Cytopathology 1991,2,137-147
ADONIS 095655079100037W
Fine needle aspiration cytology and immunocyt ochemistry of metastatic melanoma K. N A S I E L L , E. T A N 1 A N D L. S K O O G Division of Clinical Cytology, Department of Pathology, Karolinska Hospital, Stockholm, Sweden Accepted,forpublication 27 March 1991
NASIELL K., TANI E. AND SKOOG L.
(1 991) Cytopathology 2, 137-147
Fine needle aspiration cytology and immunocytochemistry of metastatic melanoma
The cytological and immunological findings of 8 1 metastatic melanomas are described. Fine needle aspiration was performed from secondary deposits in lymph nodes (38), subcutaneous and soft tissue (36), abdomen ( S ) , lung (1) from 67 patients with histologically verified malignant melanoma. One patient had disease which had spread into the cerebrospinal fluid. Cytomorphologically the cases were classified as classical (47%), carcinoma-like (22%), spindle cell type (14%), lymphoma like (6%), undifferentiated (6%), myxoid type (3%), and clear cell type (2%). All cases were immunologically characterized using antibodies to S-100, vimentin and cytokeratin. All cases were S-100 positive and the majority (96%) reacted with antibodies to vimentin. A weak heterogenous reactivity to cytokeratin antibody was detected in only eight cases. The HMB45 antibody was applied to 20 cases and 16 (80%) of these tumours were positive. In summary, we found that an immunological characterization was necessary to conclusively diagnose over 50% of metastatic melanomas which presented with an equivocal cytological picture. Keywords: FNA, metastatic melanoma, immunocytochemistry
INTRODUCTION The incidence of malignant melanoma has been increasing rapidly over the last three decades. Metastatic deposits of melanoma are relatively common and may present at almost any site in the body. Fine needle aspiration (FNA) cytology has proved to be of great value in the diagnostic work-up of patients suspected of having metastatic malignant melanoma. However, secondary lesions of melanomas show a wide variability in their cytological appearance and may mimic other neoplasms such as carcinomas, lymphomas and sarcomas'--6.The diagnostic difficulty is further compounded by the fact that as many as 20% of patients with melanoma develop a second primary tumour such as leukaemia, lymphoma, adenocarcinoma and sarcoma'. Correspondence: Dr L. Skoog, Division of Clinical Cytology, Karolinska Hospital, S-104 01 Stockholm, Sweden.
138 K. Nasiell, E. Tani & L. Skoog The application of immunological techniques to cells procured by FNA offers the possibility of corroborating objectively the cytological diagnosis and identify the cell of origin of most metastatic lesions6''. In the present article we describe the cytological and immunocytochemical findings using a panel of commercially available antibodies on FNAs from 68 patients with histologically verified melanoma. The aim was to identify morphological variants which required immunocytochemical investigation to confirm the diagnosis of metastatic melanoma. PATIENTS A N D METHODS Cases The series comprises FNAs from 8 1 metastatic malignant melanomas, from lymph node (38), soft tissue and subcutaneous (36), abdominal (3), liver (2), lung (I), and cerebrospinal fluid (1). All patients had a histological diagnosis of primary malignant melanoma which was situated in the skin (62), eye (2), nose cavity (2), vulva (1) and rectum (1). Cytological examination FNA was performed by the technique described by Zajicek. Smears were stained with May-Grunwald Giemsa (MGG) and Papanicolaou stains16. The cases were classified according to the predominating cell type and the following features were evaluated: cellular cohesiveness, cell size, polymorphism, amount of cytoplasm, cytoplasmic vacuoles, nucleoli, melanin pigment and intranuclear cytoplasmic invaginations. Using these parameters, seven different groups could be identified, namely classical, carcinoma-like, spindle cell, lymphoma-like, undifferentiated, myxoid and clear cell melanoma. Immunocytochemistry Part of the aspirate was suspended in phosphate buffered saline (PBS) and used for cytospin preparations. A three step alkaline phosphatase immunostaining was employed, as described previously 17. Antibodies to the following human antigens were used: S-100 (Dako); cytokeratin, vimentin (Dako) and HMB45 (Enzo Diagnostics Inc.). The last antibody was not available in the beginning of the study and was thus only used in 20 cases. RESULTS Cytomorphology Based on the predominant cytological cell type the cases were classified as belonging to one of the following sub-groups: classical, carcinoma-like, spindle cell, lymphoma-like, undifferentiated, myxoid, and clear cell melanoma. The classical melanoma subgroup was the largest and comprised 38 cases (47%). The cytological pattern when stained by MGG was characterized by polygonal or plasmocytoid cells with a large amount of distinct cytoplasm that exhibited vacuoles in all cases (Table 1). Binucleated sometimes bizarre giant cells were constant findings (Figure 1a). Melanin
Cytology ofmeIanoma
139
Table 1. Morphologicalcharacteristics in five morphological types* of malignant melanoma
Morphological types
Cytoplasmic
Melanin
Necrotic
vacuolization
pigment
background
Nucleoli
Intranuclear cytoplasmic inclusions
‘Classical’
100% 89%
79%
11% 44%
79% 78%
79% 50%
25% 0Yo 20%
58% 40 % 60%
25%
Carcinoma-like Spindle cell
Lymphoma-like Undifferentiated
58% 100% 80%
So%+ 42%+ 20%
+
6O%+
20%
40%
*Myxoid and clear cell type are not included +Low amount in few cells
pigment, distinct nucleoli and intranuclear cytoplasmic inclusions were common features observed in 79% of the tumours (Table I). Eighteen patients (22%) had tumours classified as carcinoma-like. Characteristically the individual cells were relatively monomorphous and showed marked cellular cohesion (Figure 2). Cytoplasmic vacuoles and nucleoli were observed in 89% and 78%, respectively (Table 1). As can be seen from this table, melanin and intranuclear invaginations were found in 50% of the cases. The third largest sub-group, spindle cell type, comprised 12 cases (14%). The majority of the melanoma cells often formed loosely cohesive clusters and the individual cells had ‘bipolar’ cytoplasm with an elongated or oval nucleus (Figure 3). Cellular features such as vacuolated cytoplasm and nucleoli were found in 58% of the smears. Melanin pigment and intranuclear inclusions were inconstant findings and detected in 42% and 25% of cases respectively (Table 1). Lymphoma like smears were obtained from five patients (6%). The cells were rounded and strikingly monotonous and had a thin rim of cytoplasm (Figure 4). Their size was approximately twice that of a mature lymphocyte. Finely granular melanin pigment was observed in a majority of the cells in four cases. Inconspicuous nucleoli were observed in two cases while intranuclear invaginations were present in a few cells from one patient (Table 1). The smears from five patients exhibited dispersed and loosely cohesive cells with moderate pleomorphism (Figure 5). These cells lacked cytological characteristics and were referred to as undifferentiated melanomas. Vacuolated cytoplasm, melanin pigment, nucleoli and intranuclear inclusions were indistinct features observed in few cells (Table 1). Myxoid substance and clear cells dominated the smears from two patients and one patient respectively. The cells belonging to the myxoid subgroup often showed a vacuolated cytoplasm which contained melanin pigment (Figure 6). In addition, both cases had distinct intranuclear inclusions. Cytoplasmic vacuoles were prominent features of most cells aspirated from a single case of clear cell melanoma (Figure 7). The cells lacked pigment and intranuclear invaginations. Immunocytochemistry The immunological staining patterns for the three most common cytological subgroups are presented in Table 2. Figure l(b,c,d) shows the immunophenotype of a classical melanoma.
140 K . Nasiell, E. Tani & L. Skoog
Cytology of melanoma
141
Figure 2. Carcinoma like melanoma, characterized by clusters of monomorphic cells sometimes showing microacinar arrangement. MGG. x 360.
Figure 3. Spindle cell type melanoma often presenting naked elongated or oval nuclei. An indistinct sometimes ‘bipolar’ cytoplasm can occasionally be observed. MGG. x 360.
Antibodies to S-100 and vimentin reacted with tumour cells from all 81 melanomas. The intensity of the staining for S-100 varied in the sub-groups. Thus, 85% of the classical melanoma were strongly positive while moderate and weak positivity was observed in 10% and 5 % , respectively. Spindle cell melanoma showed positivity for S-100 which was strong in 42% of the tumours, moderate in 25% and weak in 33% (Table 2). The corresponding figures for the carcinoma like sub-group were 55%, 39% and 6%, respectively. Antibodies to vimentin reacted with a moderate to strong intensity with a majority (96%) of the tumours from the classical, carcinoma like, and spindle cell melanoma as can be seen from Table 2. Cytokeratin reactivity was observed with a low intensity in three cases (8%) with classical melanoma morphology and in three (1 7%) of carcinoma like melanomas. The staining
142 K. Nasiell, E. Tani & L. Skoog
Figure 4. Lymphoma-like melanoma showing dispersed rounded cells with scanty cytoplasm. MGG. x 360.
Figure 5. Large pleomorphic cells of an undifferentiated melanoma. Nuclear polymorphism and prominent nucleoli are evident. MGG. x 360.
was heterogenous and found only in a minority of the cells. Two cases of classical melanoma exhibited a moderate reactivity to the cytokeratin antibody. Both these cases were strongly S-100 and vimentin positive. The spindle cell variant was cytokeratin negative (Table 2). The immunological staining pattern for the less frequently found cytological variants of melanoma is shown in Table 3. It can be seen from this table that all tumours reacted with antibodies to S-100 and vimentin. In contrast no cytokeratin positivity was noted. The antibody HMB-45 has been reported to be specific for melanocyte derived lesions. We had access to this antibody towards the end of the study and analysed aspirates from 20 cases. The results are summarized in Table 4 and showed a reactivity with HMB-45 in 16 (80%) of the tumours. Four cases were negative which were classified cytologically as classical, carcinoma like, myxoid, and clear cell melanoma. All of these were negative for cytokeratin marker but positive for vimentin and S-100.
Cytology of melanoma
143
Figure 6. Melanoma of the myxoid type with pleomorphic cells in a background of myxoid substance. MGG. x 360.
Figure 7. Clear cell melanoma presenting cells with abundant vacuolated cytoplasm. MGG. x 360.
DISCUSSION The diverse cytological presentation of both primary and metastatic melanoma has been described in several Our findings of the cytomorphology of both node based and extranodal metastases of melanoma are in good agreement with these previous reports. Based on cytological appearances we could identify seven sub-groups, namely classical, spindle cell, carcinoma like, lymphoma like, undifferentiated, clear cell, and myxoid melanoma, the classical sub-group being the largest comprising 47% of the cases. The cytological features of dissociated polymorphic cells, giant cells, melanin pigment, and intranuclear invagination make this category readily identifiable. It should be possible to diagnose cases belonging to this sub-group conclusively even without a known history of a primary melanoma. We therefore refer to this subgroup as classical melanoma instead of ‘epithelioid’ or ‘epithelial’ as suggested by
15 (83) 3 (17) -
32 (87) 3 (8) 2 (5)
-
37*(100)
18(100)
-
-
("/.I
("/.I
-
Carcinomalike
'Classical'
12 (100)
12 (100)
_ _ _ -
(Yo)
Spindlecell
20 (54) 17 (46) 37*(100)
- _ - _
(%)
'Classical'
Vimentin
-
2 (10) 3 (17) 13 (72) 18 (100)
-
("/.I
Carcinomalike
-
5
Total cases
~
5
I
-
2
~
1
-
-
1
2 -
5 -
5
-
+++
+ ++
-
Clear Myxoid cell
Lymphoma like
Undifferentiated
Reaction intensity
Cytokeratin Undifferentiated
Vimentin Lymphoma like
Table 3. Immunological analysis in the four less common cytomorphological types
*One case (cerebrospinal fluid) not analysed
Total
+ ++ +++
-
Reaction intensity
Cytokeratin
Table 2. Immunological analysis of the three most frequent cytomorphological types
Clear Myxoid cell
1 (8) 5 (42) 6 (50) 12(100)
- _
("/.I
Spindlecell
(5)
Undifferentiated
s-100
4 (10) 32 (85) 38(100)
2
_ -
("/.I
'Classical'
s-100
Lymphoma like
1 (6) 7 (39) 10 (55) 18(100)
- -
(%)
Carcinomalike
-
Clear Myxoid cell
4 (33) 3 (25) 5 (42) 12 (100)
-
("/.I
Spindlecell
e
P P
Cytology of melanoma
145
Table 4. HMB-45 analysis in 20 cases in the different cytomorphological types*
Carcinoma
Spindle
'Classical'
like
cell
Lymphoma like Myxoid
-
1
1 -
-
-
1
-
2
1
-
-
2 3
-
2 3
-
-
Total
3 3 2 9
1
1
Reaction intensity
+ ++ +++
3
Clear cell
Total
1
1
-
-
4 4 6 6 20
*No available material for HMB-45 analysis in the 'undifferentiated' type
In this study a majority, 53%, of the aspirates from metastatic melanomas had a cytomorphology which was divergent enough to suggest neoplasms other than melanoma. To indicate this resemblance to other tumours we used descriptive terms for these sub-groups such as spindle cell, lymphoma like, undifferentiated, and carcinoma like melanoma. The identification of melanin in the tumour cell should suggest the true nature of the neoplasm. However, melanin pigment was observed in less than 50% of the tumours with a morphology different from that of classical melanoma. Other cellular features such as cytoplasmic vacuoles and intranuclear invaginations are unspecific and can at the best provide circumstantial evidence of the tumour cell origin. In such cases an immunological characterization of the cells might be the only way to arrive at a conclusive diagnosis. Aspirates from suspected melanomas were immunologically characterized by a panel of antibodies to S-100 and the intermediate filaments vimentin and cytokeratin. Antibody S-100 has been reported to react with most melanocytic tumours thus exhibiting a high sensitivi ty3,7.9.~ I , 1 3 ~ 1.4In agreement with this all tumours analysed in this study were positive for S-100. However, the S-100 protein is not melanoma specific and can be found in other tumours such as gliomas, a subset of carcinomas, Schwann cell tumours, chondrosarcomas, and o s t e o s a r ~ o m a s ~ * A " ~positive '~. reaction to S- 100 excludes a diagnosis of lymphoma, malignant fibrous histiocytoma and most carcinomas. Some carcinomas, mainly ovarian and mammary, may show weak S-100 positivity. Their epithelial origin is, however, readily disclosed by a positive reaction to cytokeratin. Five of the classical and three of the carcinoma like melanomas exhibited a minor fraction of cells which reacted weakly with antibodies to cytokeratin. However, their melanocytic origin was confirmed by staining for S-100 and vimentin. The finding of three carcinoma like melanomas which expressed cytokeratin weakly, emphasizes the importance of corroborating the cytological diagnosis with more than one confirmatory antibody and whenever possible a panel of antibodies should be employed to increase the diagnostic accuracy. Spindle cell melanomas present a distinct diagnostic problem. Cytologically such tumours often lack characteristics indicating their true origin. In addition several types of mesenchyma1 neoplasms express both S-100 and vimentin9.l3.Accordingly a conclusive diagnosis of such spindle cell melanomas must include the use of a specific antibody to neoplasms derived from melanocytes. HMB-45 has been described to react almost exclusively with cells of . Only basal cell carcinomas may exhibit some cross-reactivity melanocytic rigi in^,^,' I - I 3 3 I 5
146 K. Nusiell, E. Tuni & L. Skoog with HMB-45. We found that 80% of our metastatic melanomas stained with HMB-45. This figure is in good agreement with previous reports on the sensitivity of this a n t i b ~ d y ' ~ ~ ~ ' ~ - ' ~ . ' ' . All three spindle cell melanomas tested reacted strongly with HMB-45 which allowed a conclusive diagnosis. Four tumours did not stain with HMB-45 but all were strongly S-100 and vimentin positive and cytokeratin negative. This immunological profile, in conjunction with cytology, allowed positive identification of these metastases as derived from malignant melanomas. In conclusion, we suggest that aspirates from patients with suspected secondary melanoma should be characterized immunologically using a panel of antibodies to provide positive identification of the tumour cells. This should result in a conclusive diagnosis even if the cytological presentation is equivocal.
ACKNOWLEDGEMENTS The expert technical assistance of Margot Carlsson is gratefully acknowledged. This work was supported by grants from the Cancer Society of Stockholm and the Karolinska Institute (E.T.).
REFERENCES 1 Perry MD, Gore M, Seigler HF, Johnston WW. Fine needle aspiration biopsy of metastatic melanoma: A morphologic analysis of 174 cases. Acta Cyt011986;3 0 385-96. 2 Linsk JA, Franzen S. Melanomas and skin nodules. In: Linsk JA, Franzen S, eds, Clinical Aspiration Cytology. Philadelphia: JB Lippincott, 1983;281-96. 3 Rocamora A, Carrillo R, Vives R, Solera JC. Fine needle aspiration biopsy of myxoid metastasis of malignant melanoma. Acta Cytoll988; 3 2 94-100. 4 Lindholm K, de la Torre M. Fine needle aspiration cytology of myxoid metastatic malignant melanoma. ACIUCytol1988; 32: 7 19-2 1. 5 Layfield LJ, Ostrzega N. Fine needle aspirate smear morphology in metastatic melanoma. Acta Cyt01 1989;33: 601-12. 6 Gupta SK, Rajwanshi AK, Das D. Fine needle aspiration cytology smear patterns of malignant melanoma. Acta Cytoll985; 2 9 983-8. 7 Springall DR, Gy J, Cocchcia D, Michetti F, Lebene A, Levene MM, Marangos PJ, Bloom SR, Polak JM. ThevalueofS-100immunostainingasa diagnostic tool in human malignant melanomas: A comparative study using S-100 and neuronspecific enolase antibodies. Virchows Archiv (Pathol Antat) 1983; 400:31 1 4 3 . 8 Esclamado RM, Gown AM, Vogel AM. Unique proteins defined by monoclonal antibodies specific for human melanoma. Some potential clinical applications. Am J Surg 1986; 152 37685.
9 Gown AM, Vogel AM, Hoak D, Gough F, McNutt MA. Monoclonal antibodies specific for melanocytic tumors distinguish subpopulations of melanocytes. A m J Patholl986; 123 195-203. 10 Angeli S, Koelma IA, Fleuren GJ, Van Steenis GJ. Malignant melanoma in fine needle aspirates and effusions: An immunocytochemical study using monoclonal antibodies. Acta Cytol 1988; 3 2 707-1 1. 11 Ordoiiez NG, Xiaolong J, Hickey RC. Comparison of HMB-45 monoclonal antibody and S-100 protein in the immunohistochemical diagnosis of melanoma. Am J Clin Patholl988; 90: 385-90. 2 Walts AE, Said JW, Shintaku IP. Cytodiagnosisof malignant melanoma: Immunoperoxidase staining with HMB-45 antibody as an aid to diagnosis. Am J Patholl988; 90: 77-80. 3 Abdelatif OM, Khankhanian NK, Crosby JH, Chamberlain CR Jr, Seigler MM, Tom GD. Malignant fibrous histiocytoma and malignant melanoma: The role of immunohistochemistry and electron microscopy in the differential diagnosis. Mod Patholl989; 2 477-85. 14 Shoup SA, Johnston WW, Siegler HF e f al. A panel of antibodies useful in the cytologic diagnosis of metastatic melanoma. Acra Cytol 1990; 3 4 385-92. 15 Pelosi G, Bonetti F, Colombari R, Bonzanini M, Tannuci A. Use of monoclonal antibody HMB-45 for detecting malignant melanoma cells in fine needle aspiration biopsy samples (Letter to the editor). Acfa Cytol1990; 3 4 460-2.
Cytology of melanoma 16 Zdjicek J. Aspiration biopsy cytology: Part 1. Cytology of supradiaphragmatic organs. In: Wied GL, ed., Monogruphs in Clinical Cytology, 1-29, Fourth vol. Basel: S Karger, 1974.
147
17 Tani E, Christensson B, Porwit A, Skoog L. Immunocytochemical analysis and cytomorphologic diagnosis on fine needle aspirates of lymphoproliferative diseases. Acfa Cyroll988;32:209-1 5.
ADONIS 095655079100037W
Fine needle aspiration cytology and immunocyt ochemistry of metastatic melanoma K. N A S I E L L , E. T A N 1 A N D L. S K O O G Division of Clinical Cytology, Department of Pathology, Karolinska Hospital, Stockholm, Sweden Accepted,forpublication 27 March 1991
NASIELL K., TANI E. AND SKOOG L.
(1 991) Cytopathology 2, 137-147
Fine needle aspiration cytology and immunocytochemistry of metastatic melanoma
The cytological and immunological findings of 8 1 metastatic melanomas are described. Fine needle aspiration was performed from secondary deposits in lymph nodes (38), subcutaneous and soft tissue (36), abdomen ( S ) , lung (1) from 67 patients with histologically verified malignant melanoma. One patient had disease which had spread into the cerebrospinal fluid. Cytomorphologically the cases were classified as classical (47%), carcinoma-like (22%), spindle cell type (14%), lymphoma like (6%), undifferentiated (6%), myxoid type (3%), and clear cell type (2%). All cases were immunologically characterized using antibodies to S-100, vimentin and cytokeratin. All cases were S-100 positive and the majority (96%) reacted with antibodies to vimentin. A weak heterogenous reactivity to cytokeratin antibody was detected in only eight cases. The HMB45 antibody was applied to 20 cases and 16 (80%) of these tumours were positive. In summary, we found that an immunological characterization was necessary to conclusively diagnose over 50% of metastatic melanomas which presented with an equivocal cytological picture. Keywords: FNA, metastatic melanoma, immunocytochemistry
INTRODUCTION The incidence of malignant melanoma has been increasing rapidly over the last three decades. Metastatic deposits of melanoma are relatively common and may present at almost any site in the body. Fine needle aspiration (FNA) cytology has proved to be of great value in the diagnostic work-up of patients suspected of having metastatic malignant melanoma. However, secondary lesions of melanomas show a wide variability in their cytological appearance and may mimic other neoplasms such as carcinomas, lymphomas and sarcomas'--6.The diagnostic difficulty is further compounded by the fact that as many as 20% of patients with melanoma develop a second primary tumour such as leukaemia, lymphoma, adenocarcinoma and sarcoma'. Correspondence: Dr L. Skoog, Division of Clinical Cytology, Karolinska Hospital, S-104 01 Stockholm, Sweden.
138 K. Nasiell, E. Tani & L. Skoog The application of immunological techniques to cells procured by FNA offers the possibility of corroborating objectively the cytological diagnosis and identify the cell of origin of most metastatic lesions6''. In the present article we describe the cytological and immunocytochemical findings using a panel of commercially available antibodies on FNAs from 68 patients with histologically verified melanoma. The aim was to identify morphological variants which required immunocytochemical investigation to confirm the diagnosis of metastatic melanoma. PATIENTS A N D METHODS Cases The series comprises FNAs from 8 1 metastatic malignant melanomas, from lymph node (38), soft tissue and subcutaneous (36), abdominal (3), liver (2), lung (I), and cerebrospinal fluid (1). All patients had a histological diagnosis of primary malignant melanoma which was situated in the skin (62), eye (2), nose cavity (2), vulva (1) and rectum (1). Cytological examination FNA was performed by the technique described by Zajicek. Smears were stained with May-Grunwald Giemsa (MGG) and Papanicolaou stains16. The cases were classified according to the predominating cell type and the following features were evaluated: cellular cohesiveness, cell size, polymorphism, amount of cytoplasm, cytoplasmic vacuoles, nucleoli, melanin pigment and intranuclear cytoplasmic invaginations. Using these parameters, seven different groups could be identified, namely classical, carcinoma-like, spindle cell, lymphoma-like, undifferentiated, myxoid and clear cell melanoma. Immunocytochemistry Part of the aspirate was suspended in phosphate buffered saline (PBS) and used for cytospin preparations. A three step alkaline phosphatase immunostaining was employed, as described previously 17. Antibodies to the following human antigens were used: S-100 (Dako); cytokeratin, vimentin (Dako) and HMB45 (Enzo Diagnostics Inc.). The last antibody was not available in the beginning of the study and was thus only used in 20 cases. RESULTS Cytomorphology Based on the predominant cytological cell type the cases were classified as belonging to one of the following sub-groups: classical, carcinoma-like, spindle cell, lymphoma-like, undifferentiated, myxoid, and clear cell melanoma. The classical melanoma subgroup was the largest and comprised 38 cases (47%). The cytological pattern when stained by MGG was characterized by polygonal or plasmocytoid cells with a large amount of distinct cytoplasm that exhibited vacuoles in all cases (Table 1). Binucleated sometimes bizarre giant cells were constant findings (Figure 1a). Melanin
Cytology ofmeIanoma
139
Table 1. Morphologicalcharacteristics in five morphological types* of malignant melanoma
Morphological types
Cytoplasmic
Melanin
Necrotic
vacuolization
pigment
background
Nucleoli
Intranuclear cytoplasmic inclusions
‘Classical’
100% 89%
79%
11% 44%
79% 78%
79% 50%
25% 0Yo 20%
58% 40 % 60%
25%
Carcinoma-like Spindle cell
Lymphoma-like Undifferentiated
58% 100% 80%
So%+ 42%+ 20%
+
6O%+
20%
40%
*Myxoid and clear cell type are not included +Low amount in few cells
pigment, distinct nucleoli and intranuclear cytoplasmic inclusions were common features observed in 79% of the tumours (Table I). Eighteen patients (22%) had tumours classified as carcinoma-like. Characteristically the individual cells were relatively monomorphous and showed marked cellular cohesion (Figure 2). Cytoplasmic vacuoles and nucleoli were observed in 89% and 78%, respectively (Table 1). As can be seen from this table, melanin and intranuclear invaginations were found in 50% of the cases. The third largest sub-group, spindle cell type, comprised 12 cases (14%). The majority of the melanoma cells often formed loosely cohesive clusters and the individual cells had ‘bipolar’ cytoplasm with an elongated or oval nucleus (Figure 3). Cellular features such as vacuolated cytoplasm and nucleoli were found in 58% of the smears. Melanin pigment and intranuclear inclusions were inconstant findings and detected in 42% and 25% of cases respectively (Table 1). Lymphoma like smears were obtained from five patients (6%). The cells were rounded and strikingly monotonous and had a thin rim of cytoplasm (Figure 4). Their size was approximately twice that of a mature lymphocyte. Finely granular melanin pigment was observed in a majority of the cells in four cases. Inconspicuous nucleoli were observed in two cases while intranuclear invaginations were present in a few cells from one patient (Table 1). The smears from five patients exhibited dispersed and loosely cohesive cells with moderate pleomorphism (Figure 5). These cells lacked cytological characteristics and were referred to as undifferentiated melanomas. Vacuolated cytoplasm, melanin pigment, nucleoli and intranuclear inclusions were indistinct features observed in few cells (Table 1). Myxoid substance and clear cells dominated the smears from two patients and one patient respectively. The cells belonging to the myxoid subgroup often showed a vacuolated cytoplasm which contained melanin pigment (Figure 6). In addition, both cases had distinct intranuclear inclusions. Cytoplasmic vacuoles were prominent features of most cells aspirated from a single case of clear cell melanoma (Figure 7). The cells lacked pigment and intranuclear invaginations. Immunocytochemistry The immunological staining patterns for the three most common cytological subgroups are presented in Table 2. Figure l(b,c,d) shows the immunophenotype of a classical melanoma.
140 K . Nasiell, E. Tani & L. Skoog
Cytology of melanoma
141
Figure 2. Carcinoma like melanoma, characterized by clusters of monomorphic cells sometimes showing microacinar arrangement. MGG. x 360.
Figure 3. Spindle cell type melanoma often presenting naked elongated or oval nuclei. An indistinct sometimes ‘bipolar’ cytoplasm can occasionally be observed. MGG. x 360.
Antibodies to S-100 and vimentin reacted with tumour cells from all 81 melanomas. The intensity of the staining for S-100 varied in the sub-groups. Thus, 85% of the classical melanoma were strongly positive while moderate and weak positivity was observed in 10% and 5 % , respectively. Spindle cell melanoma showed positivity for S-100 which was strong in 42% of the tumours, moderate in 25% and weak in 33% (Table 2). The corresponding figures for the carcinoma like sub-group were 55%, 39% and 6%, respectively. Antibodies to vimentin reacted with a moderate to strong intensity with a majority (96%) of the tumours from the classical, carcinoma like, and spindle cell melanoma as can be seen from Table 2. Cytokeratin reactivity was observed with a low intensity in three cases (8%) with classical melanoma morphology and in three (1 7%) of carcinoma like melanomas. The staining
142 K. Nasiell, E. Tani & L. Skoog
Figure 4. Lymphoma-like melanoma showing dispersed rounded cells with scanty cytoplasm. MGG. x 360.
Figure 5. Large pleomorphic cells of an undifferentiated melanoma. Nuclear polymorphism and prominent nucleoli are evident. MGG. x 360.
was heterogenous and found only in a minority of the cells. Two cases of classical melanoma exhibited a moderate reactivity to the cytokeratin antibody. Both these cases were strongly S-100 and vimentin positive. The spindle cell variant was cytokeratin negative (Table 2). The immunological staining pattern for the less frequently found cytological variants of melanoma is shown in Table 3. It can be seen from this table that all tumours reacted with antibodies to S-100 and vimentin. In contrast no cytokeratin positivity was noted. The antibody HMB-45 has been reported to be specific for melanocyte derived lesions. We had access to this antibody towards the end of the study and analysed aspirates from 20 cases. The results are summarized in Table 4 and showed a reactivity with HMB-45 in 16 (80%) of the tumours. Four cases were negative which were classified cytologically as classical, carcinoma like, myxoid, and clear cell melanoma. All of these were negative for cytokeratin marker but positive for vimentin and S-100.
Cytology of melanoma
143
Figure 6. Melanoma of the myxoid type with pleomorphic cells in a background of myxoid substance. MGG. x 360.
Figure 7. Clear cell melanoma presenting cells with abundant vacuolated cytoplasm. MGG. x 360.
DISCUSSION The diverse cytological presentation of both primary and metastatic melanoma has been described in several Our findings of the cytomorphology of both node based and extranodal metastases of melanoma are in good agreement with these previous reports. Based on cytological appearances we could identify seven sub-groups, namely classical, spindle cell, carcinoma like, lymphoma like, undifferentiated, clear cell, and myxoid melanoma, the classical sub-group being the largest comprising 47% of the cases. The cytological features of dissociated polymorphic cells, giant cells, melanin pigment, and intranuclear invagination make this category readily identifiable. It should be possible to diagnose cases belonging to this sub-group conclusively even without a known history of a primary melanoma. We therefore refer to this subgroup as classical melanoma instead of ‘epithelioid’ or ‘epithelial’ as suggested by
15 (83) 3 (17) -
32 (87) 3 (8) 2 (5)
-
37*(100)
18(100)
-
-
("/.I
("/.I
-
Carcinomalike
'Classical'
12 (100)
12 (100)
_ _ _ -
(Yo)
Spindlecell
20 (54) 17 (46) 37*(100)
- _ - _
(%)
'Classical'
Vimentin
-
2 (10) 3 (17) 13 (72) 18 (100)
-
("/.I
Carcinomalike
-
5
Total cases
~
5
I
-
2
~
1
-
-
1
2 -
5 -
5
-
+++
+ ++
-
Clear Myxoid cell
Lymphoma like
Undifferentiated
Reaction intensity
Cytokeratin Undifferentiated
Vimentin Lymphoma like
Table 3. Immunological analysis in the four less common cytomorphological types
*One case (cerebrospinal fluid) not analysed
Total
+ ++ +++
-
Reaction intensity
Cytokeratin
Table 2. Immunological analysis of the three most frequent cytomorphological types
Clear Myxoid cell
1 (8) 5 (42) 6 (50) 12(100)
- _
("/.I
Spindlecell
(5)
Undifferentiated
s-100
4 (10) 32 (85) 38(100)
2
_ -
("/.I
'Classical'
s-100
Lymphoma like
1 (6) 7 (39) 10 (55) 18(100)
- -
(%)
Carcinomalike
-
Clear Myxoid cell
4 (33) 3 (25) 5 (42) 12 (100)
-
("/.I
Spindlecell
e
P P
Cytology of melanoma
145
Table 4. HMB-45 analysis in 20 cases in the different cytomorphological types*
Carcinoma
Spindle
'Classical'
like
cell
Lymphoma like Myxoid
-
1
1 -
-
-
1
-
2
1
-
-
2 3
-
2 3
-
-
Total
3 3 2 9
1
1
Reaction intensity
+ ++ +++
3
Clear cell
Total
1
1
-
-
4 4 6 6 20
*No available material for HMB-45 analysis in the 'undifferentiated' type
In this study a majority, 53%, of the aspirates from metastatic melanomas had a cytomorphology which was divergent enough to suggest neoplasms other than melanoma. To indicate this resemblance to other tumours we used descriptive terms for these sub-groups such as spindle cell, lymphoma like, undifferentiated, and carcinoma like melanoma. The identification of melanin in the tumour cell should suggest the true nature of the neoplasm. However, melanin pigment was observed in less than 50% of the tumours with a morphology different from that of classical melanoma. Other cellular features such as cytoplasmic vacuoles and intranuclear invaginations are unspecific and can at the best provide circumstantial evidence of the tumour cell origin. In such cases an immunological characterization of the cells might be the only way to arrive at a conclusive diagnosis. Aspirates from suspected melanomas were immunologically characterized by a panel of antibodies to S-100 and the intermediate filaments vimentin and cytokeratin. Antibody S-100 has been reported to react with most melanocytic tumours thus exhibiting a high sensitivi ty3,7.9.~ I , 1 3 ~ 1.4In agreement with this all tumours analysed in this study were positive for S-100. However, the S-100 protein is not melanoma specific and can be found in other tumours such as gliomas, a subset of carcinomas, Schwann cell tumours, chondrosarcomas, and o s t e o s a r ~ o m a s ~ * A " ~positive '~. reaction to S- 100 excludes a diagnosis of lymphoma, malignant fibrous histiocytoma and most carcinomas. Some carcinomas, mainly ovarian and mammary, may show weak S-100 positivity. Their epithelial origin is, however, readily disclosed by a positive reaction to cytokeratin. Five of the classical and three of the carcinoma like melanomas exhibited a minor fraction of cells which reacted weakly with antibodies to cytokeratin. However, their melanocytic origin was confirmed by staining for S-100 and vimentin. The finding of three carcinoma like melanomas which expressed cytokeratin weakly, emphasizes the importance of corroborating the cytological diagnosis with more than one confirmatory antibody and whenever possible a panel of antibodies should be employed to increase the diagnostic accuracy. Spindle cell melanomas present a distinct diagnostic problem. Cytologically such tumours often lack characteristics indicating their true origin. In addition several types of mesenchyma1 neoplasms express both S-100 and vimentin9.l3.Accordingly a conclusive diagnosis of such spindle cell melanomas must include the use of a specific antibody to neoplasms derived from melanocytes. HMB-45 has been described to react almost exclusively with cells of . Only basal cell carcinomas may exhibit some cross-reactivity melanocytic rigi in^,^,' I - I 3 3 I 5
146 K. Nusiell, E. Tuni & L. Skoog with HMB-45. We found that 80% of our metastatic melanomas stained with HMB-45. This figure is in good agreement with previous reports on the sensitivity of this a n t i b ~ d y ' ~ ~ ~ ' ~ - ' ~ . ' ' . All three spindle cell melanomas tested reacted strongly with HMB-45 which allowed a conclusive diagnosis. Four tumours did not stain with HMB-45 but all were strongly S-100 and vimentin positive and cytokeratin negative. This immunological profile, in conjunction with cytology, allowed positive identification of these metastases as derived from malignant melanomas. In conclusion, we suggest that aspirates from patients with suspected secondary melanoma should be characterized immunologically using a panel of antibodies to provide positive identification of the tumour cells. This should result in a conclusive diagnosis even if the cytological presentation is equivocal.
ACKNOWLEDGEMENTS The expert technical assistance of Margot Carlsson is gratefully acknowledged. This work was supported by grants from the Cancer Society of Stockholm and the Karolinska Institute (E.T.).
REFERENCES 1 Perry MD, Gore M, Seigler HF, Johnston WW. Fine needle aspiration biopsy of metastatic melanoma: A morphologic analysis of 174 cases. Acta Cyt011986;3 0 385-96. 2 Linsk JA, Franzen S. Melanomas and skin nodules. In: Linsk JA, Franzen S, eds, Clinical Aspiration Cytology. Philadelphia: JB Lippincott, 1983;281-96. 3 Rocamora A, Carrillo R, Vives R, Solera JC. Fine needle aspiration biopsy of myxoid metastasis of malignant melanoma. Acta Cytoll988; 3 2 94-100. 4 Lindholm K, de la Torre M. Fine needle aspiration cytology of myxoid metastatic malignant melanoma. ACIUCytol1988; 32: 7 19-2 1. 5 Layfield LJ, Ostrzega N. Fine needle aspirate smear morphology in metastatic melanoma. Acta Cyt01 1989;33: 601-12. 6 Gupta SK, Rajwanshi AK, Das D. Fine needle aspiration cytology smear patterns of malignant melanoma. Acta Cytoll985; 2 9 983-8. 7 Springall DR, Gy J, Cocchcia D, Michetti F, Lebene A, Levene MM, Marangos PJ, Bloom SR, Polak JM. ThevalueofS-100immunostainingasa diagnostic tool in human malignant melanomas: A comparative study using S-100 and neuronspecific enolase antibodies. Virchows Archiv (Pathol Antat) 1983; 400:31 1 4 3 . 8 Esclamado RM, Gown AM, Vogel AM. Unique proteins defined by monoclonal antibodies specific for human melanoma. Some potential clinical applications. Am J Surg 1986; 152 37685.
9 Gown AM, Vogel AM, Hoak D, Gough F, McNutt MA. Monoclonal antibodies specific for melanocytic tumors distinguish subpopulations of melanocytes. A m J Patholl986; 123 195-203. 10 Angeli S, Koelma IA, Fleuren GJ, Van Steenis GJ. Malignant melanoma in fine needle aspirates and effusions: An immunocytochemical study using monoclonal antibodies. Acta Cytol 1988; 3 2 707-1 1. 11 Ordoiiez NG, Xiaolong J, Hickey RC. Comparison of HMB-45 monoclonal antibody and S-100 protein in the immunohistochemical diagnosis of melanoma. Am J Clin Patholl988; 90: 385-90. 2 Walts AE, Said JW, Shintaku IP. Cytodiagnosisof malignant melanoma: Immunoperoxidase staining with HMB-45 antibody as an aid to diagnosis. Am J Patholl988; 90: 77-80. 3 Abdelatif OM, Khankhanian NK, Crosby JH, Chamberlain CR Jr, Seigler MM, Tom GD. Malignant fibrous histiocytoma and malignant melanoma: The role of immunohistochemistry and electron microscopy in the differential diagnosis. Mod Patholl989; 2 477-85. 14 Shoup SA, Johnston WW, Siegler HF e f al. A panel of antibodies useful in the cytologic diagnosis of metastatic melanoma. Acra Cytol 1990; 3 4 385-92. 15 Pelosi G, Bonetti F, Colombari R, Bonzanini M, Tannuci A. Use of monoclonal antibody HMB-45 for detecting malignant melanoma cells in fine needle aspiration biopsy samples (Letter to the editor). Acfa Cytol1990; 3 4 460-2.
Cytology of melanoma 16 Zdjicek J. Aspiration biopsy cytology: Part 1. Cytology of supradiaphragmatic organs. In: Wied GL, ed., Monogruphs in Clinical Cytology, 1-29, Fourth vol. Basel: S Karger, 1974.
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17 Tani E, Christensson B, Porwit A, Skoog L. Immunocytochemical analysis and cytomorphologic diagnosis on fine needle aspirates of lymphoproliferative diseases. Acfa Cyroll988;32:209-1 5.
