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S-100 Protein Positive (Sustentacular) Cells in Pulmonary Carcinoid Tumorlets: a Quantitative Study of 24 Cases
Path. Res. Pract. 192,414-417 (1996)
S-100 Protein Positive (Sustentacular) Cells in Pulmonary Carcinoid Tumorlets: a Quantitative Study of 24 Cases M. ResP, B. Knll 2, J. Simek3 and J. Bukac 4 University Departments of Pathology', Medicine 2, Cardiovascular and Thoracic Surgery3 and Computer Technology Center4, Charles University, Hradec Kralove, Czech Republic
SUMMARY Stellate-shaped S-100 protein positive sustentacular cells (SCs) appear to correlate inversely with the degree of tumor malignancy in some neuroendocrine tumors. Therefore, the SCs have been investigated in carcinoid pulmonary tumorlets and subsequently examined quantitatively in order to provide a basis for estimation of this phenomenon in tumorlet related lesions, especially carcinoids and neuroendocrine carcinomas. Pulmonary tissue from twenty-four patients with carcinoid tumorlets was studied immunochistochemically for S-l 00 protein positive SCs together with glial fibrillary acidic protein, actin, desmin, vimentin and cytokeratins. Tumorlet SCs were calculated per 10,000 tumorlet elements. The presence of SCs was proven in 18 subjects (75%) of all examined cases with quantitative frequency between 13 and 196 SCs per 10,000 tumorlet elements. The histogram showed three separate clusters of cases. Cluster 2 and cluster 3 with strikinglyhigh frequencyofSCs, i.e. from 66 up to 196 SCs per 10,000 tumorletcells, may represent the biphasic differentiation potential of tumorlet elements. The mentioned lesions could be regarded as the possible precursors of pulmonary paragangliomas or paraganglioid carcinoids.
Introduction Pulmonary tumorlets were first described by Pagel in 1924 17 and the term tumorlet was coined by Whitwell in 1955 25 for small cellular nests formed of uniform elements with a fusiform or polygonal nuclei and a variable amount of cytoplasma. In 1977, Ranchod labeled this neuroendocrine lesion as carcinoid tumorlet, a term which expresses an association with carcinoid tumor l8 • Sustentacular cells (SCs) are a well-recognized cell population in the paraganglionar system and other tis0344-0338/96/0192-0414$3.50/0
sues, e.g. in the olfactory epithelium or adrenal medullaB, 10, 16,24. They represent the secondary elements which probably provide a growth-regulatory support for endocrine cells, but their exact histogenetic origin and function remains obscure. These stellate-shaped cells have also been demonstrated in paragangliomas and pheochromocytomass, 8,11,15, and other neuroendocrine tumours 3, 4,13. They appear to correlate inversely with the degree of tumor malignancy. In view of this fact, we have investigated quantitatively the SCs in a large series of carcinoid tumorlets in order to provide a basis for © 1996 by Gustav Fischer Verlag, Stuttgart
5-100 Protein Positive Cells in Tumorlets . 415
46 and 91 (mean 75) years. Of them, twenty-three lesions appeared as incidental microscopical findings in autopsy material, and the remaining case was found in surgical pathology material as a result of retrospective analysis of 137 surgically treated non tumorous lesions during the last 12 years. Three additional subjects from this surgical material had been rejected from the set due to insufficient amount of tumourlet elements in serial sections necessary for immunohistochemistry, i.e. the investigated lesion had been cut-up. Morphologically, all carcinoid tumorlets displayed the typical small nests of the uniform elements with a fusiform, polygonal or mixed nuclear configuration and a variable amount of the cytoplasm in the connective tissue stroma (Figs. 1, 2). All 24 subjects with tumorlets showed the cellular argyrophilia and the strong labeling for neuron specific enolase. A distinct cellular labeling for S-l 00 protein together with cytologic features was used for the SCs identification, i.e. ovoid nuclei with a small chromatin amount and long slender plasmatic processes (Fig. 3). There was no evidence of SCs in six cases (25%) out of all 24 examined cases. There was only one case (4 %) with the evidence of labeling for GFAP, and the immunoreactivity of the SCs for vimentin and desmin was 62,5% and 44,4%, respectively. In all cases the SCs were negative for cytokeratin expression. The quantitative frequency of SCs in the remaining 18 positive cases (75%) was between 13 and 196 SCs per 10,000 tumorlet elements. The frequency of SCs in individual cases, sex and age of each of the twenty four subjects is shown in Table 1. Table 4 shows the SCs number histogram of all 24 examined case. The SCs number difference investigated by cluster analysis showed three separate clusters. Cluster 1 consisting of 17 cases with a frequency of SCs up to 46 elements per 10,000 tumorlet cells, cluster 2 consisting of 5 cases with a frequency between 66 and 111 SCs per 10,000 elements, and cluster 3 consisting of 2 cases with strikingly high frequency of SCs, i.e. 189 and 196 of these elements per 10,000 tumorlet cells. These clusters can be easily identified on the attached histogram (Table 2).
Discussion
subsequent estimation of this phenomenon in tumorletrelated lesions, and to discuss their histogenetic origin. Results Twenty-four tumourlets (10 men and 14 women) have been identified in patients between the ages of
We presume that the investigated cells are identical or strongly related elements to SCs previously described in normal tissues and pathological lesions 2 5,8,10,11,13,15,16,24. We have the following reasons for the above mentioned statement: stellate cellular shape, localization of the examined elements within the lesion and their expression of S-100 protein. The results in the presented study prove the presence of S-100 protein positive (sustentacular) cells in pulmonary carcinoid tumorlets and support the premise that the SCs represent the cell population observed in the entire spectrum of the neuroendocrine pulmonary le-
416 . M. Resl et al. Table 1. The frequency of sustentacular cells per 10,000 tumorlet elements, sex and age of twenty-four cases
Case No. SCs Sex Age Case No. SCs Sex Age
1 36 F 88 13 0 M 71
2 40 M 84 14 0 F 75
3 97 M 89 15 66 F 69
4 0 F 91 16 92 M 46
5 23 F 80 17 18 F 73
6 0 M 88 18 111 F 79
7 22 F 68 19 93 M 81
8 196 F 48 20 28 F 82
9 15 F 84 21 28 F 82
10 189 M 62 22 13 M 83
11 46 F 77 23 0 M 71
12
0 M 74 24 28 M 51
F: Female, M: male, SCs: sustentacular cells Table 2. Sustentacular cell number histogram of 24 investigated cases. The abscissa expresses the SCs number, and the ordinate the number of examined cases
10 8 6 4
2 0+--+--+--+--~-+--~-r--~~-4
o
20· 40 60 80 100 120 140 160 180 200
sions, i.e. tumorlets, both types of carcinoids, paragangliomas and small cell carcinomas described elsewhere 1,3,13. Their role as an indicator of tumor malignancy is a matter of research 4 • The frequency of SCs in the examined set is 75% of all 24 subjects and the quantitative SC frequency in individual tumourlets is up to 196 SCs per 10,000 tumorlet elements. SCs have been examined in the rat carotid body, the human adrenal medulla and the paraganglionar system 9, 16, 24. Kondo et al. 12 have suggested that SCs represent homologous elements to Schwann cells for their topographic continuity with Schwann cells and the expression of S-1 00 protein. This opinion has been supported by description of the immunoreactive SCs for human nerve growth factor in 8 pheochromocytomas and 3 paragangliomas 23 • The SCs have also been found as positive elements for GFAP and S-100 protein in 16 of 18 paragangliomas 11. Ultrastructural description of SCs in one subject of low grade paraganglioma was provided by Kliewer et al. 11, however without any exact evidence of their neuroectodermal origin. It is also evident that the expression of GFAP cannot be regarded as an unambiguous support of the neuroectodermal cell origin. This marker has been found in quite histogenetically different elements, especially in myoepithelial cells of normal and tumorous tissues 23 • On the other hand, the S-100 protein is now known to be a ubiquitous antigen and its presence in myoepithelial elements is well documented 3, 19,20,22. The myoepithelial origin of SCs has been suggested by Martin et al. 14 in carcinoid tumors. In contrast, Barbareschi
et al. 1 have not found SCs immunoreactivity for actin in 12 bronchial carcinoids. The above mentioned shows that SCs histogenesis remains uncertain and that the data in the literature are often contradictory. Our results have shown a strong reactivity for S-100 protein but a very low expression of GFAP - in one subject only though an autocontrol with positive neural structures to GFAP in examined pulmonary tissue was often found. This finding remains obscure and it will be interesting to investigate this phenomenofi in tumorlet-related neuroendocrine lesions. We assume that S-100 protein positive SC cells in the presented set are of mesenchymal (myofibrocytic or myoepithelial) rather than neuroectodermal origin. The reason for this hypothesis is their low expression of GFAP and their high immunoreactivity for vimentin a desmin (62% and 44%). Finally, Schroder and Johannsen 21 have suggested that SCs in paragangliomas and phaeochromocytomas are the neoplastic elements, and tumours rich with SCs, are the biphasic lesions. The presented findings of our seven cases with tumourlets intensively infiltrated with SCs (cluster 2 and 3) seem to support this hypothesis. They may represent the lesions with the biphasic differentiation potential and could be regarded as the possible precursors of the pulmonary paraganglioma or lesions described by Barbareschi et al. 1 and labeled as paragangliod carcinoids.
Material and Methods The autopsy and surgical pathology files of the Department of Pathology, Charles University, were surveyed to retrieve samples of 24 pulmonary carcinoid tumorlets. Formalinfixed, paraffin-embedded tissue blocks were used. The argyrophil technique supplemented by immunohistochemistry in the streptavidin-biotin-peroxidase complex method, as previously described 6,7, were applied for tumorlet categorization. The argyrophil technique was performed using the Grimelius silver-impregnation method. Monoclonal antibody to neuron specific enolase (Dakopatts, Denmark, dilution 1 : 400) was used for complementary detection of tumourlet elements. SCs in tumor lets were studied by immunostaining using antibodies to S-100 protein (Dakopatts, Denmark, dilution
S-100 Protein Positive Cells in Tumorlets . 417 1 : 4000) together with monoclonal antibodies to glial fibrillary acidic protein-GFAP (BioGenex, USA, dilution 1 : 200), cytokeratins (clones AElIAE3, Boehringer, Germany, dilution 1: 200), desmin (BioGenex, USA, dilution 1: 400), vimentin (Dakopatts, Denmark, dilution 1: 50) and actin (BioGenex, USA, dilution 1: 100). Tumorlets with S-100 protein positive SCs have been photographed in high-power magnification (x 480) and the total number of tumourlet cells and the number of SCs was counted on microphotographs. The final number of the SCs was then calculated per 10,000 tumourlet elements. For statistical analysis, the number of SCs was considered by frequency histogram and the case difference in SCs number was evaluated by cluster analysis.
Acknowledgments We wish to thank Prof. Dr. Thomas Gral, UCLA-UCI, CA, YSA for reviewing the translation of this paper and Blanka Spicarova for her technical assistance.
References 1 Barbereschi M, Frigo B, Mosca L, Arrigoni GP, Leonardi E, Wi lander E, Siegel GP, Shiro BC (1990) Bronchial carcinoids with S-100 positive sustentacular cells. A comparative study with gastrointestinal carcinoids, pheochromocytomas and paragangliomas. Path Res Pract 186: 212-222 2 Bencini C, Pulera N (1991) The carotid bodies in bronchial asthma. Histopathology 18: 281-283 3 Dail DH, Hammar SP (1994) Pulmonary pathology, p 1293, 2nd edition. Springer, Heidelberg 4 Gosney JR, Denley H, Resl M, Beesley C (1994) Sustentacular cells in bronchial carcinoid tumours (Suppl). J. Pathol 173: 179 5 Granger JK, Houn HY (1990) Head and neck paragangliomas: a clinicopathologic study with DNA flow cytometric analysis. South Med J 83: 1407-1412 6 Grimelius L (1968) The argyrophil reaction in islet cells of adult human pancreas studied with a new silver nitrate procedure. Acta Soc Med Upsaliensis 73: 271-294 7 Hsu S, Raine L, Fanger H (1981) Use of avidin-biotinperoxidase complex [ABC] in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody [PAP] procedures. J Histochem Cytochem 29: 577-580 8 Johnson TL, Zabro RJ, Lloyd RV, Crissman JD (1988) Paragangliomas of the head and neck: immunohistochemical, neuroendocrine and intermediate filament typing. Mod Pathol18: 216-223 9 Kahn JH, Marks A, Thorn H, Baumal R (1983) Role of antibody to S-100 protein in diagnostic pathology. Am J Clin Pathol 79: 341-347
10 Khan Q, Smith P, Heath D (1990) The distribution of enkephalins in human carotid bodies showing cellular proliferation and chronic glomi tis. Arch Pathol Lab Med 114: 1232-1235 11 Kliewer KE, Wen DR, Cancilla PA (1989) Paragangliomas: assesment of prognosis by histologic, immunohistochemical, and ultrastructural techniques. Hum Pathol 20: 29-39 12 Kondo H, Iwanaga T, Nakajima T (1982) Immunohistochemical study on the localization of neuron-specific enolase and S-100 protein in the carotid body of rats. Cell Tissue Res 227: 291-295 13 Lunqvist M, Wilander E (1987) A study of the histopathogenesis of carcinoid tumors of the'small intestine and appendix. Cancer 60: 201-206 14 Martin JME, Maung RT (1987) Differential immunohistochemical reaction of carcinoid tumors. Hum Pathol 18: 941-945 15 Miettinen M, Saari A (1988) Pheochromocytoma combined with malignant schwannoma: unusual neoplasm of the adrenal medulla. Ultrastruct Pathol12: 513-527 16 Nakajima T, Watanabe S, Sato Y, Kameya T, Hirota T, Shimosato Y (1982) An immunoperoxidase study of S-100 protein distribution in normal and neoplastic tissues. Am J Sur~ Pathol 6: 715 -727 1 Pagel W (1926) Dber den Zusammenhang von ungewohnlichen Wucherungen atypischen und ortsfremden Epithels der Bronchien mit Bronchiektasien, Virchows Arch Pathol Anat 262: 583-594 18 Ranchod M (1977) The histogenesis and development of pulmonary tumorlets. Cancer 39: 1135 -1145 19 Rosai J (1989) Ackerman's surgical pathology, 7th ed, p 41. CV Mosby, Washington, D.C. 20 Seifert G (1991) Histological typing of salivary gland tumours. World Health Organization, International Histological Classification of Tumours, 2nd ed, pp 6-28. Springer, Heidelberg 21 Shroder HD, Johannsen L (1986) Demonstration of S100 protein in sustentacular cells of phaechromocytomas and paragangliomas. Histopathology 10: 1023-1033 22 Tavassoli FA (1991) Myoepithelial lesions of the breast. Am J Surg Pathol 15: 554-568 23 Thompson SJ, Schatteman GC, Gown AM, Bothwell M (1989) A monoclonal antibody against nerve growth factor receptor. Immunohistochemical analysis of normal and neoplastic human tissue. Am J Clin Pathol 92: 415-423 24 Weiss L (1988) Cell and tissue biology, 6th ed, p 756. Urban-Schwarzenberg, Baltimore 25 Whitwell F (1955) Tumorlets of the lung. J Pathol Bact 70: 529-541
Received November 13, 1995 . Accepted in revised form January 4, 1996
Key words: Lung - Neuroendocrine tumors - Carcinoid - Tumorlet - Paraganglioma - Sustentacular cell- S-l 00 protein - Glial fibrillary acidic protein MUDr. Milan Resl, CSc, Department of Pathology, Faculty Hospital of Charles University, CZ 500 36 Hradec Kralove, Czech Republic, Fax: 049-2435
S-100 Protein Positive (Sustentacular) Cells in Pulmonary Carcinoid Tumorlets: a Quantitative Study of 24 Cases M. ResP, B. Knll 2, J. Simek3 and J. Bukac 4 University Departments of Pathology', Medicine 2, Cardiovascular and Thoracic Surgery3 and Computer Technology Center4, Charles University, Hradec Kralove, Czech Republic
SUMMARY Stellate-shaped S-100 protein positive sustentacular cells (SCs) appear to correlate inversely with the degree of tumor malignancy in some neuroendocrine tumors. Therefore, the SCs have been investigated in carcinoid pulmonary tumorlets and subsequently examined quantitatively in order to provide a basis for estimation of this phenomenon in tumorlet related lesions, especially carcinoids and neuroendocrine carcinomas. Pulmonary tissue from twenty-four patients with carcinoid tumorlets was studied immunochistochemically for S-l 00 protein positive SCs together with glial fibrillary acidic protein, actin, desmin, vimentin and cytokeratins. Tumorlet SCs were calculated per 10,000 tumorlet elements. The presence of SCs was proven in 18 subjects (75%) of all examined cases with quantitative frequency between 13 and 196 SCs per 10,000 tumorlet elements. The histogram showed three separate clusters of cases. Cluster 2 and cluster 3 with strikinglyhigh frequencyofSCs, i.e. from 66 up to 196 SCs per 10,000 tumorletcells, may represent the biphasic differentiation potential of tumorlet elements. The mentioned lesions could be regarded as the possible precursors of pulmonary paragangliomas or paraganglioid carcinoids.
Introduction Pulmonary tumorlets were first described by Pagel in 1924 17 and the term tumorlet was coined by Whitwell in 1955 25 for small cellular nests formed of uniform elements with a fusiform or polygonal nuclei and a variable amount of cytoplasma. In 1977, Ranchod labeled this neuroendocrine lesion as carcinoid tumorlet, a term which expresses an association with carcinoid tumor l8 • Sustentacular cells (SCs) are a well-recognized cell population in the paraganglionar system and other tis0344-0338/96/0192-0414$3.50/0
sues, e.g. in the olfactory epithelium or adrenal medullaB, 10, 16,24. They represent the secondary elements which probably provide a growth-regulatory support for endocrine cells, but their exact histogenetic origin and function remains obscure. These stellate-shaped cells have also been demonstrated in paragangliomas and pheochromocytomass, 8,11,15, and other neuroendocrine tumours 3, 4,13. They appear to correlate inversely with the degree of tumor malignancy. In view of this fact, we have investigated quantitatively the SCs in a large series of carcinoid tumorlets in order to provide a basis for © 1996 by Gustav Fischer Verlag, Stuttgart
5-100 Protein Positive Cells in Tumorlets . 415
46 and 91 (mean 75) years. Of them, twenty-three lesions appeared as incidental microscopical findings in autopsy material, and the remaining case was found in surgical pathology material as a result of retrospective analysis of 137 surgically treated non tumorous lesions during the last 12 years. Three additional subjects from this surgical material had been rejected from the set due to insufficient amount of tumourlet elements in serial sections necessary for immunohistochemistry, i.e. the investigated lesion had been cut-up. Morphologically, all carcinoid tumorlets displayed the typical small nests of the uniform elements with a fusiform, polygonal or mixed nuclear configuration and a variable amount of the cytoplasm in the connective tissue stroma (Figs. 1, 2). All 24 subjects with tumorlets showed the cellular argyrophilia and the strong labeling for neuron specific enolase. A distinct cellular labeling for S-l 00 protein together with cytologic features was used for the SCs identification, i.e. ovoid nuclei with a small chromatin amount and long slender plasmatic processes (Fig. 3). There was no evidence of SCs in six cases (25%) out of all 24 examined cases. There was only one case (4 %) with the evidence of labeling for GFAP, and the immunoreactivity of the SCs for vimentin and desmin was 62,5% and 44,4%, respectively. In all cases the SCs were negative for cytokeratin expression. The quantitative frequency of SCs in the remaining 18 positive cases (75%) was between 13 and 196 SCs per 10,000 tumorlet elements. The frequency of SCs in individual cases, sex and age of each of the twenty four subjects is shown in Table 1. Table 4 shows the SCs number histogram of all 24 examined case. The SCs number difference investigated by cluster analysis showed three separate clusters. Cluster 1 consisting of 17 cases with a frequency of SCs up to 46 elements per 10,000 tumorlet cells, cluster 2 consisting of 5 cases with a frequency between 66 and 111 SCs per 10,000 elements, and cluster 3 consisting of 2 cases with strikingly high frequency of SCs, i.e. 189 and 196 of these elements per 10,000 tumorlet cells. These clusters can be easily identified on the attached histogram (Table 2).
Discussion
subsequent estimation of this phenomenon in tumorletrelated lesions, and to discuss their histogenetic origin. Results Twenty-four tumourlets (10 men and 14 women) have been identified in patients between the ages of
We presume that the investigated cells are identical or strongly related elements to SCs previously described in normal tissues and pathological lesions 2 5,8,10,11,13,15,16,24. We have the following reasons for the above mentioned statement: stellate cellular shape, localization of the examined elements within the lesion and their expression of S-100 protein. The results in the presented study prove the presence of S-100 protein positive (sustentacular) cells in pulmonary carcinoid tumorlets and support the premise that the SCs represent the cell population observed in the entire spectrum of the neuroendocrine pulmonary le-
416 . M. Resl et al. Table 1. The frequency of sustentacular cells per 10,000 tumorlet elements, sex and age of twenty-four cases
Case No. SCs Sex Age Case No. SCs Sex Age
1 36 F 88 13 0 M 71
2 40 M 84 14 0 F 75
3 97 M 89 15 66 F 69
4 0 F 91 16 92 M 46
5 23 F 80 17 18 F 73
6 0 M 88 18 111 F 79
7 22 F 68 19 93 M 81
8 196 F 48 20 28 F 82
9 15 F 84 21 28 F 82
10 189 M 62 22 13 M 83
11 46 F 77 23 0 M 71
12
0 M 74 24 28 M 51
F: Female, M: male, SCs: sustentacular cells Table 2. Sustentacular cell number histogram of 24 investigated cases. The abscissa expresses the SCs number, and the ordinate the number of examined cases
10 8 6 4
2 0+--+--+--+--~-+--~-r--~~-4
o
20· 40 60 80 100 120 140 160 180 200
sions, i.e. tumorlets, both types of carcinoids, paragangliomas and small cell carcinomas described elsewhere 1,3,13. Their role as an indicator of tumor malignancy is a matter of research 4 • The frequency of SCs in the examined set is 75% of all 24 subjects and the quantitative SC frequency in individual tumourlets is up to 196 SCs per 10,000 tumorlet elements. SCs have been examined in the rat carotid body, the human adrenal medulla and the paraganglionar system 9, 16, 24. Kondo et al. 12 have suggested that SCs represent homologous elements to Schwann cells for their topographic continuity with Schwann cells and the expression of S-1 00 protein. This opinion has been supported by description of the immunoreactive SCs for human nerve growth factor in 8 pheochromocytomas and 3 paragangliomas 23 • The SCs have also been found as positive elements for GFAP and S-100 protein in 16 of 18 paragangliomas 11. Ultrastructural description of SCs in one subject of low grade paraganglioma was provided by Kliewer et al. 11, however without any exact evidence of their neuroectodermal origin. It is also evident that the expression of GFAP cannot be regarded as an unambiguous support of the neuroectodermal cell origin. This marker has been found in quite histogenetically different elements, especially in myoepithelial cells of normal and tumorous tissues 23 • On the other hand, the S-100 protein is now known to be a ubiquitous antigen and its presence in myoepithelial elements is well documented 3, 19,20,22. The myoepithelial origin of SCs has been suggested by Martin et al. 14 in carcinoid tumors. In contrast, Barbareschi
et al. 1 have not found SCs immunoreactivity for actin in 12 bronchial carcinoids. The above mentioned shows that SCs histogenesis remains uncertain and that the data in the literature are often contradictory. Our results have shown a strong reactivity for S-100 protein but a very low expression of GFAP - in one subject only though an autocontrol with positive neural structures to GFAP in examined pulmonary tissue was often found. This finding remains obscure and it will be interesting to investigate this phenomenofi in tumorlet-related neuroendocrine lesions. We assume that S-100 protein positive SC cells in the presented set are of mesenchymal (myofibrocytic or myoepithelial) rather than neuroectodermal origin. The reason for this hypothesis is their low expression of GFAP and their high immunoreactivity for vimentin a desmin (62% and 44%). Finally, Schroder and Johannsen 21 have suggested that SCs in paragangliomas and phaeochromocytomas are the neoplastic elements, and tumours rich with SCs, are the biphasic lesions. The presented findings of our seven cases with tumourlets intensively infiltrated with SCs (cluster 2 and 3) seem to support this hypothesis. They may represent the lesions with the biphasic differentiation potential and could be regarded as the possible precursors of the pulmonary paraganglioma or lesions described by Barbareschi et al. 1 and labeled as paragangliod carcinoids.
Material and Methods The autopsy and surgical pathology files of the Department of Pathology, Charles University, were surveyed to retrieve samples of 24 pulmonary carcinoid tumorlets. Formalinfixed, paraffin-embedded tissue blocks were used. The argyrophil technique supplemented by immunohistochemistry in the streptavidin-biotin-peroxidase complex method, as previously described 6,7, were applied for tumorlet categorization. The argyrophil technique was performed using the Grimelius silver-impregnation method. Monoclonal antibody to neuron specific enolase (Dakopatts, Denmark, dilution 1 : 400) was used for complementary detection of tumourlet elements. SCs in tumor lets were studied by immunostaining using antibodies to S-100 protein (Dakopatts, Denmark, dilution
S-100 Protein Positive Cells in Tumorlets . 417 1 : 4000) together with monoclonal antibodies to glial fibrillary acidic protein-GFAP (BioGenex, USA, dilution 1 : 200), cytokeratins (clones AElIAE3, Boehringer, Germany, dilution 1: 200), desmin (BioGenex, USA, dilution 1: 400), vimentin (Dakopatts, Denmark, dilution 1: 50) and actin (BioGenex, USA, dilution 1: 100). Tumorlets with S-100 protein positive SCs have been photographed in high-power magnification (x 480) and the total number of tumourlet cells and the number of SCs was counted on microphotographs. The final number of the SCs was then calculated per 10,000 tumourlet elements. For statistical analysis, the number of SCs was considered by frequency histogram and the case difference in SCs number was evaluated by cluster analysis.
Acknowledgments We wish to thank Prof. Dr. Thomas Gral, UCLA-UCI, CA, YSA for reviewing the translation of this paper and Blanka Spicarova for her technical assistance.
References 1 Barbereschi M, Frigo B, Mosca L, Arrigoni GP, Leonardi E, Wi lander E, Siegel GP, Shiro BC (1990) Bronchial carcinoids with S-100 positive sustentacular cells. A comparative study with gastrointestinal carcinoids, pheochromocytomas and paragangliomas. Path Res Pract 186: 212-222 2 Bencini C, Pulera N (1991) The carotid bodies in bronchial asthma. Histopathology 18: 281-283 3 Dail DH, Hammar SP (1994) Pulmonary pathology, p 1293, 2nd edition. Springer, Heidelberg 4 Gosney JR, Denley H, Resl M, Beesley C (1994) Sustentacular cells in bronchial carcinoid tumours (Suppl). J. Pathol 173: 179 5 Granger JK, Houn HY (1990) Head and neck paragangliomas: a clinicopathologic study with DNA flow cytometric analysis. South Med J 83: 1407-1412 6 Grimelius L (1968) The argyrophil reaction in islet cells of adult human pancreas studied with a new silver nitrate procedure. Acta Soc Med Upsaliensis 73: 271-294 7 Hsu S, Raine L, Fanger H (1981) Use of avidin-biotinperoxidase complex [ABC] in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody [PAP] procedures. J Histochem Cytochem 29: 577-580 8 Johnson TL, Zabro RJ, Lloyd RV, Crissman JD (1988) Paragangliomas of the head and neck: immunohistochemical, neuroendocrine and intermediate filament typing. Mod Pathol18: 216-223 9 Kahn JH, Marks A, Thorn H, Baumal R (1983) Role of antibody to S-100 protein in diagnostic pathology. Am J Clin Pathol 79: 341-347
10 Khan Q, Smith P, Heath D (1990) The distribution of enkephalins in human carotid bodies showing cellular proliferation and chronic glomi tis. Arch Pathol Lab Med 114: 1232-1235 11 Kliewer KE, Wen DR, Cancilla PA (1989) Paragangliomas: assesment of prognosis by histologic, immunohistochemical, and ultrastructural techniques. Hum Pathol 20: 29-39 12 Kondo H, Iwanaga T, Nakajima T (1982) Immunohistochemical study on the localization of neuron-specific enolase and S-100 protein in the carotid body of rats. Cell Tissue Res 227: 291-295 13 Lunqvist M, Wilander E (1987) A study of the histopathogenesis of carcinoid tumors of the'small intestine and appendix. Cancer 60: 201-206 14 Martin JME, Maung RT (1987) Differential immunohistochemical reaction of carcinoid tumors. Hum Pathol 18: 941-945 15 Miettinen M, Saari A (1988) Pheochromocytoma combined with malignant schwannoma: unusual neoplasm of the adrenal medulla. Ultrastruct Pathol12: 513-527 16 Nakajima T, Watanabe S, Sato Y, Kameya T, Hirota T, Shimosato Y (1982) An immunoperoxidase study of S-100 protein distribution in normal and neoplastic tissues. Am J Sur~ Pathol 6: 715 -727 1 Pagel W (1926) Dber den Zusammenhang von ungewohnlichen Wucherungen atypischen und ortsfremden Epithels der Bronchien mit Bronchiektasien, Virchows Arch Pathol Anat 262: 583-594 18 Ranchod M (1977) The histogenesis and development of pulmonary tumorlets. Cancer 39: 1135 -1145 19 Rosai J (1989) Ackerman's surgical pathology, 7th ed, p 41. CV Mosby, Washington, D.C. 20 Seifert G (1991) Histological typing of salivary gland tumours. World Health Organization, International Histological Classification of Tumours, 2nd ed, pp 6-28. Springer, Heidelberg 21 Shroder HD, Johannsen L (1986) Demonstration of S100 protein in sustentacular cells of phaechromocytomas and paragangliomas. Histopathology 10: 1023-1033 22 Tavassoli FA (1991) Myoepithelial lesions of the breast. Am J Surg Pathol 15: 554-568 23 Thompson SJ, Schatteman GC, Gown AM, Bothwell M (1989) A monoclonal antibody against nerve growth factor receptor. Immunohistochemical analysis of normal and neoplastic human tissue. Am J Clin Pathol 92: 415-423 24 Weiss L (1988) Cell and tissue biology, 6th ed, p 756. Urban-Schwarzenberg, Baltimore 25 Whitwell F (1955) Tumorlets of the lung. J Pathol Bact 70: 529-541
Received November 13, 1995 . Accepted in revised form January 4, 1996
Key words: Lung - Neuroendocrine tumors - Carcinoid - Tumorlet - Paraganglioma - Sustentacular cell- S-l 00 protein - Glial fibrillary acidic protein MUDr. Milan Resl, CSc, Department of Pathology, Faculty Hospital of Charles University, CZ 500 36 Hradec Kralove, Czech Republic, Fax: 049-2435