaminobenzidine (DAB) or 3-amino-9-methylcarbazole in N,N-dimethylformamide (AEC) was used as a chromogen and the slides counterstained with hematoxylin. Positive and negative control slides were prepared, and all slides were immunostained concurrently for each antibody to control for variation in staining technique.
Results The clinical features of all the cases are summarized in Table I. Twelve of the 15 patients aspirated had a documented history of malignant melanoma. In the remaining three cases, the diagnosis was confirmed by the presence of melanin pigment identified on the H&E sections and
Clinical Features of Patients Studied With Metastatic Melanoma
Case
Age/sex
FNA site
Site of primary
Surgical pathology'
1 2 3 4 5 6 7 8 9
61/F 53/F 72/F 63/F 38/F 40/F 5 1/F 26/F 20/M 32/F 68/M 44/M 72/M 70/M 85/M
Inguinal lymph node (LN) Shoulder nodule Paraspinous mass Inguinal LN Inguinal LN Iliac LN Extradural mass Neck LN Axillary LN Ovarian mass Axillary LN Hepatic nodules Inguinal LN Hepatic nodules Inguinal LN
Anal verge Scalp Unknownb Foot Foot Leg Back Shoulder Unknown' Leg Arm Unknownb Leg Foot Anal verge
Yes Yes No Yes Yes Yes No Yes Yes Yes No No Yes No Yes
10 11
12 13 14 15
aSurgical specimen available for immunohistochemical evaluation. In cases 1, 2, 4, and 15, the surgical specimen was from the primary site. In the remaining cases, the surgical specimen was from the metastasis. bAbundant melanin pigment present in FNA. 'Electron microscopy of biopsy showed premelanosomes. Diagnostic Cytopathology, Vol 7, No 4
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SIMMONS AND MARTIN
confirmed by special (Fontana) stains (cases 3 and 12) or premelanosomes (case 9) within tumor cells. In 9/15 cases the metastatic tumor was aspirated from a lymph node (LN); in 3 cases, from a soft tissue mass; in 2 cases, from liver; and in 1 case, from ovary. Surgical specimens were available for comparison in 10/15 cases. The cytologic features of the FNA specimens are compared in Table 11. The tumor cell population in the aspirates varied from primarily cohesive groups of cells (5 cases, see Fig. l a ) to predominantly single cells (4 cases, see Fig. lb), with an even mixture of clusters and individual cells in 6 cases. In 12 cases, the cells had an epithelial appearance. In case 15, many of the cells assumed a signet ring appearance, mimicking the cytologic appearance of an adenocarcinoma (see Fig. C-1A and C-1B). In three cases, a combined epithelioid and spindle-cell pattern or a predominantly spindle-cell pattern was seen. Melanin pigment was identified in six cases, with abundant intracellular and extracellular melanin pigment in three cases (See Fig. 2a) and focal scanty cytoplasmic melanin pigment in three cases. Nucleoli were present in 14/15 cases and prominent in 6 cases. Multinucleated tumor cells and intranuclear cytoplasmic inclusions were observed in 6/ 15 cases (see Fig. 2b). Immunocytochemical analysis of all FNA specimens and ten surgical cases is shown in Table 111. As expected, all of the aspirates and surgical specimens were negative for keratin and CLA and positive for vimentin. HMB45 was positive in 13/15 aspirates and in 9/10 surgical specimens. Immunostains for polyclonal anti-S- 100 were positive in 9/10 surgical specimens but only 5/15 FNA specimens. Immunostaining with monoclonal anti-S- 100 showed positive staining in only 1/15 alcohol-fixed FNA specimens, while 7/10 of the corresponding surgical specimens were positive. A typical immunostaining pattern for
MS-100 and HMB-45 is illustrated in Figure C-2B and C-2C. Interestingly, in case 2, while the FNA of the metastatic lesion was negative for HMB-45 and S-100, the primary tumor was positive for both of these antigens. While this may indicate the existence of a second primary, i.e., a sarcoma, the morphologic appearance of the cells in the tumor lesions is identical, and the differential staining most likely represents antigenic variation between the primary and metastatic malignant melanoma.
Discussion Fine-needle cytodiagnosis of recurrent malignant melanoma has been used successfully for a number of years. 3-6 In a patient with a previous history of cutaneous melanoma, recurrent or metastatic melanoma in lymph nodes, subcutaneous nodules, and other sites can be diagnosed by FNA with high accuracy. 3,5 Patients presenting with metastatic disease without a previous history of primary melanoma may be more of a diagnostic challenge. If gross extracellular or focal intracytoplasmic melanin granules are seen in smears, the diagnosis is easily made. Unfortunately, a significant number of melanomas are amelanotic or contain only focal melanotic areas. Melanin pigment was identified in 43% of our cases, a finding comparable to that of Hadju and Savino, who reported melanin pigment to be present in 50% of the FNA specimens of malignant melanoma they studied. In the absence of melanin pigment, the use of cytologic features such as prominent nucleoli, intranuclear cytoplasmic inclusions, cellular discohesion, and the presence of multinucleated cells can suggest the diagnosis of melanoma. 3,5*6 None of these features alone, however, is specific for melanoma. Immunohistochemical studies have been useful in the histologic diagnosis of malignant melanoma. One of the most widely used immunohistochemical markers of ma-
Table 11. Cytologic Features of Melanoma in FNA Case
Cohesion"
Pattern
1 2
Single cells Cohesive Mixed Cohesive Mixed Single cells Mixed Single cells Single cells Cohesive Cohesive Cohesive Mixed Mixed Mixed
Epithelial Spindle EpitheliaVspindle Epithelial Epithelial Epithelial Epithelial Epithelial Epithelial Epithelial Epithelial Epithelial/spindle Epithelial Epithelial Epithelial
3 4 5
6 7 8 9 10 11 12
13 14 15
Pigment
C -
EC -
EC EC -
C
-
C -
Nucleoli
+ + ++ + ++ + + ++ ++ + ++ + ++ +
Multinucleated cells
Nuclear inclusions
-
+ + + + +
-
-
+,
Diagnostic Cytopathology, Vol 7, No 4
-
-
-
-
-
-
-
-
-
-
-
-
-
+
+
C, focal cytoplasmic melanin pigment; EC, abundant cytoplasmic and extracellular melanin; +, present; + prominent; -, absent. aSpecimens could be classified as predominantly single cells, predominantly cohesive clusters of cells, or a combination of both (mixed).
382
+ + + + + -
Fig. C-1A
Fig. C-1B
Fig. C-2A
Fig. C-2B
Fig. C-2C Figs. C-1-C-6 Fig. C-1. Smears of FNA of inguinal mass in patient (case 15) with primary rectal melanoma. (A) Cells demonstrate nucleoli and pale cytoplasm without pigmentation. Note “cell-within-cell” arrangement (Papanicolaou stain, original magnification X 250). (B) Cell demonstrating vacuolated cytoplasm containing condensed material resembling inspissated mucin (Papanicolaou stain, original magnification X 250). Fig. C-2. Sections of alcohol-fixed cell block of same aspirate. (A) Many cells show vacuolated cytoplasm and resemble signet ring cells (H&E, original magnification X 100). (B) Immunostains for S-100 protein using monoclonal antibody MoAb 079 shows no positive staining (original magnification X 100). (C) Immunostaining for melanoma cytoplasmic antigen using monoclonal antibody HMB-45 shows strong positive specific granular staining of cytoplasm of majority of cells (original magnification, x 100).
SIMMONS AND MARTIN
Fig. 1. (a) FNA specimen of malignant melanoma showing cohesive cluster of malignant cells (H&E of cell block section, X 200). (b) FNA specimen of malignant melanoma showing predominantly single cells (H&E of cell block section, X400)
lignant melanoma is S-100 protein. While S-100 protein is not specific for melanoma and is, in fact, present in many normal tissues as well as in a variety of neoplasms, 9,10*14 most series cite close to 100% immunoreactivity to S-100 protein in malignant melanoma using formalin-fixed, paraffin-embedded tissues and polyclonal anti-S- 100 antisera. 9-11,15 Monoclonal antibodies to S-100 protein have shown a lower incidence of positive staining. 16-18 In a recent study of 40 mucosal, cutaneous, and metastatic malignant melanomas, we found that 85% stained positively for polyclonal S-100 and 70% stained positively for the monoclonal anti-S- 100 reagent. l 8 In the present study, both monoclonal and polyclonal anti-S- 100 reagents reacted with a substantially smaller percentage of FNA biopsies of malignant melanoma than of surgical specimens from the same patients. Only 1/15 aspirates (7%) stained with monoclonal S-100 as compared with 7/10 (70%) surgical specimens from these patients. Only 5/15 (33%) aspirates were positive with polyclonal S-100 compared with 9/10 (90%) surgical specimens. While several factors could have contributed to this discrepancy, we believe that alcohol fixation of the FNA specimens is the primary etiologic factor. It has been 384
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noted that S-100, an acidic protein soluble in saturated ammonium sulfate, loses immunoreactivity secondary to solubility in alcohol and acetone. l 9 The small size of the tissue fragments in FNA specimens is likely to allow rapid penetration by the alcohol and may facilitate loss of S-100 protein from the cells. While we have not evaluated immunostaining of alcohol-fixed smears of melanoma, such staining may also reflect loss of this antigen. In this regard, it may be advisable to utilize formalin fixation in cases in which there is a suspected diagnosis of malignant melanoma based on either clinical history or preliminary, onsite evaluation of the aspiration. In practice, however, we have found it difficult to implement usage of a panel of different fixatives as opposed to a single fixative for cell block preparations. In contrast to our findings with immunostaining of FNA of melanoma for S-100 protein, immunostaining of these FNA for keratin, vimentin, and HMB-45 did not show significant discrepancies between the aspirates and the surgical specimens. None of the aspirates or surgical specimens stained with keratin, whereas all of the specimens stained with vimentin. The fact that malignant melanoma does not stain with keratin has been noted be-
FNA BIOPSY OF MELANOMA
Fig. 2. (a) F N A of malignant melanoma showing extensive melanin pigment (H&E section of cell block, X 1,000). (b) F N A specimen of malignant melanoma showing intranuclear inclusions, prominent nucleoli, and multinucleated cells (H&E section of cell block, X 1,000).
fore73s,10,11 and can be very useful in distinguishing poorly differentiated carcinoma from melanoma. Positive staining of melanoma for vimentin intermediate filaments is also well documented.8 HMB-45 is a relatively recent marker of malignant melanoma developed by Gown et al. We have found this antibody to be a sensitive and useful marker for melanoma, staining approximately 90% of all melanomas. l 8 The value of immunohistochemistry is well illustrated in case 15. This most unusual variant of malignant melanoma, called signet ring cell melanoma, 2o is at best a diagnostic dilemma, especially given the primary location in this case of the anal verge. Strong positive staining for HMB-45, globular cytoplasmic staining for vimentin, and negative staining for keratin support a diagnosis of melanoma as opposed to adenocarcinoma. This study has demonstrated that immunocytochemistry can be very helpful in the accurate and definitive cytologic diagnosis of malignant melanoma in FNA specimens. The use of a single immunostain, however, is not prudent. Rather, a panel of antibodies, including keratin, vimentin, CLA, and HMB-45, should be used to separate melanoma from other malignancies when melanin pig-
ment is not present. We have found that s-100 is not a useful marker of malignant melanoma in alcohol-fixed material.
Acknowledgment The authors thank Ms. Sandra Teasley for her technical assistance in performing the immunostains and Ms. Gina Madrid for assistance in preparation of the manuscript.
References 1. Kopf AN, Bart RS, Rodriguez-Sains RS. Malignant melanoma: A review. J Dermatol Surg Oncol 1977; 3:43. 2. Das Gupta T, Brasfield R. Metastatic melanoma: A clinicopathologic study. Cancer 1964; 17:1323-9. 3. Perry PD, Seigler HF, Johnston WW. Diagnosis of metastatic malignant melanoma by fine needle aspiration biopsy: A clinical and pathologic correlation of 298 cases. JNCI 1986; 77:1013-9. 4. Kline TS, Kannan V. Aspiration biopsy and melanoma. Am J Clin Pathol 1982; 77597-601. 5. Hafstrom L, Nugander A, Jonsson PE, Lindberg LG. Fine needle aspiration cytodiagnosis of recurrent malignant melanoma. J Surg Oncol 1980; 15:229-34. 6. Hadju SI, Savino A. Cytologic diagnosis of malignant melanoma. Acta Cytol (Baltimore) 1973;17:320-7. 7. Battifora H. Recent progress in the immunohistochemistry of solid tumors. Semin Diagn Pathol 1984; 1:251-71. Diagnostic Cytopathology, Vol 7,No 4
385
SIMMONS AND MARTIN Table 111. Comparison of Immunocytochemical Profiles of FNA and Corresponding Surgical Specimens in Malignant Melanomaa
Cuse
Keratin
1 2 3 4
-/-//NA -/-/-/- /NA -/-
5
6 7 8 9 lo 11 12 13 14 15
-/-/- /NA -/NA -/- /NA -/-
Totals FNA Sureical
0/15 0/10
Vimentin
HMB-45
+ +/+ + + +/+ + + +/NA + +/+ + + +/+ + + +/+ + + +/NA + +/+ +/+ + + +/+ + +/NA +/+ + +/NA + +/+ +
+ +/+ + -/+ + +/NA +/+ + +/+ + + +/+ /NA + +/+ +/++/+ + +/NA ++/NA + +/+ ++/NA + +/+ +
15/15 10/10
13/15 9/10
+/NA
-
MS- I00
PS- I00
-/+ + -/+ - /NA -/+ + -/+ +
Common leu koeyte antigen
-//NA -/-
-//NA
- /NA - /NA
/NA -/NA
-/+ + - /NA +/+
-/-/-/NA -/-/-/-/NA -/-/- /-/NA - /NA -/-/NA -/-
1/15 7/10
5/15 9/10
0/15 0/10
-/-
-/+ /NA -/+ + -/+ + -
+
-/+ -/+
-/+ - /NA -/+
+
-/+ +/+ +/+ +
aResults are expressed as (immunostaining of FNA)/(immunostaining of surgical). NA, surgical specimen not available for immunohistochemical evaluation; HMB-45, monoclonal antibody to melanoma cytoplasmic antigen; MS-100, monoclonal S-100; PS-100, polyclonal S-100; +, weakly positive; + +, strongly positive; -, negative. 8. Taylor CR. Immunomicroscopy: A diagnostic tool for the surgical pathologist. Philadelphia: WB Saunders, 1986:282-6. 9. Kahn HJ, Marks A, Thom H, Baumal R. Role of antibody to S-100 protein in diagnostic pathology. Am J Clin Pathol 1983; 79:341-7. 10. Drier JK, Swanson PE, Cherwitz DL, Wick MR. S-100 protein immunoreactivity in poorly differentiated carcinomas. Arch Pathol Lab Med 1987; 11 1:447-52. 11. Gown AM, Vogen AM, Hoak D, Gough F, McNutt MA. Monoclonal antibodies specific for melanocytic tumors distinguish subpopulation of melanocytes. Am J Pathol 1986; 123:195-203. 12. Walts AE, Said JW, Shintaku P: Cytodiagnosis of malignant melanoma: Immunoperoxidase staining with HMB-45 antibody as an aid to diagnosis. Am J Clin Pathol 1988; 90:77-80. 13. Hsu S, Raine L, Fanger H. Use of avidin-biotin-immunoperoxidase complex (ABC) in immunoperoxidase techniques: A comparison between ABC and unlabelled antibody (PAP) procedures. J Histochem Cytochem 1981; 29577-80. 14. Nakajima T, Watanabe S, Sato Y, Kameja T, Hirota T, Shimasato Y. An immunoperoxidase study of SlOO protein distribution in normal and neoplastic tissues. Am J Surg Pathol 1982; 6:715-27.
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15. Kindblom LG, Lodding P, Rosengren L, Baudier J, Haglid K. S-100
16.
17.
18.
19. 20.
protein in melanocytic tumors. Acta Pathol Microbiol Immunol Scand 1984; 92:219-30. Gatter KC, Ralfiiaen E, Skinner J, Brown D, Heryet A, Pulford KAF, Hor-Jensen K , Mason DV. An immunocytochemical study of malignant melanoma and its differential diagnosis from other malignant tumours. J Clin Pathol 1985; 38:1353-7. Loeffel SC, Gillespie GY, Mirmiran SA, Miller EW, Golden P, Askin FB, Seigal GP. Cellular immunolocalization of S-100 protein within fixed tissue sections by monoclonal antibodies. Arch Pathol Lab Med 1985; 109:117-22. Fitzgibbons PL, Chaurushiya P, Nichols PW, Chandrasoma PT, Martin SE. Primary mucosal malignant melanoma: An immunohistochemical study of 12 cases with comparison to cutaneous and metastatic melanomas. Hum Pathol 1989; 20:269-72. Takahashi K. S-100 immunoreactivity in normal human peripheral blood lymphocytes. Am J Clin Pathol 1988; 89:453. Sheibani K, Battifora H. Signet-ring cell melanoma: A rare morphologic variant of malignant melanoma. Am J Surg Pathol 1988; 12: 28-34.
