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� � ��������� Vol. 33, pp. 393�402, 2005
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� �� ��������� �WHO ��� grade IV� � �!�"��# ���$%&'()"*��+,(� ���"�-#./0� �123�� �4�3�� �567�3� 89:�;-3� $<=>,0/($� ?"�@-���AB#9CDEFG�3"H�I$�JK..L(� �AB M���@����N�O�/0P,>"Q3"�RS�OTUV� 9 "� �WX#���GYG" glial fibrillary acidic protein* ��GYG" laminin"Z[�\]^OT/� _`!aGbGc"dO�/0efKV$#g$O_�h'( �;-7�� �Si+,�jUV� kV� 5 "��WXj> microdissection ��%� �%� &��%O�l0mnK� 4o"�GYG �D3S1284, D7S507, D10S226, D17S786�O�/0 mi-crosatellite pqOTUV� 3 "� � ���%* �% rst�UV microsatellite in-stability $'u>,� �567�3� Ovw'(xy*z=>,V$� 2 � �%#"{microsatellite instability${>, �;-3� #(|K�/*)}+,V� ~m"S�j>� �" ����� #9C7�*+$t�(�,�$��+,V�
��������� � �� _`!aGbGc"d� microdissection, microsatellite
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� � �gliosarcoma: GS� �����*�-���$%&'(���"WX��G�O.�*'(����� � WHO ��1� ��!� �glioblas-toma: GB, grade IV� "��#��+,(� GS "�-��u0� � �"/0� GB "1 2� ���r�"1 0.2*2�+,0/(2�3�� GS "������# GB ��+,� �-����3%WX#{>,(�4� ���� �� �����WX��� 3� �� � �� �� �5�#��'(4��7�� GS "6�� 1895# Stroebe8� $�u0�/V$� 7���"�&��8�+,0/V� K
jK� 1955� Feigin and Gross9� � 3 " GS O2�K� P"�9� �GB :#� '(��12$ �;KV)"� L¡¢*£fKV� �"H�GS �%¤��*K0'�+,� ���"7�*+Op;'(Vu#� ¥¦s"��WX��S�10��13�§7�<=O�/VAB14�15�$T¨,0©V�GS :" ��-#./0h&k # 4."3$
><+,0/(16�� ª��«?�� �123� Feigin and Gross
9� ¬H� ¦s"2�$�+,0/(10�11� �Fig. 1a�� �8 �123� �7�� ����12:@#*+'(*K0/V$� �"H"ABj>12®7�$ �"A¯*z=>,(9¢#�UV17�18�� ª�� �4�3� GB $°±KV4�WX$ �;'(*/¢)" L($19� �Fig.1b�� P"3�4�j>B,0�-'( GS O3; ©²CD�#EK/� ª³� �567�3� �
1 �������� ´}���2 µF�¶·¸ ¹º�G»��G H¼S½
393
37
����������������� �� ������20�21� �Fig. 1c�� ��� GB ��������������� ���� ����� ����Fig. 1d�� ����� �� GB ���� !"#$%&' ��������(�)���*14�15�
�+,-.�� /.0��1 �23�� � �4��� � GS � 2���567)������ �bi-clonal tumor� ��89:� /����;< �=�>?��@A ��16�� B8� GS ��� �/���C6�� ?�� ����� � ������ GS � 1D������ �monoclonal tumor� �E�89:� �23�� F �4��� ��GHI����/.0���JKL�M�8NOPIQRS0�T�6-.8C)�10�11�� 17��19�� UV��W��PIXY�Z: GS�[\� monoclonal tumor���/���]�678C)� Boerman 022�� fluore-
scence in situ hybridization �FISH� � comparativegenomic hybridization �CGH� ���� GS ������������6^_`5a�b0.��*�)� -0�� Birnet 023�� GS �������?� p53cdW�5������ Reis024������� p53�b60e�?� PTEN�5F p16 �CDKN
2A�fg9ZhMDM2� CDK4��i��@A ��*�)�GS ���jk�-0�l�)m�� �XY��
9��� ���8 ����� � ����� ��no �p7)� qrPIo ��st�u^_��vwx y z!{���� ������"|�#},~�����o���b)� �W��PIo��� microdissection $������������%�� microsatellite �MS� ���8�� ���IM&�o �)�
�����
��1997�2004 '�K�����)��� 9 ��o
���)�L 7�� �L 2�� '( 35�73��)� 56.3 ��� ��-.)o`� 10 �������"���� 2�3*���� ������+,�� -.qr���)m��"� ¡��¢£¤��H&E� ^_��/¥¦§¨�^_17��p7)� 69� �XY�©"�ª�«¬�P�01.®¯°� ±�²³´�cdW��XY�°�µ2�38p¶.)� 939·��
Fig. 1. Hypotheses for the histogenesis of gliosarcoma �GS�. a. Vascular theory. Sarcomacells �green� arise from vascular pericytes in glioblastoma �GB, red�. b. Meninxtheory. Sarcoma arises from mesenchymal cells that are transformed from
meningeal cells. c. Precursor cell theory. Both GB and sarcoma cells arise from a
common precursor cell. d. Metaplasia theory. GB cells undergo mesenchymal
metaplasia �yellow�� leading to the development of sarcoma.
¸¹ºl »¼½4 0394
38
�����������3 mm ��������� 0.3� � ����������� fibronectin ����������������� �0.1� trypsin, 37�C� 30 ���glial fibrillary acidic protein: GFAP, laminin, CD10�; proteinase K solution �Dako, Carpinteria,USA, !"�� #$� 5� �CD31�; 0.05� prote-
inase, 37�C� 15� �collagen IV��� %&���'�GFAP ()*+,-.�� �Dako, !"�� �laminin /0*+,-.�� �LAM-89, Novocas-tra, Newcastle, UK, 50�� � fibronectin ()*+,-.�� �Dako, 500�� � collagen IV /0*+,-.�� �CIV22, Dako, !"�� � CD10/0*+,-.�� �56C6, Novocastra, 100� 123� CD31 /0*+,-.�� �HM57, Novo-castra,200� ��4� 56��7 4�C8 169�:;<�� Phosphate-bu#ered saline �PBS� 8=��>?��4� peroxidase-polymer @A&���Envisionuniversal, Dako� 7#$8 30��:;<� diaminobenzidine BC��4D�E����� ���������������������� �0.1� trypsin,
37�C� 30��������� F�����GHD�GFAP ()*+,-.��7:;<� PBS �2I>?�� rhodamine JKL�MNO IgG ���Dako, 50� 7#$8 30 ��:;<�� &��P����� laminin/0*+,-.��QI4'� b-III tubulin /0*+,-.�� �Promega,Madison, USA, 2000� 7 4�C8 169�:;<PBS 8>?���� biotin JKL�RMS IgG
�� �Dako, 500� 123 fluorescein isothiocy-
anate �FITC�@A streptavidin �Dako,25�7#$8T 30 ��@A;<�� UVE�' TO-PRO-3iodide �Molecular Probes, Eugene, USA; 2 mM� ��4�25�� VE �'��W�X �Axiovert 200 M,Carl Zeiss, Jena, Germany� 8YZ�� 30�mW [.\�� 1�mW �])M^_`�� 5�mW a])M^_`���b��: 488�� 543�� 633�nm���cIde�f,g,hSi^�Dj�kl�����LSM510 Meta, Carl Zeiss��MS�����������mVE��4 microdissec-
tion �Leica Microsystems, Wetzlar, Germany� �D
�n�� �n�123�n�������� Pop�56� proteinase K bu#er �50 mM Tris-HCl, pH 8.0; 1 mM ethylene diamine tetraacetic
acid �EDTA�; 0.5� Tween 20; proteinase K �250 mg
�ml�� �8 55�C� 72 9����� q�%��!r7�D��s^t�uR, PCR ��4v DNA�"#����26�� &�� $po�w%�!r�4x� MS R,y, �D3S1284, D7S507, D10S226,D17S786
22�; Invitrogen, Frederick, USA� 123 b-
globin z&{� PCR ��4 �denature: 95�C� 1�� annealing: 52�C� 2�� extension: 72�C� 2��45 cycle; final extension: 72�C� 7��� DNA|%�12� ()[*).[}~�.�'��(�� �����M^8�.VE����� �(�'�(kl���M�[ �Kodak digital science 1D, ver. 2.0.2,New Haven, USA� ��4D)�*�+����
� �
����HEVE8'� vD��,�14D GB7�n�
-���������L,��.cI/01�56��.�� �Fig. 2a�� GB�'�234���5r��6�n��28�;o� 7����"w7%�8'���8�D4�� %9� �n�8':���2�"����8� ���n ¡����56¢n�;£���<�¤�D4�� =mVE8'� GB �8'�2-�2���>���¥�¦?��L,��¤����@�� �n�8'A6��2����§po� -¨�J��B©��ª��Fig. 2b���������n/«1��8QI GFAP27�' GS v,� GB��¬C��¤�� �nD:���2��®'E¯�°�� �Fig. 3a�� �n��F±56²1³´��¯�BGHR,y,� laminin, fibronectin123 IVr collagen
19�28� 7��µ�2R,y,8QI CD1029� �VE����� Laminin, fibronec-tin 7 IVr collagen 'vD��BGH7:���n�2�C��¤���� ¶¨�VE��=mVE��nI´�L,��·¸AJ�� �Fig. 3b, c��CD10'�n�2�%���§C�8� =mVE�L,�7'%J��°���@¹ºK�� »�� ��L?R,y,� CD3130� �F±VE¸����� ��n�2�'v��®'§po�°�� �Fig. 3d��
��n��w¼½ 395
39
��������������� GFAP �rhodamine ��� ���
����� laminin �FITC ��� ���� ���� ��������������������� 9� GS�� GB �� !� ����� !� "#�$� GFAP � laminin ���%&'( �� )*#+,�� �Fig. 4�� +-�./�0���� GFAP �12������ b-
III tubulin31� ���� ��� �3��%&'
( �$45"#� 67�8�9$: "#��Fig. 5��MS ��GS 4;,* microdissection !����� ��
�-<=������>� ?��@ADNA�B50�� DNA ��C4���)!���! b-globin
D�E� PCR �����FG� 4� GS ���H#�@A,*IJ�KLM$�*#H� NOPQLRSTUV�DNA�W$X��Y+04Z,*[\0��]^_��� `a 5������� MS
Wb�]^� Fig. 6 !c&� d 1�����!D3S1284� D10S226�� ���! D7S507� D17S786�MS instability �MSI�$ )*#� d 2�����!�YD7S507�MSI$Y*#�� e��d 3�����!D17S786�MSI$fa� ���!�D3S1284�MSI�D7S507� LOH �loss ofheterozygosity� $ )*#�� d 4 �����!D7S507�MSI� D10S226�gh$fa� ��!� D3S1284, D7S507 � D10S226 � MSI $: "#�� d 5�4;!�����$ie#+,��$� D3S1284, D7S507, D10S226 ����������j� MS Nk�L�c0� D17S786 ����� MSI ��0����
�
GS � GB ���$lm&' n+@Ao,*pq3�r!�stu>� ��v���wxy!u��z{,*"|$+"#�t�10��13�� 19�� z#�!�GS �\$%v� �GB� �V$%v�����,*+'l}���~�*#� ���������%��!&'0 GB ��()0��w&'�"#���17�18�� 0,0� �*��Ew+��pq!<aGS� monoclonal tumor ���v�� GB f'�� GB �������!&'&'� ��,W$�eauuf'22��24�� ��0� ��w�� � ������ ��H#$�0��,���-.�� ���F���W.!�>����pq�����eH� GS !� GB ,*�%����&' ��w�� $��!�m&'�,�/�@A��!400�� ��� ��w�� � GB ������V�!��0� �3� n��������%&'� ���� ��)�� F#!¡¢t GFAP �laminin ���� ����� +-� £¤¥;1!<'40�� laminin �¦§US�¨©¥;� �fibronectin �ª«US�¨©¥;��2¬� !30���45Nk�L�c0�$ �Fig. 3b, c�� ��� !� GFAP �ª«US�¨©¥;� ��@Y}®¯$°5+ laminin ¥;�±²0�� 9 �GS �� GB �� �GFAP- rhodamine�� ����� �laminin- FITC� !� "#�$� �3���%&'( ����45"#+,���Fig. 4�� F�]^!��u�W³$°5�f'�eH´µ! ��w�� �¶a�~�'F�$�t'� ������ !·�¸� GB �����
Fig. 2. Histology of gliosarcoma. a. Bimorphic ap-
pearance of a gliosarcoma in which the uniform
spindle component is a fibrosarcoma �H& E��b. Reticulin stain. Reticulin invests individual
mesencymal cells, while the glial component is
devoid of reticulin. G, glial component. S, sar-
comatous component.
6¹�º »¼½7 *396
40
Fig. 3. Immunohistochemical characteristics of gliosarcoma. a. GFAP is strongly positive
in glial component, but negative in sarcomatous part. b, c. Lamin �b� andfibronectin �c� highlighted sarcomatous component, but were absent in glial area.d. No CD31 immunoreactivity is detected in gliosarcoma. Arrows indicate CD31-
positive veins. G, glial component. S, sarcomatous component.
Fig. 4. Double immunofluorescent staining of GFAP and laminin
in a gliosarcoma. a. Laminin �FITC�� b. GFAP �rhoda-mine�. c. Nuclei �TO-PRO-3�� d. Merged image. �600.
������� 397
41
��������� ������� ��������������� �� !"#�$%&' �( GS )*+,�-� �./�01��Fig. 1c�� 23� GB �(24GFAP 567�89���:;*<=� ��> ���?� *@
�ABCDE�FG� HI*JK- GB ��GFAP L"�MN��>OP3��Q ���R&���� Q ST� U� VWX?�YZF� [\]�A3^F� ��� �� _`�aE������� bc d>*� efghi �j*�kl
Fig. 5. Double immunofluorescent staining of GFAP and b-III
tubulin in a gliosarcoma. a. b-III tubulin �FITC�� b.
GFAP �rhodamine�� c. Nuclei �TO-PRO-3�� d. Mergedimage. Tumor cells co-expressing GFAP and b-III tubulin
were labeled in yellow. �600.
Fig. 6. Microsatellite analysis on genetic progression of gliosarcoma. N, non-tumoral brain. G,
glial component. S, sarcomatous component. �� microsatellite instability.
mnop qrst �398
42
��� ����� � ����� � �������������������������� � !�� MS "#�$�%�&'()��GS ��*���*���+�,-��� MS ��%�&�.�/ 01��2�345 �1�4bp� �6� ����7�� �8����8��9allele :;<�=>����?� �heterozygous��MSI �@ABCDE�FG�=H I� MS ��J�����KL9� M�N� ON� &PN� GB�?Q�*� �R���32�33�� *��STUVW��9�XYZ� =[�\]^_�`���� �a�����b�c�9%�&=H�de���IXY� *���=>X���fg� MSI ��h���� I�� �i�j� +�klm!�>�32�� !��GB\GS9=H�?��J��n 3�7� 10� 17opqr� MS BVsV22��"�Y"#�$X�� >t� Boerman�22��u*�DNA�"�YGS�MS"#�$XYt�� microdissection9*�v#�#wY MS xyVW��h����$z{�|RY9��� }% 1� 3� 49��*���*� ~&�� MSI \ LOH ����� �Fig.6�� �i�'b�� ����:�TUVW����� ����� ��Q�������>X��b(� }% 2 � 5 9��*� ��� 1 �)�MSI ��R��� �*� ���>�%�&=H��* *�J�Y��� ������*� GB��+���m!_���� ����� ,��>�������� ���� ��%�&�/9��*�v#�=>X� MSI xyVW���m!_Z�-J�� ��� ?�)� MS BVsV�"��,-�./�J���GS ����� +��z{�*�01� �
2�����9�>Q� ��34\5��34�6�>�� Z���������� !��J� }%��� ����� � ����� � ���������"�����
� �
GS ���+��#&01� ,7��� 8���� � GB ��*�9��: �¡ ������ ��R���� �*�v#�~&� ���� ��;����klJ��� '<>��� MS"#9Z=����� b�� GS 9��*v#�GB �� ����m!_Z>�9¢>�X�� �
��XY� GS ��*v#�}% I������=>���Z����� �?�=� �J� ?Q�*��"��,7�£R����
� �
$¤¥�~¦@'V§V¨A©�ª" «�� ¬7B� ¬®�C��$�"D�¯° E±²FG³ �´���µ�� >t� ��?H�/¶�n94·I$01�¸¹¸�2005J 4º 15I� »K� t�Y+L���
� �
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�������� 401
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Abstract
Histogenesis of Gliosarcoma�A Molecular Pathological Analysis
Masatomo Doi1, Hirotaka Koizumi1, Hiroyoshi Suzuki2,
and Mamoru Tadokoro1
Gliosarcoma is a rare malignant brain tumor �WHO classification, grade IV� that is defined as aglioblastoma admixed with a sarcomatous component. Thus far, four hypotheses have been postulated to
account for the cellular origin of sarcoma, including vascular, meninx, precursor cell, and metaplasia
theories, whereas recent molecular biological studies have suggested the latter two to be most likely. In this
study, we compared these two hypotheses using molecular pathological approaches to determine which
pathway contributes to the histogenesis of gliosarcomas. Double immunofluorescent staining of glial
fibrillary acidic protein �GFAP�� a glial marker, and laminin, a sarcoma marker, was performed in nine
archival gliosarcoma tissues. Consequent laser confocal microscopy detected no “metaplastic” tumor cells
thought to co-express GFAP and laminin. Microsatellite analyses on four loci �D3S1284� D7S507� D10S226� D17S786� were further carried out in five gliosacomas in which glial and sarcomatous components andnon-tumoral area were separately microdissected. Glial and sarcomatous regions of three tumors exhibited
distinct patterns of microsatellite instability, lending strong support to the “precursor cell theory”.In the
other two tumors, however, only the sarcomatous component showed microsatellite instability of one locus,
which could be consistent with the “metaplasia theory”. These collective results raise the possibility that the
cellular origin of sarcomatous component in gliosarcoma may di#er from case to case.
Key words
brain tumor, gliosarcoma, laser confocal microscopy, microdissection, microsatellite
1 Department of Diagnostic Pathology, St. Marianna University School of Medicine
2 Department of Clinical Laboratory, Sendai Medical Center
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