Med. Microbiol. Immunol. 161, 15--21 (1975) 9 by Springer-Verlag 1975
Attempts to Detect Virus-Specific DNA Sequences in Human Tumors
I I I . E p s t e i n - B a r r Viral D N A
in N o n - L y m p h o i d N a s o p h a r y n g e a l C a r c i n o m a Cells
H a n s Wolf*, t t a r a l d zur Hausen*, George Klein**, Volker Becker***, Gertrude ttenle +, and Werner Henle +
Received January 2, 1975
Abstract. Fourteen tumors of the nasopharyngeal region were analyzed for the presence of Epstein-Barr virus-specific DNA by DNA-eRNA hybridization. These data were compared to the histology of the respective tumors and the seroreactivity of the tumor-bearing patient against EBV-related antigens. With one exception, all tumor pieces containing nasopharyngeal carcinoma cells hybridized significantly with EBV-cRNA. Tumors of predominantly epithelial morphology annealed in the highest range. In situ-hybridizations of freeze sections from a tumor containing almost equal amounts of tumor cells and lymphocytes revealed hybridizing DNA within nuclei of non-lymphoid cells. Although these data do not exclude the presence of EBV-DNA within lymphoid cells, they clearly demonstrate that in nasopharyngeal carcinomas the vast majority of EBV-specific DNA rests within non-lymphoid cells.
Introduction
The regular association of Epstein-Barr virus (EBV) with nasopharyngeal carcinoma (NPC) has been demonstrated repeatedly. P a t i e n t s with n a s o p h a r y n - geal carcinomas reveal high a n t i b o d y titers against EBV-specific antigens (Old et al., 1966; de Schryvcr et al., 1969; Henle et al., 1970; de Thd et al., 1973) and EBV-specific nucleic acids have been observed in m o s t biopsies examined b y nucleic acid hybridizations (zur Hausen et al., 1970; N o n o y a m a and Pagano, 1973 ; W o l f et al., 1973; zur Hauscn et al., 1974b).
Since nasopharyngeal carcinomas represent tumors containing varying admix- tures of l y m p h o c y t e s and connective tissue (reviewed b y S h a n m u g a r a t n a m , 1972) it is of obvious interest to localize and to characterize those cells which h a r b o r EBV-genetic information. T h e presence of E B V - D N A in a v a r i e t y of lympho- blastoid cells (zur H a u s e n and Schulte-Holthausen, 1970 ; N o n o y a m a a n d Pagano, 1971 ; zur H a u s e n et al., 1972) suggested to some investigators a passenger role of E B V in ~qPC b y assuming the presence of viral D N A within infiltrating lympho- cytes r a t h e r t h a n within the epithelial t u m o r cells (MeAllister, 1973).
I n a previous report (Wolf et al., 1973) we reported the presence of a p p a r e n t l y EBV-specific D N A in epithelial cells of two t u m o r s as d e m o n s t r a t e d b y in situ- hybridizations a n d a n t i - c o m p l e m e n t a r y immunofluorescence. The following is an
* Institut fiir Klinische Virologic der Universit~t Erlangen-~Ifirnberg, Erlangen.
** Department of Tumor Biology, Karolinska Institutet, Stockholm.
*** Pathologisch-Anatomischcs Institut der Universit~t Erlangen-Niirnberg, Erlangen.
+ Division of Virology, The Children's Hospital of Philadelphia, Philadelphia, Pennsyl- vania 19104.
16 H. Wolf et al.
e x t e n s i o n o f t h e s e studies. A d d i t i o n a l t u m o r b i o p s i e s were a n a l y z e d h i s t o l o g i c a l l y a n d t h e i r h i s t o l o g y was c o m p a r e d t o filter h y b r i d i z a t i o n s w i t h E B V - s p e c i f i c com- p l e m e n t a r y ]~NA (cl~NA). i n a d d i t i o n , s o m e o f t h e m were s u b j e c t e d t o i n situ- h y b r i d i z a t i o n s . T h e r e s u l t s c o n f i r m t h e i n t e r p r e t a t i o n o f p r e v i o u s d a t a : E B v i r a l D N A r e s t s a t l e a s t p r e d o m i n a n t l y w i t h i n t h e n u c l e i o f n o n - l y m p h o i d n a s o p h a r y n - g e a l t u m o r cells.
M a t e r i a l s a n d ~Iethods
The preparation of DNA from tumor biopsies has been described (zur Hausen et al., 1970;
Wolf et at., 1973). Nucleic acid hybridizations were performed as reported elsewhere (zur Hausen et al., 1974a).
I n s i t u . H y b r i d i z a t i o n
The procedure of Gall and Pardue (1971) was followed with minor modifications. Freeze sections (10 ~m) of biopsy material were air dried and fixed for 30 rain in a solution of 250/0 acetic acid and 750/0 methanol. After fixation the slides were transferred for 2 min into 45%
acetic acid and kept twice for 10 min each in ethanol. After air drying the sections were covered by RNase solution (100 fzg/ml in 2 • SSC) and kept for 30 min at 34~ then washed four times in 2• SSC for 10 min each and in addition for 10 min in 700/0 ethanol and then in 100~ ethanol. After additional air drying they were treated with 0.07N KOH for 3.5 min, washed twice for 5 min each with phosphate-citrate (PC)-buffer (pH 5.75} and then again for 5 min in a 1 : 1 mixture of ethanol-PC-buffer. Thereafter they were transferred into 100~
ethanol for 5 min and then placed into a fresh solution of ethanol for additional l0 min.
Hybridization with EBV-specific cRNA (zur Hausen et al., 1974b) was performed with 50000 cpm of cRNA in 50 ~zl of a solution containing 2.5 • SSC, 50~ formamide, and 0.050/0 sodium-dodeeylsulfate under a coverslip (24• mm) for 4 days at 45~ Thereafter the coverslip was washed off with 2 • SSC and the slide then washed 4 times with 2 • SSC (5 min each), treated with RNase (30 tzg/ml in 2 • SSC) at 34~ for 30 min, and washed again 5 times with 2 • SSC. After 2 consecutive washings with destilled water for 1 min, the slides were kept in 700/0 ethanol (5 rain), 100~ ethanol (5 rain), and again in 100~ ethanol (10 min).
After air drying the slides were exposed to autoradiography by using an Ilford G 5 emulsion.
Tim procedure has been described before (Wolf et at., 1973). After 2 weeks of exposure the slides were developed and stained as described before.
I n d i r e c t I m m u n o f l u o r e s c e n e e
Methods to determine EBV-specific antibodies against viral capsid antigens (VCA) and R- and D-antigens of the early antigen (EA)-complex have been described (Henle et at., 1971).
Results
T a b l e 1 s u m m a r i z e s t h e h i s t o l o g i c a l c h a r a c t c r i s t i c s o f 14 A f r i c a n t u m o r s w h i c h were d e r i v e d f r o m t h e n a s o p h a r y n g e a l r e g i o n a n d c o m p a r e s t h e nucleic a c i d h y b r i - d i z a t i o n d a t a o b t a i n e d b y filter h y b r i d i z a t i o n o f 50 ~g o f t u m o r D N A w i t h 50000 c p m o f E B V - c R N A w i t h t h e h i s t o l o g y o f t h e r e s p e c t i v e t u m o r s a n d t h e s e r o r e a c t i v i t y o f t h e t u m o r - b e a r i n g p a t i e n t s a g a i n s t E B V - s p e c i f i c a n t i g e n s . T h e t u m o r s r e p r e s e n t u n s e l e c t e d b i o p s i e s o f w h i c h pieces were h y b r i d i z e d before h i s t o - logical e x a m i n a t i o n o f t h e r e m a i n i n g p a r t .
A l l h i s t o l o g i c a l l y t y p i c a l n a s o p h a r y n g e a l c a r c i n o m a s ( t u m o r s No. 1, 3, 5, 10, 13) h y b r i d i z e c l e a r l y a b o v e t h e r a n g e o f n o n - N P C - t u m o r s ( t u m o r s No. 2, 6 - - 8 , 14) w i t h t h e e x c e p t i o n o f t u m o r No. l 1. i n t h i s case t h e h y b r i d i z a t i o n was r a t h e r low d e s p i t e t h e p r e d o m i n a n c e o f n o n - l y m p h o i d c a r c i n o m a cells. A l l o t h e r h i g h l y h y b r i d i z i n g n a s o p h a r y n g e a l c a r c i n o m a s r e p r e s e n t in t h e i r m a j o r i t y e p i t h e l i a l
E B V - D N A in Nasopharyngeal Carcinomas. I I I 17 Table 1. Comparison of t u m o r histology w i t h h y b r i d i z a t i o n d a t a of t u m o r D N A w i t h E B V -
e R N A a n d t h e serology of the t u m o r - b e a r i n g p a t i e n t against EBV-speeific antigens T u m o r No. Histological diagnosis cpm hy-
bridized
EBV-serology of p a t i e n t VCA E A
D R
I
KCC 1496 2 KCC 1499 3 KCC 1497 4 KCC 1497
5
KCC 1377 6 40316 7 43632 8 KCC 1519 9 36187 10 46306 11 KCC 845 12 KCC 845
13 KCC 1528 14 KCC 1535 H u m a n e m b r y - onic lung fi- broblasts
Nasopharyngeal carcinoma, approx. 1740 480 a 60 a N.T.
600/0 of epithelial morphology, re- m a i n i n g cells m a i n l y lymphoe.
Squameous epithelium, r a t h e r ca. 62 160 0 0 in situ, w i t h some i n f l t r a t i o n s into
deeper layers.
NPC, considerable c o n t e n t of eonnee- 830 1280 320 N.T.
t i v e tissue, few l y m p h o c y t e s only.
Same p a t i e n t as No. 3. This piece 84 1280 320 N.T.
contains exclusively s a l i v a r y g l a n d tissue. A p p a r e n t l y p a r a t u m o r o u s tissue of post-nasal space.
NPC. P r e d o m i n a n t l y epithelial, a b o u t 940 320 160 N.T.
200/0 lyrnphocytes. Some salivary g l a n d tissue.
Squameous cell carcinoma No kera- 69 1070 20 210 tinization. No lymph, infiltration 80
L y m p h a t i c tissue w i t h o u t ca.--cells. 68 N.T. N.T. N.T.
N u m e r o u s g i a n t cells a t the periphery.
Squameous cell carcinoma. Some 55 40 0 0
k e r a t i n i z a t i o n , no lymph, infiltrat.
Only necrotic a n d connect, tissue. 77 640 40 160 No t u m o r cells detectable
NPC. Approx. equal a m o u n t s of 374 640 320 N.T.
epithel, a n d lymph, cells. Some 460 g i a n t cell formation.
NPC. P r e d o m i n a n t l y epithelial. Lyre- 101 640 20 N.T.
p h a t i c infiltration only a t the periphery.
Same p a t i e n t as No. 11. This piece 74 640 20 N.T.
contains a l m o s t n o t u m o r tissue.
E x t e n s i v e scarification. P r o b a b l y previous irradiation.
P r e d o m i n a n t l y epith, t u m o r w i t h 273 1280 640 N.T.
almost no lymph, infiltration. Almost 273 p u r e carcinoma.
No NPC. Contains some g l a n d u l a r 55 80 0 0 tissue. Reticulosis, l y m p h o m a .
n o r m a l tissue, control 50 - - - - - - 57
a Geometric m e a n of three tests performed w i t h different sera of t h e same p a t i e n t . 2 ~fed. ]Kicroblol. Immunol., u 161
18 H. Wolf et al.
Fig. 1. Serial sections of a nasopharyngeal carcinoma biopsy. The right picture represents a freeze section without denaturation, the left figure the corresponding area after denaturation and in situ-hybridization with EBV-cRNA. Giemsa staining
~v ~7 0~ C~ O
20 H. Wolf et al.
t u m o r cells. The sera of all patients with t u m o r s which hybridized with E B V - D N A reacted a t high titers against EBV-specific antigens.
F o r in 8itu-hybridizations a t u m o r was selected which contained almost cqual a m o u n t s of epithelial a n d l y m p h a t i c cells (Tumors No. 10). A non-hybridizing t u m o r (tumor ~qo. 14) was t r e a t e d identically a n d served as control.
The result is shown in Fig. 1. This figure shows the same histological region in consecutive sections before and after d e n a t u r a t i o n and hybridization with cRNA.
I t is obvious t h a t regions showing cells with large pale nuclei are preferentially labelled which correspond to non-lymphoid cells in the non-denatured section.
No labelling was observed in sections of the non-hybridizing control tumor.
Discussion
I n a previous publication (Wolf eta[., 1973) we reported results which showed the specific association of E B viral genomes with epithelial nasopharyngeal carci- n o m a cells. The present s t u d y confirms and further clarifies this association. The labelling of non-lymphoid cells appears to be confined to the nuclear region, although the denaturation procedure affects the m o r p h o l o g y of the cells. The d a t a do n o t exclude t h a t some of the l y m p h o i d cells in nasopharyngeal t u m o r material also carry viral genomes. T h e y show, however, t h a t m o s t of the virus-specific D N A which has been revealed b y filter hybridizations is located within n o n - l y m p h o i d cells. These results are also supported b y filter hybridization experiments with D N A fl'om Ficoll-separated l y m p h o c y t e s and epithelial cells from NPC-materials (Desgranges et al., 1974) in which the preferential annealing of lymphocyte- depleted cells w a s obvious, whereas isolated l y m p h o c y t e D N A did n o t anneal significantly.
The presence of E B V - D N A within the non-lymphoid carcinoma cells of N P C t u m o r s substantially supports the specific role of E B V in this malignancy (zur Hausen, 1975). The regular presence of viral D N A within this t u m o r has been confirmed repeatedly (zur H a u s e n et al., 1970; N o n o y a m a a n d Pagano, 1973; zur H a u s e n et al., 1974b; W o l f et al., 1975).
The presence of viral D N A in epithelial t u m o r cells raises questions on the mode of infection of these cells. Until now E B V h a d only been observed to infect B - t y p e lymphocytes. Besides the possibility t h a t specialized epithelial cells of the n a s o p h a r y n x which cannot be grown in vitro up to now, bear receptors for EBV, fusion of epithelial cells with EBV-genome carrying lymphoblasts (Glaser a n d R a p p , 1972) still remains a possible mechanism.
Acknowledgements. We gratefully acknowledge the generous help of ~Ir. Surjit Singh in supplying the biopsy materials tested in this study. This work was supported by the Deutsche Forsehungsgemeinschaft, Bad Godesberg (SFB 118), by contract No. NOI CP 33316 within the Virus Cancer Program of the National Cancer Institute, by research grant CA 04568, and by contract No. NOI CP 33,272 from the National Cancer Institute, U.S. Public Health Service. W.H. is a recipient of Career Award 5K6-AI-22,683, National Institutes of Health, U.S. Public Health Service.
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