727
0 1992 The Japanese Society
of Pathology
Nucleolar Organizer Regions in Malignant Salivary Gland Tumors
Shuichi Fujita, Hiroshi Takahashi, and Haruo Okabe Prollferative activity of carcinomas arising from salivary glands was analyzed by enumeration of argyrophilic nucleolar organizer regions (AgNORs). The mean numbers of AgNORs in the various tumors were as follows: mucoepidermoid carcinoma, 2.20 : acinic cell carcinoma, 2.51 ; adenoid cystic carcinoma (ACC), 2.57 ; carcinoma in pleomorphic adenoma, 1.00 (benign component) and 3.99 (cancer-bearing area) ; salivary duct carcinoma, 4.49 ; polymorphous low-grade adenocarcinoma, 3.37 ; sebaceous carcinoma, 2.57 : oncocytic carcinoma, 4.63 ; adenocarcinoma, 4.53. Cells of most tumors showed heterogeneous activity within the same tumor. In mucoepidermoid carcinoma, the mucous cells had suppressed activity in comparison with the epidermoid cells and intermediate cells. In ACC, the activity of the tumor cells increased according to growth pattern in the order tubular, glandular and solid. In carcinoma in pleomorphic adenoma, vigorous proliferative activity was observed in the malignant component, whereas less active cells were seen in the myxoid or chondroid matrix. AgNOR staining was useful for distinguishing benign from malignant regions in carcinoma in pleomorphic adenoma. Our results suggest that mucoeg idermoid carcinoma, acinic cell carcinoma and ACC, except for that with a solid growth pattern, may be considered as low-grade malignancies, whereas solid-type ACC, the cancer component in carcinoma in pleomorphic adenoma and some of the other carcinomas have high-grade malignant behavior. Acta Pathol Jpn 42 : 727-733, 1992. Key words : AgNOR, Cell proliferation, Malignant salivary gland tumor
A simple, silver colloid staining method for nucleolar organizer regions (NORs) has been applied to paraffin sections of tumors in studies of oncogenesis. Benign and malignant tumors show a clear difference in their numbers of AgNORs (1-4),and a relationship between Received March 25, 1992. Accepted for publication July 13, 1992. Department of Oral Pathology, Nagasaki University School of Dentistry, Nagasaki. Mailing address : Shuichi Fujita, Dapartment of Oral Pathology, Nagasaki University School of Dentistry, 1-7-1 Sakamoto, Nagasaki 852, Japan.
Ag NOR count and prognosis has been emphasized (5-7). Ultrastructurally, AgNORs are found in the fibrillar center and the dense fibrillar component of the nucleolus in the interphasic nucleus (8-1l), and are considered to repre sent RNA polymerase I , C23, 823, l O O K and 80K protein in the nucleus (12). These proteins reflect the activity of NOR, that is, loops of ribosomal DNA transcribing rRNA. The AgNOR count per nucleus is thought to represent cellular proliferation, as is also the case for Ki-67 immunoreactivity (13,14) and bromodeoxyuridine (BrdU) labeling index (15). Most tumors arising from the salivary glands show various histological appearances, owing to the diverse shapes and arrangements of the tumor cells and different kinds of extracellular matrix. Carcinoma in pleomorphic adenoma in particular exhibits a complicated structure containing carcinoma-bearing areas and benign pleomorphic adenoma within the same tumor (16). Morgan e t a/. (17)and Matsumura et a/. (18)have reported AgNORs in salivary gland tumors. However, they did not discuss the proliferative activity of the cell types composing the tumors. In this study, the proliferative abilities of the histologic components of malignant salivary gland tumors were investigated using the AgNOR technique. Furthermore, in carcinoma in pleomorphic adenoma, the differences in proliferative activity between the benign and malignant components were clarified.
MATERIALS AND METHODS Thirty-one cases of mucoepidermoid carcinoma, 4 acinic cell carcinomas, 67 adenoid cystic carcinomas (ACCs), 1 1 carcinomas in pleomorphic adenoma, 3 salivary duct carcinomas, 1 polymorphous low-grade adenocarcinoma, 1 sebaceous carcinoma, 1 oncocytic carcinoma and 9 adenocarcinomas were examined. The materials were obtained from the files of the Department of Oral Pathology, Nagasaki University School of Dentistry, and the Pathology Division, Central Diagnostic Laboratory, Nagasaki University Hospital. Some cases
728
NORs in Malignant Salivary Gland Tumors (Fujita et a/.)
were culled from the consultation files of the authors. All samples were fixed in neutral buffered formalin for approximately 2 4 h and embedded in paraffin. Sections 3.5 ,um thick were stained with hematoxylin and eosin for reclassification of histological type. For the AgNOR staining, our modified one-step method (7, 19) was employed. The paraffin sections were dewaxed in xylene, rehydrated through an ethanol series and washed with deionized distilled water. The specimens were immersed in an AgNOR staining solution for 40 min under darkroom conditions a t room temperature. The AgNOR staining solution was a mixture of 2% gelatin dissolved in 1% aqueous formic acid mixed with 50% aqueous silver nitrate in a proportion of 1 : 2. The slides were washed with deionized distilled water. For gold toning, they were immersed in 0.2% aqueous gold chloride for 1 0 min (20). After washing, the sections were placed in 1: 5-diluted photographic fixing solution for 5 min in order to prevent discoloration of the AgNOR dots. The counterstain was Mayer's hematoxylin. The dehydrated sections were mounted in a synthetic medium. The prepared sections were observed using a light microscope equipped with a x 100 oil-immersion lens. The number of AgNOR dots per 100 nuclei of each undermentioned cell type was counted per section by one of the authors. The mean number per nucleus and standard deviation of the AgNORs were then calculated. The results were analyzed by Student's t test. Tumor cell types based on morpholog ica I c ha racteristics were as follows.
Mucoepidermoid carcinoma : mucous cells, epidermoid cells and intermediate cells. The tumors examined were divided into 14 low-grade and 1 7 high-grade malignancies based on the WHO classification (21). Acinic cell carcinoma : classification of tumor cells was not done because the number of cases examined was too low for statistical validity. ACC : These were divided histologically into three growth patterns : tubular, glandular and solid. Tumor cell types in the glandular pattern were cyst-lining cells and small angular cells, and those in the solid pattern were small angular cells. Carcinoma in pleomorphic adenoma : benign component and cancer-bearing area. Classification of detailed histological parts was not performed because the number of cases was not large. Other carcinomas : Classification of tumor cells was not done.
RESULTS The mean numbers of AgNOR per nucleus and the standard deviations in malignant salivary gland tumors are listed in Table 1. Mucoepidermoid
carcinoma
Mucous cells in mucoepidermoid carcinoma formed cystic structures containing a mucous pool in the lumen, or were intermingled with nests of epidermoid cells. They possessed abundant cytoplasm containing mucous
Table 1. Mean Numbers of AgNORs in Malignant Salivary Gland Tumors Histologic component (*)
Histologic Diagnosis (*) Mucoepidermoid carcinoma (31)
Mucous cells (22) Epidermoid cells (30) Intermediate cells (8) Acinic cell carcinoma (4) Adenoid cystic carcinoma (67) Tubular pattern (19) Glandular pattern (51) Cyst-lining cells (51) Small angular cells (51) Solid pattern Small angular cells (7)
Mean AgNOR number-(S.D.) 2.20 (0.60) 1.05 (0.23)'.* 2.64 (0.50)' 2.63 (0.39)' 2.51 (0.71) 2.57 (0.68) 2.07 (0.64)3 2.67 (0.63)3,4 2.59 (0.65) 2.74 (0.74) 3.29 (0.49)'
Carcinoma in pleomorphic adenoma (1 1) Benign region (11) Cancerous region (11)
~.
Salivary duct carcinoma (3) Polymorphous low-grade adenocarcinoma (1) Sebaceous carcinoma (1) Oncocytic carcinoma (1) Adenoca rci no ma (9)
* Number of cases examined
1.00 (0.18)5 3.99 (1.42)5 4.49 (1.61) 3.37 2.57 4.63 4.53 (2.19)
p
0 1992 The Japanese Society
of Pathology
Nucleolar Organizer Regions in Malignant Salivary Gland Tumors
Shuichi Fujita, Hiroshi Takahashi, and Haruo Okabe Prollferative activity of carcinomas arising from salivary glands was analyzed by enumeration of argyrophilic nucleolar organizer regions (AgNORs). The mean numbers of AgNORs in the various tumors were as follows: mucoepidermoid carcinoma, 2.20 : acinic cell carcinoma, 2.51 ; adenoid cystic carcinoma (ACC), 2.57 ; carcinoma in pleomorphic adenoma, 1.00 (benign component) and 3.99 (cancer-bearing area) ; salivary duct carcinoma, 4.49 ; polymorphous low-grade adenocarcinoma, 3.37 ; sebaceous carcinoma, 2.57 : oncocytic carcinoma, 4.63 ; adenocarcinoma, 4.53. Cells of most tumors showed heterogeneous activity within the same tumor. In mucoepidermoid carcinoma, the mucous cells had suppressed activity in comparison with the epidermoid cells and intermediate cells. In ACC, the activity of the tumor cells increased according to growth pattern in the order tubular, glandular and solid. In carcinoma in pleomorphic adenoma, vigorous proliferative activity was observed in the malignant component, whereas less active cells were seen in the myxoid or chondroid matrix. AgNOR staining was useful for distinguishing benign from malignant regions in carcinoma in pleomorphic adenoma. Our results suggest that mucoeg idermoid carcinoma, acinic cell carcinoma and ACC, except for that with a solid growth pattern, may be considered as low-grade malignancies, whereas solid-type ACC, the cancer component in carcinoma in pleomorphic adenoma and some of the other carcinomas have high-grade malignant behavior. Acta Pathol Jpn 42 : 727-733, 1992. Key words : AgNOR, Cell proliferation, Malignant salivary gland tumor
A simple, silver colloid staining method for nucleolar organizer regions (NORs) has been applied to paraffin sections of tumors in studies of oncogenesis. Benign and malignant tumors show a clear difference in their numbers of AgNORs (1-4),and a relationship between Received March 25, 1992. Accepted for publication July 13, 1992. Department of Oral Pathology, Nagasaki University School of Dentistry, Nagasaki. Mailing address : Shuichi Fujita, Dapartment of Oral Pathology, Nagasaki University School of Dentistry, 1-7-1 Sakamoto, Nagasaki 852, Japan.
Ag NOR count and prognosis has been emphasized (5-7). Ultrastructurally, AgNORs are found in the fibrillar center and the dense fibrillar component of the nucleolus in the interphasic nucleus (8-1l), and are considered to repre sent RNA polymerase I , C23, 823, l O O K and 80K protein in the nucleus (12). These proteins reflect the activity of NOR, that is, loops of ribosomal DNA transcribing rRNA. The AgNOR count per nucleus is thought to represent cellular proliferation, as is also the case for Ki-67 immunoreactivity (13,14) and bromodeoxyuridine (BrdU) labeling index (15). Most tumors arising from the salivary glands show various histological appearances, owing to the diverse shapes and arrangements of the tumor cells and different kinds of extracellular matrix. Carcinoma in pleomorphic adenoma in particular exhibits a complicated structure containing carcinoma-bearing areas and benign pleomorphic adenoma within the same tumor (16). Morgan e t a/. (17)and Matsumura et a/. (18)have reported AgNORs in salivary gland tumors. However, they did not discuss the proliferative activity of the cell types composing the tumors. In this study, the proliferative abilities of the histologic components of malignant salivary gland tumors were investigated using the AgNOR technique. Furthermore, in carcinoma in pleomorphic adenoma, the differences in proliferative activity between the benign and malignant components were clarified.
MATERIALS AND METHODS Thirty-one cases of mucoepidermoid carcinoma, 4 acinic cell carcinomas, 67 adenoid cystic carcinomas (ACCs), 1 1 carcinomas in pleomorphic adenoma, 3 salivary duct carcinomas, 1 polymorphous low-grade adenocarcinoma, 1 sebaceous carcinoma, 1 oncocytic carcinoma and 9 adenocarcinomas were examined. The materials were obtained from the files of the Department of Oral Pathology, Nagasaki University School of Dentistry, and the Pathology Division, Central Diagnostic Laboratory, Nagasaki University Hospital. Some cases
728
NORs in Malignant Salivary Gland Tumors (Fujita et a/.)
were culled from the consultation files of the authors. All samples were fixed in neutral buffered formalin for approximately 2 4 h and embedded in paraffin. Sections 3.5 ,um thick were stained with hematoxylin and eosin for reclassification of histological type. For the AgNOR staining, our modified one-step method (7, 19) was employed. The paraffin sections were dewaxed in xylene, rehydrated through an ethanol series and washed with deionized distilled water. The specimens were immersed in an AgNOR staining solution for 40 min under darkroom conditions a t room temperature. The AgNOR staining solution was a mixture of 2% gelatin dissolved in 1% aqueous formic acid mixed with 50% aqueous silver nitrate in a proportion of 1 : 2. The slides were washed with deionized distilled water. For gold toning, they were immersed in 0.2% aqueous gold chloride for 1 0 min (20). After washing, the sections were placed in 1: 5-diluted photographic fixing solution for 5 min in order to prevent discoloration of the AgNOR dots. The counterstain was Mayer's hematoxylin. The dehydrated sections were mounted in a synthetic medium. The prepared sections were observed using a light microscope equipped with a x 100 oil-immersion lens. The number of AgNOR dots per 100 nuclei of each undermentioned cell type was counted per section by one of the authors. The mean number per nucleus and standard deviation of the AgNORs were then calculated. The results were analyzed by Student's t test. Tumor cell types based on morpholog ica I c ha racteristics were as follows.
Mucoepidermoid carcinoma : mucous cells, epidermoid cells and intermediate cells. The tumors examined were divided into 14 low-grade and 1 7 high-grade malignancies based on the WHO classification (21). Acinic cell carcinoma : classification of tumor cells was not done because the number of cases examined was too low for statistical validity. ACC : These were divided histologically into three growth patterns : tubular, glandular and solid. Tumor cell types in the glandular pattern were cyst-lining cells and small angular cells, and those in the solid pattern were small angular cells. Carcinoma in pleomorphic adenoma : benign component and cancer-bearing area. Classification of detailed histological parts was not performed because the number of cases was not large. Other carcinomas : Classification of tumor cells was not done.
RESULTS The mean numbers of AgNOR per nucleus and the standard deviations in malignant salivary gland tumors are listed in Table 1. Mucoepidermoid
carcinoma
Mucous cells in mucoepidermoid carcinoma formed cystic structures containing a mucous pool in the lumen, or were intermingled with nests of epidermoid cells. They possessed abundant cytoplasm containing mucous
Table 1. Mean Numbers of AgNORs in Malignant Salivary Gland Tumors Histologic component (*)
Histologic Diagnosis (*) Mucoepidermoid carcinoma (31)
Mucous cells (22) Epidermoid cells (30) Intermediate cells (8) Acinic cell carcinoma (4) Adenoid cystic carcinoma (67) Tubular pattern (19) Glandular pattern (51) Cyst-lining cells (51) Small angular cells (51) Solid pattern Small angular cells (7)
Mean AgNOR number-(S.D.) 2.20 (0.60) 1.05 (0.23)'.* 2.64 (0.50)' 2.63 (0.39)' 2.51 (0.71) 2.57 (0.68) 2.07 (0.64)3 2.67 (0.63)3,4 2.59 (0.65) 2.74 (0.74) 3.29 (0.49)'
Carcinoma in pleomorphic adenoma (1 1) Benign region (11) Cancerous region (11)
~.
Salivary duct carcinoma (3) Polymorphous low-grade adenocarcinoma (1) Sebaceous carcinoma (1) Oncocytic carcinoma (1) Adenoca rci no ma (9)
* Number of cases examined
1.00 (0.18)5 3.99 (1.42)5 4.49 (1.61) 3.37 2.57 4.63 4.53 (2.19)
p
