:Acta . NTeurochlrurgica

Acta Neurochir (Wien) (1992) 117:178-181

9 Springer-Verlag 1992 Printed in Austria

Proliferating Cell Nuclear Antigen (PCNA) Immunostaining as an Alternative to Bromodeoxyuridine (BrdU) Immunostaining for Brain Tumours in Paraffin Embedded Sections A. Sasaki 1, H. Naganuma ~, R. Kimura 1, S. Isoe 1, S. Nakano 1, H. Nukui l, K. Suzuki 2, and A. Kawaoi 2 Department of Neurosurgery and 2Second Department of Pathology, Yamanashi Medical College, Yamanashi, Japan

Summary Immunohistochemical staining for proliferating cell nuclear antigen (PCNA) and BrdU using anti-PCNA and anti-BrdU monoclonal antibodies, respectively, was performed in 16 human brain tumours, including 3 gtioblastomas multiforme, 2 anaplastic astrocytomas, 1 cerebellar astrocytoma, 2 recurrent meningiomas, 4 nonrecurrent meningiomas, 3 neurinomas and 1 medulloblastoma. Patients with brain tumours received an injection of bromodeoxyuridine (BrdU) intravenously during surgery, and tumour specimens were fixed in 70% ethanol and embedded in paraffin. The percentage of positive cells for PCNA was compared with a BrdU labelling index using adjacent paraffin-embedded sections. The percentage of PCNA-positive cells was correlated with the BrdU labelling index and the histological malignancy of the brain tumours. The correlation coefficient was 0.84. This suggests that the immunohistochemical staining for PCNA in paraffin sections is a good alternative to the BrdU labelling index. Keywords: Brain tumours; proliferating cell nuclear antigen; bromodeoxyuridine; immunohistochemistry.

Introduction The biological m a l i g n a n c y o f b r a i n t u m o u r s is est a b l i s h e d u s u a l l y histologically. The analysis o f the p r o liferating activity o f b r a i n t u m o u r s can be e v a l u a t e d b y a u t o r a d i o g r a p h y using 1-3H ] - t h y m i d i n e 5 or i m m u n o h i s t o c h e m i c a l staining using a n t i b o d i e s for B r d U 8, D N A p o l y m e r a s e a a n d m o n o c l o n a l a n t i b o d y Ki-676. But these m e t h o d s have n o t been used r o u t i n e l y because they require p r e t r e a t m e n t by r a d i o a c t i v e c o m p o u n d s o r D N A - b a s e a n a l o g u e s a n d special techniques preventing the i n a c t i v a t i o n o f antigenicities. P r o l i f e r a t i n g cell n u c l e a r antigen ( P C N A ) is one o f the n o n - h i s t o n e acidic n u c l e a r p r o t e i n s expressed in p r o l i f e r a t i n g cells. Recently it has been f o u n d t h a t it is an auxiliary p r o t e i n o f D N A p o l y m e r a s e ~1, 10. I n 1987, O g a t a et al. d e v e l o p e d m o n o c l o n a l a n t i b o d i e s for

P C N A 9, a n d R o b b i n s 11 a n d G a r c i a 4 r e p o r t e d t h a t P C N A was d e m o n s t r a b l e in p a r a f f i n - e m b e d d e d tissues. W e e x a m i n e d fixation c o n d i t i o n s a n d p r e t r e a t m e n t p r o c e d u r e s for P C N A i m m u n o s t a i n i n g on v a r i o u s kinds o f r a t a n d h u m a n tissue sections e m b e d d e d in p a r a f f i n using a n t i - P C N A m o n o c l o n a l antibodies, a n d d e m o n s t r a t e d a wide range o f P C N A - p o s i t i v e nuclei in p h y s i o l o g i c a l l y g r o w i n g cells a n d in n e o p l a s t i c cells 12. I n this study, we e x a m i n e d w h e t h e r P C N A was dem o n s t r a b l e in ethanol-fixed, p a r a f f i n - e m b e d d e d tissues a n d w h e t h e r the p e r c e n t a g e o f P C N A - p o s i t i v e cells was c o r r e l a t e d with the b i o l o g i c a l malignancies o f the tum o u r s a n d with b r o m o d e - o x y u r i d i n e ( B r d U ) labelling indexes.

Materials and Methods Brain tumour tissues were obtained from 16 patients. The brain tumours included three glioblastomas multiforme, two anaplastic astrocytomas, one cerebellar astrocytoma, six meningiomas, three neurinomas and one medulloblastoma (Table 1). All patients were given 150-200 mg/sqm of BrdU intravenously for 30 to 60 minutes at the time of craniotomy. Tumour specimens were fixed in 70% ethanol and embedded in paraffin. Thin sections were cut, and for BrdU immunostaining, the sections were treated with 2N HC1 for 30 minutes then neutralized with 0.1 M Na2B407 (pH 8.5). The sections were incubated for 1 hour with anti-PCNA monoclonal antibody (IgM class) (Coulter Immunology, Hialeah, Florida) or anti-BrdU monoclonal antibody (Becton-Dickinson, Mountain view, California). The sections were washed with PBS, and treated with biotynyl anti-mouse immunoglobulin for 10 minutes, then washed with PBS again and treated with peroxydase labelled streptavidin for 5 minutes, and incubated in 3, 3 '-diaminobenzidine (DAB) solution, then counterstained with methylgreen for PCNA immunostaining and with haematoxyline for BrdU immunostaining. As a negative control, normal mouse serum was used instead of the specific antibodies. The percentages of PCNA- and

A. Sasaki et al.: Proliferating Cell Nucelar Antigen (PCNA) Immunostaining as an Alternative

179

Table 1. Pathological Diagnosis and Percentages of Cells Positive for BrdU and PCNA Case

Tumour

Percent positive cells for BrdU

Percent positive cells for PCNA

1 2 3

glioblastoma multiforme glioblastoma multiforme glioblastoma multiforme mean 4- SD

8.10 4.01 3.53 5.21 4- 2.05

5.25 3.21 4.42 4.29 4- 0.84

4 5

anaplastic astrocytoma anaplastic astrocytoma mean 4- SD

4.52 2.81 3.68 • 0.86

4.97 1.77 3.37 4- 1.60

6

cerebellar astrocytoma

0.60

< 0.1

7 8

meningioma, rec(+) ~ meningioma, rec(+) 1 mean 4- SD

3.62 8.63 6.13 4- 2.51

6.99 14.2 10.6 -4-3.63

9 10 11 12

meningioma, rec(-) 2 meningioma, rec( - )2 meningioma, rec(- )2 meningioma, rec(-) 2 mean 4- SD

0.58 0.67 1.07 0.73 0.76 • 0.19

1.39 0.74 2.79 0.50 1.36 • 0.89

13 14 15

neurinoma neurinoma neurinoma mean • SD

0.47 1.03 0.93 0.81 -4-0.24

0.91 < 0.1 0.74 0.58 -4-0.35

16

medulloblastoma

15.0

10.6

1rec(+ ) = recurrent. zrec(-- ) = non-recurrent.

BrdU-positive cells were determined by counting 300 to 1500 cells in at least two microphotographs of each section.

Results The percentages o f P C N A - or B r d U - p o s i t v e cells are s h o w n in T a b l e 1. They were well correlated with the histological m a l i g n a n c y , except in two o f the men i n g i o m a s (cases 7 a n d 8) which showed high percentages of P C N A - or BrdU-positive cells. I n case 7, the p a t i e n t was operated u p o n twice because of a recurrence a n d after the second o p e r a t i o n he received local i r r a d i a t i o n with a dose o f 50 Gy. I n case 8, the p a t i e n t was operated u p o n four times because of 3 recurrences. A l t h o u g h after the third o p e r a t i o n she received i r r a d i a t i o n with a dose of 50 Gy, the t u m o u r recurred 15 m o n t h s later. The histological diagnosis was m e n i n g o t h e l i a l m e n i n g i o m a . The percentage of P C N A - p o s i t i v e cells was correlated with that o f B r d U (Fig. 1). Also, there was a correlation between the percentages o f P C N A a n d

B r d U in each category o f the histological diagnosis (Fig. 2). The intensity of i m m u n o s t a i n i n g for P C N A was less t h a n for B r d U (Fig. 3).

% PCNA Q

10

0

5

i0

% BrdU

Fig. 1. The correlation between the percentages of cells positive for BrdU and PCNA in each case. The correlation coefficient is 0.84. In cases 6 and 14, the percent positive cells for PCNA are calculated as 0.1

180

A. Sasaki et al.: Proliferating Cell Nuclear Antigen (PCNA) Immunostaining as an Alternative

% PCNA 10

.f"

,

Mg,ree(+)

...... "~

...... | Ned

.s" j,~ j.s" 5

f-

.f " "

-'-"'*

GM

...........-I'AA Mg-~-~e e ( - ) .......... . N e u 0 CA

5

l0

% BrdU

Fig. 2. The correlation between the percentages of cells positive for BrdU and PCNA in each category of brain tumours. The correlation coefficient is 0.86. GM glioblastoma multiforme, AA anaplastic astrocytoma, CA cerebellar astrocytoma, Mg. rec( + ) = recurrent meningioma, Mg. rec(-) = non-recurrent meningioma, Neu. = neurinoma, Med.= medulloblastoma

Discussion Recently t u m o u r proliferating activity has been assessed by immunohistochemical methods using anti-

bodies for BrdU, D N A polymerase a, P C N A and m o n o c l o n a l a n t i o b o d y Ki-67. These methods, however, have not been used routinely because they require pretreatment by B r d U or special techniques preventing inactivation of antigenicities. I m m u n o s t a i n i n g with a n t i b o d y for D N A polymerase, a, P C N A and m o n o c l o n a l antibody Ki-67 has been done in frozen sections. P C N A , however, can be demonstrated in the usual paraffin-embedded sections by polyclonal or m o n o c l o n a l a n t i - P C N A antibodies 4' 11, 12 In the present study, we c o m p a r e d the percentage of cells positive for B r d U with that for P C N A in percentage paraffin-embedded sections o f ethanol-fixed t u m o u r specimens. O u r results showed a g o o d correlation between them and suggested that P C N A immunostaining is a g o o d alternative to B r d U immunostaining. P C N A has been shown to be a cell-cycle-related nuclear protein that is expressed strongly in the late

Fig. 3. Case 8. Recurrent meningioma. (A) HE stain, x 200. (B) Immunoperoxidase staining for BrdU, x 200. (C) Immunoperoxidase staining for PCNA, x 200

A. Sasaki et al.: Proliferating Cell Nucelar Antigen (PCNA) Immunostaining as an Alternative

G 1 to S phase of the cell cycle7, and is an auxiliary protein of DNA polymerase ill. 10. Therefore, the percentage of cells positive for PCNA should be higher than that for BrdU. But the S-phase cells with antiPCNA antibodies have been preferentially stained in ethanol-fixed specimens 3. That is why the percentage of cells positive for PCNA was not so different from that for BrdU in our cases. As fixatives in this study, we used 70% ethanol in order to examine the BrdU labelling of the tumour cells. Galand et al. 3 used several kinds of fixatives for 18 hours at 4-8 ~ and reported that methanol-fixed specimens showed the most intense staining. In our recent studies 12 using various fixation conditions PCNA was demonstrated not only in methanol-fixed specimens but also in routinely prepared, formalinfixed, paraffin-embedded tissues. This method needs no prior labelling procedure either in vivo or in vitro, which previous studies using anti-BrdU monoclonal antibodies needed, and can be used with paraffinembedded sections. In conclusion, we suggest the use of PCNA immunostaining in paraffin-embedded sections as an alternative to BrdU immunostaining to evaluate the proliferative activity or the biological malignancy of brain tumours.

3.

4.

5. 6.

7.

8.

9.

10.

11.

Acknowledgements The authors thank Drs. H. Yamazaki and T. Wakao for providing tumour samples.

References 1. Bravo R, Frank R, Blundell PA, Bravo HM (1987) Cyclin/ PCNA is the auxiliary protein of DNA polymerase-6. Nature 326:515-517 2. Celis J, Celis A (1985) Cell cycle-dependent variations in the

12.

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distribution of the nuclear protein cyclin proliferating cell nuclear antigen in cultured cells: subdivision of S phase. Proc Natl Acad Sci USA. 82:3262-3266 Galand P, Degraef C (1989) Cyclin/PCNA immunostaining as an alternativeto tritiated thymidine pulse labelling for marking S phase cells in paraffin sections from animal and human tissues. Cell Tissue Kinet 22:383-392 Garcia RL, Coltera MD, Gown AM (1989) Analysis of proliferative grade using anti-PCNA monoclonal antibodies in fixed, embedded tissue. Am J Pathol 134:733-739 Hoshino T, Wilson CB (1979) Cell kinetic analysis of human malignant brain tumours (gliomas). Cancer 44:956-962 Kunishio K, Mishima N, Matsuhisa T, Tuno K, Matsumi N, Satoh T, Matsumoto K, Furuta T, Nishimoto A, Shiraishi T (1990) Immunohistochemical demonstration of DNA polymerase c~in brain-tumour cells. J Neurosurg 72:268-272 Kurki P, Vanderlaan M, Dolbeare F, Gray J, Tan EM (1986) Expression of proliferating cell nuclear antigen (PCNA)/cyclin during the ceil cycle. Exp Cell Res 166:209-219 Nagashima T, DeArmond SJ, Murovic J, Hoshino T (1985) Immunocytochemical demonstration of S-phase cells by antibromode-oxyuridine monoclonal antibody in human brain tumour tissues. Acta Neuropathol (Berl) 67:155-159 Ogata K, Kurki P, Celis JE, Nakamura RM, Tan EM (1987) Monoclonal antibodies to a nuclear protein (PCNA/Cyclin) associated with DNA replication. Exp Cell Res 168:475-486 Prelich G, Tan CK, Kostura M, Mathews MB, So AG, Downey KM, Stillman B (1987) Functional identity of proliferating cell nuclear antigen and a DNA polymerase-5 auxiliary protein. Nature 326:517-520 Robbins BA, de la Vega D, Ogata K, Tan EM, Nakamura RM (1987) Immunohistochemical detection of proliferating cell nuclear antigen in solid human malignancies. Arch Pathol Lab Med 111:841-845 Suzuki K, Katoh R, Kawaoi (1992) A Immunohistochemical demonstration of proliferating cell nuclear antigen (PCNA) in formalin-fixed, paraffin-embedded sections from rat and human tissues. Acta Histochem Cytochem 25:13-21

Correspondence and Reprints: Atsushi Sasaki, M.D., Department of Neurosurgery, Yamanashi Medical College, 1110, Shimokato, Tamaho-machi, Nakakoma-gun, 409-38 Yamanashi, Japan.

Proliferating cell nuclear antigen (PCNA) immunostaining as an alternative to bromodeoxyuridine (BrdU) immunostaining for brain tumours in paraffin embedded sections.

Immunohistochemical staining for proliferating cell nuclear antigen (PCNA) and BrdU using anti-PCNA and anti-BrdU monoclonal antibodies, respectively,...
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