Path. Res. Pract. 187, 530-533 (1991)
Expression of Glycoprotein Hormones and Intracytoplasmic Distribution of Cytokeratin in Growth Hormone-Producing Pituitary Adenomas T. Sano and T. Ohshima 1 Departments of Pathology and lNeurological Surgery, University of Tokushima School of Medicine, Tokushima, Japan
S.Yamada Department of Neurosurgery, Toranomon Hospital, Tokyo, Japan
SUMMARY
Sixteen growth hormone (GH)-producing pituitary adenomas were studied for the expression of glycoprotein hormone subunits and cytokeratin by light microscopic immunohistochemistry. Cytokeratin immunoreactivity was demonstrated in all adenomas, but its intracytoplasmic distribution showed two distinct patterns: a prominent, dot-like pattern and a diffuse, perinuclear pattern. Seven adenomas (type 1) were exclusively composed of cells with cytokeratin in a dot-like pattern, whereas 9 adenomas (type 2) comprised of cells with cytokeratin of perinuclear distribution. The expression of a-subunit of glycoprotein hormone was significantly different between the two types of adenomas; 8 of9 adenomas of type 2 contained many a-subunit immunoreactive cells but none of type 1 adenomas showed any immunoreactivity. Only a small number of adenoma cells were positive for ~-subunit of thyrotropin stimulating hormone in 3 adenomas of type 2. ~-subunits of follicle stimulation hormone and luteinizing hormone were negative in all adenomas. These findings suggest that the expression of glycoprotein hormone subunits in GH-producing adenomas may be closely linked to their types distinguishable by the cytokeratin distribution pattern.
IDtroduction Growth hormone (GH)-producing pituitary adenomas are classified into several subtypes based on ultrastructural and immunohistochemical observations6 • Among them sparsely granulated somatotroph adenomas are known to harbor perinuclear filamentous globular bodies named fibrous bodies, which are composed of type 2 filaments and contain cytokeratin3,9. Immunohistochemically, GH-producing adenomas have been shown to express glycoprotein hormones and their subunits; a-subunit of glycoprotein hormones is 0344-0338/91/0187-0530$3.50/0
often observed in GH-producing adenomas 1, 7, 8, 13, and thyroid stimulating hormone (ISH), follicle stimulating hormone (FSH), and luteinizing hormone (LH) are also reportedly demonstrated8, 10, 11. However, since some of the antisera against glycoprotein hormones may contain contaminated antibodies against a-subunit as has been pointed out2,12, a careful evaluation of immunoreactivity is of importance. We studied immunohistochemically the intracytoplasmic distribution of cytokeratin and the expression of glycoprotein hormones in GH-producing adenomas by use of preabsorbed antisera with a-subunit, and found that © 1991 by Gustav Fischer Verlag, Stuttgart
Glycoprotein hormones and cytokeratin in GH-producing adenomas· 531
there are at least two types of adenomas; one is rich in cytokeratin-positive, dot-like globules but devoid of asubunit expression, whereas another shows perinuclear, diffuse distribution of cytokeratin and mostly positive a-subunit immunoreactivity. Material and Methods Pituitary adenomas removed surgically from 16 patients with acromegaly were studied by light microscopic immunohistochemistry. All specimens examined were fixed in formalin and embedded in paraffin. Immunohistochemistry was performed by the avidin-biotin-peroxidase complex (ABC) method using an ABC kit (Vector Lab). The following primary antisera were used: anti-GH (DAKO, 1: 800), anti-prolactin (DAKO, 1: 2000), anti-B-hTSH (NIDDK, 1 : 1200), anti-BhFSH (NIDDK, 1 : 8000) and anti-BhLH (NIDDK, 1: 10000). Monoclonal antibodies against a-subunit of human chorionic gonadotropin (ICN Immuno Biologicals, 1 : 1000) and against human cytokeratin CAM 5.2 (Becton-Dickinson, 25 ftg/ml) were also utilized. CAM 5.2 is specific for cytokeratins 8 and 18. Incubation of the sections with the primary antibodies was done overnight at 4 dc. Before incubation with CAM 5.2, the sections were digested by trypsin for 30 min. The specificity of the antibodies against glycoprotein hormone subunits was verified by preabsorption tests using a-subunit at a concentration of 1.0 ftg/1.0 ml of finally diluted antibodies.
Results GH-producing pituitary adenomas could be divided into two types according to immunohistochemical findings
Fig. 1. GH-producing adenoma, type 1 (Case 6). Cytokeratin immunoreactivity exhibits a prominent, dotlike appearance (A). aSubunit is totally negative (B). ABC method, counterstained with methylgreen, x 530.
Table 1. Immunohistochemical findings of GH-producing adenomas GH type 1 1. 36F 2. 40F 3. 37M 4.40M 5. 48F 6.40F 7. 70F
++ +++ ++ ++ ++ ++ ++
type 2 8. 67F 9. 33F 10. SSM 11. 47M 12. 56F 13. 61F 14. 35F 15. 62M 16. 65F
++++ ++++ ++++ ++++ ++++ +++ +++ +++ ++++
PRL
BTSH
aSU
Dot Dot Dot Dot Dot Dot Dot
+ + + ++ + + +
cytokeratin
+ + + + Perinuclear + + +
+++ ++ ++++ +++ ++++ +++ ++++
Perinuclear Perinuclear Perinuclear Peri->Dot Peri->Dot Peri->Dot Peri->Dot Peri->Dot
++++ = most tumor cells are positive; +++ = many tumor ++ = frequent tumor cells; + = occasional tumor cells;
cells;
- = no positive cells.
(Table 1). Type 1 adenomas were exclusively composed of cells with prominent, dot-like distribution of cytokeratin immunoreactivity (Fig. 1A). The number of GH immunoreactive cells was varying from case to case, but in general smaller than that in type 2 adenomas. Interestingly, there
532 . T. Sano, T. Ohshima and S. Yamada
Fig. 2. GH-producing adenoma, type 2 (Case 10). Cytokeratin is distributed in a diffuse, perinuclear pattern (S). a-Subunit is demonstrated in many tumor cells (S). ABC method, counterstained with methylgreen, X
was no type 1 adenoma that contained immunoreactive cells for a-subunit of glycoprotein hormones or ~TSH (Fig. 1B). In type 2 adenomas, immunoreactive cytokeratin was located in the perinuclear zone to surround the nucleus either partially or completely (Fig. 2A). In a few cases of type 2 adenomas, a minor cell component contained cytokeratin with a dot-like appearance, whereas the majority showed a perinuclear diffuse pattern of cytokeratin immunoreactivity. Almost all tumor cells in type 2 adenomas were positive for GH. In contrast to type 1 adenomas, all but one type 2 adenomas (89%) contained many a-subunit immunoreactive cells (Fig. 2B), and a few adenomas (33%) contained occasional cells reacting for ~TSH. a-Subunit positivity was abolished by preabsorption with a-subunit, but ~TSH positivity was not. No ~FSH or ~LH immunoreactive cells were observed in a1l16 adenomas examined. Discussion Immunohistochemistry using monoclonal antibody CAM 5.2 clearly demonstrated cytokeratin distribution in tumor cells of GH-producing pituitary adenomas. This antibody seems most appropriate for the detection of cytokeratin in pituitary adenomas, because the endocrine cells of the human pituitary have been reported to express only cytokeratins 8 and 18S, and CAM 5.2 is specific for these two cytokeratins. Two distinct patterns of intracytoplasmic distribution of cytokeratin were recognized: prominent, dot-like pattern and diffuse, perinuclear pattern. Although the former dot-like appearance has often
530.
been documented and considered to be identical with fibrous bodies3,5,9, the latter has not been well-described in GH-producing adenomas so far. Two types of GH-producing adenomas could be distinguished according to the pattern of cytokeratin distribution as well as to the immunoreactivity for GH. Type 1 adenomas characterized by dot-like cytokeratin localization and fewer GH immunoreactive cells seem to correspond to sparsely granulated somatotroph adenomas 3,6, while most of type 2 adenomas with perinuclear cytokeratin and numerous GH cells are likely to be consistent with densely granulated somatotroph adenomas 3,6. The frequency of the expression of a-subunit of glycoprotein hormones in GH-producing adenomas has been reported to be approximately 50-60% 7,8,13, but its relation to tumor types has been little known 4 . The present study revealed that 8 of 16 adenomas (50%) contained a-subunit immunoreactive cells and that type 1 adenomas were devoid of a-subunit expression, whereas type 2 adenomas were mostly positive for a-subunit. However, it remains to be clarified why type 1 adenomas spare asubunit expression. The granular density and the concentration of GH in tumor cells are unlikely to explain it, because type 1 adenomas are totally negative for a-subunit although the intensity of GH immunoreactivity vary from cell to cell within individual adenomas. Thus, the reason may be qualitative rather than quantative; the regulatory mechanism of a-subunit expression may be significantly different between types 1 and 2 adenomas. As for ~-subunits of glycoprotein hormones, no ~FSH or ~LH immunoreactive cell was demonstrated in GHproducing adenomas examined. Only a small number of
Glycoprotein hormones and cytokeratin in GH-producing adenomas· 533 ~TSH immunoreactive cells were observed in a few adenomas, specificity of which was verified by absorption tests with a-subunit. These findings are in accordance with those by Landolt et al. 7 and T rouillas et al.13. However, the reported incidence of the expression of glycoprotein hormones of intact forms or their ~-subunits is considerably different among the authors 7, 8,10,11,13. Since more than a half of GH-producing adenomas express a-subunit and some antisera against ~-subunits may contain antibodies against a-subunit2,12, it is likely that immunohistochemistry by use of such antisera may give positive reaction due to antibodies against a-subunit but not ~-subunits.
Acknowledgement The authors wish to thank National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) for providing antisera against ~hTSH, ~hFSH and ~hLH and the center for Population Research of NICHD, NIH for supply a-subunit of HCG.
References 1 Giannattasio G, Bassetti M (1990) Human pituitary adenomas. Recent advances in morphological studies. J Endocrinol Invest 13: 435-454 2 Girod C, Trouillas J, Claustrat B (1986) The human thyrotropic adenoma: pathologic diagnosis in five cases and critical review of the literature. Semin Diag Pathol 3: 58-68
3 Horvath E, Kovacs K (1978) Morphogenesis and significance of fibrous bodies in human pituitary adenomas. Virchows Arch Cell Path 27: 69-78 4 Jautzke G (1988) Simultaneous production of the alphasubunit of glycoprotein hormones and other hormones in pituitary adenomas. Path Res Pract 183: 601-605 5 Kasper M, Stosiek P, van Muijen GNP, Moll R (1989) Cell type heterogeneity of intermediate filament expression in epithelia of the human pituitary gland. Histochemistry 93: 93-103 6 Kovacs K, Horvath E (1986) Pathology of growth hormoneproducing tumors of the human pituitary. Semin Diag Pathol3: 18-33 7 Landolt AM, Heitz PU, Zenklusen HR (1988) Production of the a-subunit of glycoprotein hormones by pituitary adenomas. Path Res Pract 183: 610-612 8 Osamura RY, Watanabe K (1987) Immunohistochemical co localization of growth hormone (GH) and a-subunit in human GH secreting pituitary adenomas. Virchows Arch A 411: 323-330 9 Neumann PE, GoldmanJE, Horoupian DS, Hess MA (1985) Fibrous bodies in growth hormone-secreting adenomas contain cytokeratin filaments. Arch Pathol Lab Med 109: 505-508 10 Riedel M, Saeger W, Ludecke DK (1985) Grading of pituitary adenomas in acromegaly. Comparison of light microscopical, immunocytochemical, and clinical data. Virchows Arch Pathol Anat 407: 83-95 11 Scheithauer BW, Horvath E, Kovacs K, Laws ERJr, Randall RV, Ryan N (1986) Plurihormonal pituitary adenomas. Semin Diag Pathol3: 69-82 12 Trouillas J, Girod C, Sassolas G, Claustrat B (1986) The human gonadotropic adenoma: pathologic diagnosis and hormonal correlations in 26 tumors. Semin Diag Pathol 3: 42-57 13 TrouillasJ, Girod C, Loras B, Claustrat B, Sassolas G, Perrin G, Buonaguidi R (1988) The TSH secretion in the human pituitary adenomas. Path Res Pract 183: 596-600
Received December 15, 1990 . Accepted January 16, 1991
Key words: Pituitary adenoma - Growth hormone - Cytokeratin - Glycoprotein hormone - Fibrous body Toshiaki Sano, M.D., Department of Pathology, University of Tokushima School of Medicine, 3-18-15 Kuramoto-cho, Tokushima 770, Japan
Expression of Glycoprotein Hormones and Intracytoplasmic Distribution of Cytokeratin in Growth Hormone-Producing Pituitary Adenomas T. Sano and T. Ohshima 1 Departments of Pathology and lNeurological Surgery, University of Tokushima School of Medicine, Tokushima, Japan
S.Yamada Department of Neurosurgery, Toranomon Hospital, Tokyo, Japan
SUMMARY
Sixteen growth hormone (GH)-producing pituitary adenomas were studied for the expression of glycoprotein hormone subunits and cytokeratin by light microscopic immunohistochemistry. Cytokeratin immunoreactivity was demonstrated in all adenomas, but its intracytoplasmic distribution showed two distinct patterns: a prominent, dot-like pattern and a diffuse, perinuclear pattern. Seven adenomas (type 1) were exclusively composed of cells with cytokeratin in a dot-like pattern, whereas 9 adenomas (type 2) comprised of cells with cytokeratin of perinuclear distribution. The expression of a-subunit of glycoprotein hormone was significantly different between the two types of adenomas; 8 of9 adenomas of type 2 contained many a-subunit immunoreactive cells but none of type 1 adenomas showed any immunoreactivity. Only a small number of adenoma cells were positive for ~-subunit of thyrotropin stimulating hormone in 3 adenomas of type 2. ~-subunits of follicle stimulation hormone and luteinizing hormone were negative in all adenomas. These findings suggest that the expression of glycoprotein hormone subunits in GH-producing adenomas may be closely linked to their types distinguishable by the cytokeratin distribution pattern.
IDtroduction Growth hormone (GH)-producing pituitary adenomas are classified into several subtypes based on ultrastructural and immunohistochemical observations6 • Among them sparsely granulated somatotroph adenomas are known to harbor perinuclear filamentous globular bodies named fibrous bodies, which are composed of type 2 filaments and contain cytokeratin3,9. Immunohistochemically, GH-producing adenomas have been shown to express glycoprotein hormones and their subunits; a-subunit of glycoprotein hormones is 0344-0338/91/0187-0530$3.50/0
often observed in GH-producing adenomas 1, 7, 8, 13, and thyroid stimulating hormone (ISH), follicle stimulating hormone (FSH), and luteinizing hormone (LH) are also reportedly demonstrated8, 10, 11. However, since some of the antisera against glycoprotein hormones may contain contaminated antibodies against a-subunit as has been pointed out2,12, a careful evaluation of immunoreactivity is of importance. We studied immunohistochemically the intracytoplasmic distribution of cytokeratin and the expression of glycoprotein hormones in GH-producing adenomas by use of preabsorbed antisera with a-subunit, and found that © 1991 by Gustav Fischer Verlag, Stuttgart
Glycoprotein hormones and cytokeratin in GH-producing adenomas· 531
there are at least two types of adenomas; one is rich in cytokeratin-positive, dot-like globules but devoid of asubunit expression, whereas another shows perinuclear, diffuse distribution of cytokeratin and mostly positive a-subunit immunoreactivity. Material and Methods Pituitary adenomas removed surgically from 16 patients with acromegaly were studied by light microscopic immunohistochemistry. All specimens examined were fixed in formalin and embedded in paraffin. Immunohistochemistry was performed by the avidin-biotin-peroxidase complex (ABC) method using an ABC kit (Vector Lab). The following primary antisera were used: anti-GH (DAKO, 1: 800), anti-prolactin (DAKO, 1: 2000), anti-B-hTSH (NIDDK, 1 : 1200), anti-BhFSH (NIDDK, 1 : 8000) and anti-BhLH (NIDDK, 1: 10000). Monoclonal antibodies against a-subunit of human chorionic gonadotropin (ICN Immuno Biologicals, 1 : 1000) and against human cytokeratin CAM 5.2 (Becton-Dickinson, 25 ftg/ml) were also utilized. CAM 5.2 is specific for cytokeratins 8 and 18. Incubation of the sections with the primary antibodies was done overnight at 4 dc. Before incubation with CAM 5.2, the sections were digested by trypsin for 30 min. The specificity of the antibodies against glycoprotein hormone subunits was verified by preabsorption tests using a-subunit at a concentration of 1.0 ftg/1.0 ml of finally diluted antibodies.
Results GH-producing pituitary adenomas could be divided into two types according to immunohistochemical findings
Fig. 1. GH-producing adenoma, type 1 (Case 6). Cytokeratin immunoreactivity exhibits a prominent, dotlike appearance (A). aSubunit is totally negative (B). ABC method, counterstained with methylgreen, x 530.
Table 1. Immunohistochemical findings of GH-producing adenomas GH type 1 1. 36F 2. 40F 3. 37M 4.40M 5. 48F 6.40F 7. 70F
++ +++ ++ ++ ++ ++ ++
type 2 8. 67F 9. 33F 10. SSM 11. 47M 12. 56F 13. 61F 14. 35F 15. 62M 16. 65F
++++ ++++ ++++ ++++ ++++ +++ +++ +++ ++++
PRL
BTSH
aSU
Dot Dot Dot Dot Dot Dot Dot
+ + + ++ + + +
cytokeratin
+ + + + Perinuclear + + +
+++ ++ ++++ +++ ++++ +++ ++++
Perinuclear Perinuclear Perinuclear Peri->Dot Peri->Dot Peri->Dot Peri->Dot Peri->Dot
++++ = most tumor cells are positive; +++ = many tumor ++ = frequent tumor cells; + = occasional tumor cells;
cells;
- = no positive cells.
(Table 1). Type 1 adenomas were exclusively composed of cells with prominent, dot-like distribution of cytokeratin immunoreactivity (Fig. 1A). The number of GH immunoreactive cells was varying from case to case, but in general smaller than that in type 2 adenomas. Interestingly, there
532 . T. Sano, T. Ohshima and S. Yamada
Fig. 2. GH-producing adenoma, type 2 (Case 10). Cytokeratin is distributed in a diffuse, perinuclear pattern (S). a-Subunit is demonstrated in many tumor cells (S). ABC method, counterstained with methylgreen, X
was no type 1 adenoma that contained immunoreactive cells for a-subunit of glycoprotein hormones or ~TSH (Fig. 1B). In type 2 adenomas, immunoreactive cytokeratin was located in the perinuclear zone to surround the nucleus either partially or completely (Fig. 2A). In a few cases of type 2 adenomas, a minor cell component contained cytokeratin with a dot-like appearance, whereas the majority showed a perinuclear diffuse pattern of cytokeratin immunoreactivity. Almost all tumor cells in type 2 adenomas were positive for GH. In contrast to type 1 adenomas, all but one type 2 adenomas (89%) contained many a-subunit immunoreactive cells (Fig. 2B), and a few adenomas (33%) contained occasional cells reacting for ~TSH. a-Subunit positivity was abolished by preabsorption with a-subunit, but ~TSH positivity was not. No ~FSH or ~LH immunoreactive cells were observed in a1l16 adenomas examined. Discussion Immunohistochemistry using monoclonal antibody CAM 5.2 clearly demonstrated cytokeratin distribution in tumor cells of GH-producing pituitary adenomas. This antibody seems most appropriate for the detection of cytokeratin in pituitary adenomas, because the endocrine cells of the human pituitary have been reported to express only cytokeratins 8 and 18S, and CAM 5.2 is specific for these two cytokeratins. Two distinct patterns of intracytoplasmic distribution of cytokeratin were recognized: prominent, dot-like pattern and diffuse, perinuclear pattern. Although the former dot-like appearance has often
530.
been documented and considered to be identical with fibrous bodies3,5,9, the latter has not been well-described in GH-producing adenomas so far. Two types of GH-producing adenomas could be distinguished according to the pattern of cytokeratin distribution as well as to the immunoreactivity for GH. Type 1 adenomas characterized by dot-like cytokeratin localization and fewer GH immunoreactive cells seem to correspond to sparsely granulated somatotroph adenomas 3,6, while most of type 2 adenomas with perinuclear cytokeratin and numerous GH cells are likely to be consistent with densely granulated somatotroph adenomas 3,6. The frequency of the expression of a-subunit of glycoprotein hormones in GH-producing adenomas has been reported to be approximately 50-60% 7,8,13, but its relation to tumor types has been little known 4 . The present study revealed that 8 of 16 adenomas (50%) contained a-subunit immunoreactive cells and that type 1 adenomas were devoid of a-subunit expression, whereas type 2 adenomas were mostly positive for a-subunit. However, it remains to be clarified why type 1 adenomas spare asubunit expression. The granular density and the concentration of GH in tumor cells are unlikely to explain it, because type 1 adenomas are totally negative for a-subunit although the intensity of GH immunoreactivity vary from cell to cell within individual adenomas. Thus, the reason may be qualitative rather than quantative; the regulatory mechanism of a-subunit expression may be significantly different between types 1 and 2 adenomas. As for ~-subunits of glycoprotein hormones, no ~FSH or ~LH immunoreactive cell was demonstrated in GHproducing adenomas examined. Only a small number of
Glycoprotein hormones and cytokeratin in GH-producing adenomas· 533 ~TSH immunoreactive cells were observed in a few adenomas, specificity of which was verified by absorption tests with a-subunit. These findings are in accordance with those by Landolt et al. 7 and T rouillas et al.13. However, the reported incidence of the expression of glycoprotein hormones of intact forms or their ~-subunits is considerably different among the authors 7, 8,10,11,13. Since more than a half of GH-producing adenomas express a-subunit and some antisera against ~-subunits may contain antibodies against a-subunit2,12, it is likely that immunohistochemistry by use of such antisera may give positive reaction due to antibodies against a-subunit but not ~-subunits.
Acknowledgement The authors wish to thank National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) for providing antisera against ~hTSH, ~hFSH and ~hLH and the center for Population Research of NICHD, NIH for supply a-subunit of HCG.
References 1 Giannattasio G, Bassetti M (1990) Human pituitary adenomas. Recent advances in morphological studies. J Endocrinol Invest 13: 435-454 2 Girod C, Trouillas J, Claustrat B (1986) The human thyrotropic adenoma: pathologic diagnosis in five cases and critical review of the literature. Semin Diag Pathol 3: 58-68
3 Horvath E, Kovacs K (1978) Morphogenesis and significance of fibrous bodies in human pituitary adenomas. Virchows Arch Cell Path 27: 69-78 4 Jautzke G (1988) Simultaneous production of the alphasubunit of glycoprotein hormones and other hormones in pituitary adenomas. Path Res Pract 183: 601-605 5 Kasper M, Stosiek P, van Muijen GNP, Moll R (1989) Cell type heterogeneity of intermediate filament expression in epithelia of the human pituitary gland. Histochemistry 93: 93-103 6 Kovacs K, Horvath E (1986) Pathology of growth hormoneproducing tumors of the human pituitary. Semin Diag Pathol3: 18-33 7 Landolt AM, Heitz PU, Zenklusen HR (1988) Production of the a-subunit of glycoprotein hormones by pituitary adenomas. Path Res Pract 183: 610-612 8 Osamura RY, Watanabe K (1987) Immunohistochemical co localization of growth hormone (GH) and a-subunit in human GH secreting pituitary adenomas. Virchows Arch A 411: 323-330 9 Neumann PE, GoldmanJE, Horoupian DS, Hess MA (1985) Fibrous bodies in growth hormone-secreting adenomas contain cytokeratin filaments. Arch Pathol Lab Med 109: 505-508 10 Riedel M, Saeger W, Ludecke DK (1985) Grading of pituitary adenomas in acromegaly. Comparison of light microscopical, immunocytochemical, and clinical data. Virchows Arch Pathol Anat 407: 83-95 11 Scheithauer BW, Horvath E, Kovacs K, Laws ERJr, Randall RV, Ryan N (1986) Plurihormonal pituitary adenomas. Semin Diag Pathol3: 69-82 12 Trouillas J, Girod C, Sassolas G, Claustrat B (1986) The human gonadotropic adenoma: pathologic diagnosis and hormonal correlations in 26 tumors. Semin Diag Pathol 3: 42-57 13 TrouillasJ, Girod C, Loras B, Claustrat B, Sassolas G, Perrin G, Buonaguidi R (1988) The TSH secretion in the human pituitary adenomas. Path Res Pract 183: 596-600
Received December 15, 1990 . Accepted January 16, 1991
Key words: Pituitary adenoma - Growth hormone - Cytokeratin - Glycoprotein hormone - Fibrous body Toshiaki Sano, M.D., Department of Pathology, University of Tokushima School of Medicine, 3-18-15 Kuramoto-cho, Tokushima 770, Japan
