TANAKA Shigeo Nippon Medical School, Department of Surgery II,Professor, 医学部, 教授 (70089720)
WATANABE Hidehiro Nippon Medical School, Department of Surgery II,Clinical instructor, 医学部, 助手 (40191788)
IWAMURA Taro Nippon Medical School, Department of Surgery II,Clinical instructor, 医学部, 助手
NAGAHAMA Mitsuji Nippon Medical School, Department of Surgery II,Clinical instructor, 医学部, 助手 (30241102)
名取 穣二 日本医科大学, 医学部, 助手
|Budget Amount *help
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1996: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1995: ¥1,100,000 (Direct Cost: ¥1,100,000)
We studied immunohistochemical and biochemical analysis on neuroendocrine and non-neuroendocrine tumors using various kinds of monoclonal antibodies such as A2B5,4F2, PKK-1 and HISL-19. Among these antibodies, A2B5 and HISL-19 showed similar reactivity to only neuroendocrine cells and their tumors in immunohistochemical investigation. HISL-19, especially, obtained numerously remarkable outcomes because of the advantages that it can vbe applied for formalin-fixed, paraffin-embedded permanent specimens. We, herein, report our results of 2 years study with the HISL-19 as central figure.
[Materials and Methods]
1) Immunohistochemical study : Materials were mainly used formalin-fixed, paraffin embedded surgical specimens of neuroendocrine, and non-neuroendocrine tumors. Indirect immunofluorescence andor SAB procedures were applied for ataining.
2) Biochemical study : Cellular protein from 1 g of cryopreserved surgical specimen of medullary carcinoma of the thyroid (MCT) obtained
at the time of surgery were extracted. Western blotting procedure was applied to determine the molecular weight of HISL-19 antigenic determinant.
3) Immunoelectron microscopic study : Conventional paraffin specimens for light microscope were first immunostained with HISL-19. Positive area was selected and applied for electron microscopic study. Electron microscopic specimens were processed to pre-embedding procedure and viewed with electron microscope with lead staining or no staining.
1) HISL-19 reacted only with MCT in the thyroid neoplasms. In adrenal tumors, pheochromocytoma was positive while adrenocortical tumors were negative to it. In Pancreas, islet cell tumors were positive while pancreatic acinar cells were negative. In gut organ, carcinoid, gastric cell carcinoma were negative, but other esophageal, gastric, rectal, colonic carcinoma were negative. Interestingly, pathologically diagnosed endocrine cell carcinoma from gall bladder were positive to HISL-19. Malignant pheochromocytoma and MCT revealed strongly positive conpared with benign pheochromocytoma in virtue of SAB procedure.
2) Biochemical analysis using Western blotting procedure revealed new antigen HISL-19 band around 42 kDa in benighn pheochromocytoma and MCT which is immunohistochemically positive to HISL-19. Furtheremore, other two bands with mulecular weitght of 55 and 65 kDa were recognized in MCT which showed similar reactivity to malignant pheochromocytoma and did not show in benighn pheochromocytoma.
3) Immunoelectron microscopic procedure demonstrated HISL-19-positive substances which numerously exist in the secretory granules in cytoplasm of the MCT.These positive granules were densely aggregated closed to nucleus and showed a tendency of getting scattered toward apical surface of the tumor cells.
Monoclonal antibody HISL-19 is an clinically useful tool as a apecific marker for neuroendocrine cell tumors described above. Furthermore, malignant endocrine cell tumors appeared as newly synthesized protein. These were demonstrated as two different molecule bands such as 55 and 60 kDa which were not recognized in benign pheochromocytoma (HISL-19-positive : molecular weight of 42 kDa) and other HISL-19-negative tumors. These HISL-19 positive-proteins were located in nuerous secretory granules suggesting unfeasible cell regulation producing endless cell proliveration. Clinically, preoperative immunohistochemical diagnosis using HISL-19 proved to be claryfy the grade of malignancy, estimate the prognosis, make a decision of operative prcedure including extent of resection and lymphnode dissection. Less