EBV SEROLOGY AND NASOPHARYNGEAL CARCINOMA

CYNTHIA MILLER, M.D,MARCH 28, 1989

EBV SEROLOGY AMD NASOPHARYNGEAL f!APf!TMr»fv;ft

Introduction

Epstein Barr virus (EBV) is a DNA virus of the Herpes

fami ly. Infect ion in infancy and chi ldhood is of ten

asymptomatic. Primary infection in adolescence and

adulthood is generally manifested as infectiousmononucleosis. In addition to its involvement in acute

viral syndromes, EBV has been associated with malignancy.

It has been implicated in both Burkitt's Lymphoma and

4f*t Nasopharyngeal Carcinoma (NPC). In vitro studies show theIpresence of viral DNA, as well as antigen expression, in

lymphoid tissue and nasopharyngeal epithelium.

Nasopharyngeal carcinoma is an uncommon tumor in NorthAmerica, but is one of the most common malignancies seen insome parts of China and Southeast Asia. There are three

classifications of NPC: Squamous Cell Carcinoma,

Nonkeratinizing Squamous Cell Carcinoma, andUndifferentiated Carcinoma. The last two groups are

associated with EBV. Since these tumors often present at a

late stage or as an occult primary, it would be useful tohave a test to detect them. EBV serology has proved to be a

useful diagnostic and prognostic test in some sett ings. In

f^ populations where there is a high prevalence of NPC, EBVserology has been used as a screening test.

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Severa l d i f fe ren t an t ibod ies a re measured in

re la t ionsh ip to NPC. Bo th IgG and IgA to v i ra l caps id

ant igen (VCA) and ear ly ant igen/di ffuse CEA/D) are assayed.

In an acute infect ion, IgM and IgG anti- VCA are produced.

IgM disappears in about four weeks, but IgG persists at low

leve ls . In NPC, IgG is present a t much h igher leve ls than

w i t h a n a c u t e i n f e c t i o n . I n a d d i t i o n , I g A a n t i - V C A , w h i c h

is no t seen w i th an acu te in fec t ion , i s p resen t a t h igh

levels with NPC.

Ant ibod ies to ear ly ant igen (EA) show both a d i f fuse

(cy top lasmic and nuc lea r ) and a res t r i c ted (cy top lasmic

o n l y ) p a t t e r n . T h e s e a r e p r e s e n t t r a n s i e n t l y w i t h a c u t e

i n f e c t i o n s a n d r e a c t i v a t i o n s . H i g h t i t e r s o f a n t i - E A / R

( res t r i c ted ) have been assoc ia ted w i th Burk i t t ' s l ymphoma.

Anti-EA/D, both IgG and IgA, are associated with NPC.

Methodology

Ant i -VCA

Smears are prepared wi th the P3-HR1 cel l l ine, wi th

10-15% of the cel ls expressing VCA. A drop of a cel l

suspension is p laced on a g lass s l ide and fixed wi th

acetone. Acetone fixed smears can be used to measure both

Ig G a n d I g A . Fo u r f o l d d i l u t i o n s o f t h e p a t i e n t s ' se ru m a re

i n c u b a t e d w i t h t h e fi x e d c e l l s . F l u o r e s c e i n i s o t h i o c y a n a t e

conjugated antiserum to human Ig subclasses is then added

and the presence of fluoresence is measured.

/ fi i t e j y

An t i -EA/D

To express EA, the Raj i ce l l l ine must be e i ther

super in fec ted w i th in fec t ious v i rus f rom a P3-HR1 ce l l

cu l tu re , o r i nduced by t rea tment w i th a va r ie ty o f agen ts .

Once th is is accompl ished, cel l suspensions are used to

p repare ace tone fixed smears . An ind i rec t immunofluorescen t

assay s im i la r to tha t done fo r an t i -VCA is run .

Acetone fixed smears can detect both d i f fuse (speck led

s t a i n i n g s e e n t h r o u g h o u t t h e c e l l ) a n d r e s t r i c t e d ( s t a i n i n g

o f t he cy top lasm on l y ) pa t te rns . Me thano l fixed smears

pre fe ren t ia l l y remove the EA/R.

D iscuss ion

The association between NPC and EBV has'been recognized

for many years. NPC has a high prevalence in some parts of

t he wo r l d . I t o f t en p resen t s a t a l a t e s tage o r as an

occu l t p r imary. These fac t s make a read i l y ava i l ab le marke r

such as v i ra l se ro logy idea l fo r d iagnos is and p rognos is .

In fact, numerous studies have shown that EBV serology is

useful in regards to NPC.

Titers to VCA and to EA/D have proven to be the most

use fu l in NPC. Near ly a l l pa t ien ts w i th NPC have e leva ted

V C A - I g G t i t e r s . H o w e v e r , t h i s i s n o t s p e c i fi c , s i n c e t h i s

a n t i b o d y c a n p e r s i s t f o l l o w i n g a p r i m a r y i n f e c t i o n . V C A - I g A

is fa r more spec ific <9-18% o f hea l thy pa t ien ts a re

p o s i t i v e ) , t o u t m u c h l ^ s s s e n s i t i v e . T h e s e n s i t i v i t y o f

this test var ies from 65-89% depending on the study and

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c l a s s i fi c a t i o n o f t h e N P C . I t h a s t h e h i g h e s t s e n s i t i v i t y

for WHO type 2 and 3 (nonkerat in iz ing and undi fferent ia ted)

N P C . Ti t e r s t o E A / D h a v e s i m i l a r p r o fi l e s . 9 4 - 1 0 0 % o f p a \

t ients have posi t ive t i ters when both IgG and IgA are taken

i n t o a c c o u n t . I g A t i t e r s a r e m o s t s p e c i fi c < ~ 9 8 % ) , b u t

l e a s t s e n s i t i v e , b e i n g p r e s e n t i n 4 7 % o f p a t i e n t s . I g G

t i t e r s a re l ess spec i fic <~65%) , bu t more sens i t i ve (p resen t

in 83-94% of pat ients) .it-

Ta k e n i n d i v i d u a l l y, n o s i n g l e t i t e r w o u l d b e u s e f u l f o r

d i agnos i s . Howeve r, when used as a p rofi l e , e l eva ted t i t e r s

a re h i gh l y sugges t i ve o f NPC. I n pa r t i cu l a r, t he p resence

of VCA-IgA, EA/D-IgG and EA/D-IgA together, makes the

probab i l i t y tha t NPC is p resent 12 t imes more l i ke ly than

t h a t o f o t h e r r e g i o n a l m a l i g n a n c i e s . I n N o r t h A m e r i c a ,

where the inc idence o f th is ma l ignancy is low (1 -2 /100,000) ,

t h e d i a g n o s t i c u t i l i t y o f t h e s e t e s t s i s m o s t i m p o r t a n t i n

the pat ient present ing wi th cervical nodes and an unknown

pr imary. However, i n a reas o f h igh inc idence , such as

s o u t h e r n C h i n a < 1 8 / 1 0 0 , 0 0 0 ) , t h e s e n s i t i v i t y a n d s p e c i fi c i t y

of VCA-IgA in detecting NPC becomes much higher (93-95% and

9 7 % , r e s p e c t i v e l y ) . I n t h i s s i t u a t i o n t h e t e s t c a n b e u s e d

success fu l l y as a screen.

EBV serology also appears to have prognost ic value in

NPC. Several studies have shown that the height of the VCA

a P * a n d E A / D t i t e r s c o r r e l a t e w i t h t h e t u m o r b u r d e n . S e v e r a l

s t u d i e s a l s o i n d i c a t e t h a t f o l l o w i n g t h e s e t i t e r s m a y b e

u s e f u l i n p r e d i c t i n g r e l a p s e a f t e r t r e a t m e n t .

Ant ibodies to nuclear ant igens (EBNA) are a lso present

in NPC but, have not been as well characterized and do not

appear to be as usefu l . A la tent membrane prote in ant ibody

a lso appears to have some prognos t i c s ign ificance ( i t s

presence be ing cor re la ted wi th a good prognos is ) . However,

th is i s measured by an an t ibody dependent ce l lu la r tox ic i t y

a s s a y w h i c h l i m i t s i t s u t i l i t y .

In conclusion, EBV serology has diagnost ic and

p rognos t i c u t i l i t y i n NPC. I n a reas whe re t he re i s a h i gh

incidence of the disease, VCA-IgA assays are a useful

screen ing tes t . In o ther areas, such as Nor th Amer ica where

the disease is uncommon, measuring a panel of VCA-IgA,

EA/D-IgG and EA/D-IgA is a useful a id to diagnosis in the

pa t i en t w i t h a h i gh i ndex o f susp i c i on f o r NPC. I t a l so has

u t i l i t y i n f o l l o w i n g t h e c o u r s e o f t h e d i s e a s e .

References

1) Fedder, M., and Gonzalez, M. F., "NasopharyngealCarcinoma", American Journal of Medicine.79C=n »<res-Q, 1985.

2) Smith, T. F. , "Nasopharyngeal carc inoma due toE p s t e i n - B a r r v i r u s : s e r o l o g i c t e s t s " , M a v o C I i n i cProceedings. 60<3):205-6, 1985.

3) Nee l , H. B. , "Nasopharyngea l carc inoma" , Oto laryngo log icCl in ics o f Nor th Amer ica . 18<3) :479-90, 1985.

4) R ich tsmeier, W. J . , Wi t te ls , E . G. , and Mazur, E . M. ,"Epste in-Barr v i rus associated ma 1 ignancies" , CRC Cr i t ica lRev iews in C l in ica l Labora tory Sc iences . 2502) :105-36, 1987.

5 ) L e v l n e , P. H . , e t a l . , " E p s t e i n - B a r r v i r u s s e r o l o g y i nthe contro l o f nasopharyngeal carc inoma", Cancer Detect ionand Prevent ion. 12<1-6) :357-62, 1988.

j fl l f e y

6) Lynn, T. C, Tu, S. M., and Kawamura, A. Jr., "Long-termfollow-up of IgG and IgA antibodies against viral capsidantigens of Epstein-Barr virus in nasopharyngeal carcinoma",Journal of Laryngology and Otology. 99(6):567-72, 1985.

7) Rowe, M., et al., "Characterization of the serologicalresponse in man to the latent membrane protein and the sixnuclear antigens encoded by Epstein-Barr virus", Journal ofGeneral Virology. 69(6):1217-28, 1988.

8) De-Vathaire, F., et al., "Prognostic Value of EBV MarkersIn The Clinical Management of Nasopharyngeal Carcinoma(NPC): A Multicenter Follow-up Study", Int. Journal ofCancer. 42(2):176-81m 1988.