Upsala J Med Sci 84: 259-274, 1979 Serum Concentrations of Estrone, Androstenedione, Testosterone and Sex-hormone- binding Globulin in Postmenopausal Women with Breast Cancer and in Age-matched Controls Hans-Olov Adami, M.D.', Elof D. B. Johansson, M.D.', Jan Vegelius, Ph.D.3 and Arne Victor, M.D.' From the Department3 of Surgery, Obstetrics & Gynecology, UniverJity Hospital, Uppsala, Sweden and Department ttf Statistics, Uppsalu University, Uppsala, Sweden ABSTRACT The c o n c e n t r a t i o n s o f e s t r o n e ( E l ) , a n d r o s t e n e d i o n e ( A ) , t e s t o s t e r o n e ( T ) a n d s e x - h o r m o n e - b i n d i n g g l o b u l i n (SHBG) i n t h e s e r u m were d e t e r m i n e d i n 1 2 2 p o s t m e n o p a u s a l women, u n s e l e c t e d w i t h r e s p e c t t o a g e a n d s t a g e o f d i s e a s e a n d w i t h a n e w l y d i a g n o s e d b r e a s t c a n c e r . The r e s u l t s were c o m p a r e d w i t h t h o s e i n 1 2 2 a g e - m a t c h e d women w i t h o u t b r e a s t c a n c e r , s e l e c t e d f r o m t h e p o p u l a t i o n r e g i s - t e r . The p a t i e n t s were f o u n d t o h a v e a s i g n i f i c a n t l y h i g h e r mean l e v e l t h a n t h e c o n t r o l s o f E l ( 1 3 2 a n d 108 p m o l / l ) , A ( 2 . 5 a n d 1.6 n m o l / l ) a n d ' T ( 1 . 5 4 a n d 1.38 n m o l / l ) a n d a lower l e v e l of SHBG ( 4 0 . 2 a n d 4 7 . 3 n m o l / l ) i n t h e s e r u m . A mul- t i p l e r e g r e s s i o n a n a l y s i s r e v e a l e d i n t h e c o n t r o l g r o u p t h a t t h e s e r u m l e v e l o f El w a s s i g n i f i c a n t l y c o r r e l a t e d t o A (r=0.48, p < 0.001) a n d T ( r = 0 . 4 5 , p < 0 . 0 0 1 ) . I n t h e p a t i e n t g r o u p E l w a s o n l y s l i g h t l y c o r r e l a t e d t o T ( r = 0 . 2 5 , p < 0 . 0 1 ) a n d n o t t o A ( r = 0 . 1 0 , p > 0 . 0 5 ) . A s i g n i f i c a n t n e g a t i v e c o r r e l a t i o n was f o u n d b e t w e e n SHBG a n d w e i g h t i n b o t h g r o u p s . O t h e r w i s e n o s i g n i f i c a n t c o r r e l a - t i o n s were f o u n d b e t w e e n a n y o f t h e hormone l e v e l s a n d a g e , s t a g e o f d i s e a s e o r w e i g h t . I t w a s c o n c l u d e d t h a t a n i n c r e a s e d a v a i l a b i l i t y o f A a n d T I l e a d i n g t o a n i n c r e a s e d a n d r o g e n i c s t i m u l a t i o n - a n d t h e r e f o r e d e c r e a s e d SHBG - a n d a n i n - c r e a s e d E l l e v e l , i s t h e most r e a s o n a b l e e x p l a n a t i o n f o r t h e f i n d i n g s . The l a c k of c o r r e l a t i o n b e t w e e n E l a n d A i n t h e p a t i e n t g r o u p i s however d i f f i c u l t to e x p l a i n a n d t h e r e s u l t s d o n o t seem t o f i t i n t o a d e f i n i t e h y p o t h e s i s . INTRODUCTION E p i d e m i o l o g i c a l a n d c l i n i c a l e v i d e n c e t h a t t h e r i s k f o r b r e a s t c a n c e r is i n - f l u e n c e d by e n d o c r i n e f a c t o r s ( 4 0 ) h a s i n i t i a t e d numerous s t u d i e s d e s i g n e d t o d e f i n e a s p e c i f i c e n d o c r i n e e n v i r o n m e n t t h a t p r e d i s p o s e s t o b r e a s t c a n c e r . Major i n t e r e s t h a s b e e n p a i d t o t h e u r i n a r y e x c r e t i o n o f a n d r o g e n a n d e s t r o g e n m e t a b o l i t e s . A l t h o u t h c o n t r a d i c t o r y i n many i n s t a n c e s , t h e r e s u l t s from t h e s e s t u d i e s h a v e i n i t i a t e d s e v e r a l h y p o t h e s e s a n d t w o o f them h a v e a t t r a c t e d s p e c i a l i n t e r e s t . 8-792856 259 According to the first of them a decreased urinary excretion of etiochola- bolone and androsterone was associated with an increased risk of breast cancer and in the manifest disease with a poor prognosis and a poor response to endo- crine ablative surgery (cf. 19). Contradictory results have however been re- ported (cf. 62) and the androgen excretion has also been shown to be decreased as a nonspecific consequence of illness (61). Secondly, the "estriol hypothesis" was derived from the fact that carcino- genic potential has been demonstrated for estrone (El) and estradial ( E 2 ) in experimental research but not for estriol (E3) and that E3 has the capacity to impede certain estrogenic activities of El and E 2 (33). The relative amount of E3 in relation to El and E 2 was therefore suggested as an important determinant for breast cancer risk. This hypothesis was supported when population studies revealed a parallelism between the urinary estriol ratio and a high, intermedi- ate and low breast cancer risk, respectively (13, 41). The difference was most marked in young women, a finding combined with the previous finding of a reduced breast cancer risk due to an early age at first birth (40). Recent research has revealed several lines of data which has seriously brought into challenge the concepts that the pattern of excretion of the urinary androgens and estrogens is causally related to the etiology of breast cancer (cf. 32, 6 2 ) . Results derived from determinations of the steroid hormones in serum are very few and have not resulted in any convincing alternative hypo- thesis (32). The design of the present investigation was not primarily aimed at analysis of steroid hormones and their binding globuline. The availability of serum within the frame of an ongoing epidemiologic breast cancer study which included an assessment of thyroid function and an access to recently developed radio- immunoassays did, however, enable the study. The aim was therefore to compare the serum concentrations of El, androstenedione (A), testosterone (T) and sex- hormone-binding globulin (SHBG) in an unselected series of postmenopausal women with a newly diagnosed breast cancer with those in a group of age-matched, non-hospitalized, postmenopausal women without breast cancer. SUBJECTS AND METHODS Patients This study was based on 149 postmenopausal women included in a series of 179 women with breast cancer registered consecutively during five months in four Swedish counties. The population within this area was uniform as to race and nationality. Only two of the breast cancer patients who were diagnosed during the observation period (2/181) refused therapy and participation in the study. Hospital records were written on a special form and all patients answered a comprehensive questionnaire concerning their reproductive history, gynecologic 260 d i s e a s e s , h e i g h t , w e i g h t , d r u g consumption and o t h e r f a c t o r s of e p i d e m i o l o g i c i n t e r e s t . A l l p a t i e n t s were c l a s s i f i e d a c c o r d i n g t o t h e T N M - c l a s s i f i c a t i o n ( 2 3 ) ( T a b l e 1 ) . Two blood s a m p l e s were drawn f o r a n a l y s i s of SHBG and hormones i n t h e serum. The s e r a were d i r e c t l y s e n t t o Uppsala and a r r i v e d t h e same e v e n i n g or t h e n e x t morning and were f r o z e n and s t o r e d a t -9OOC u n t i l a n a l y z e d . The f i r s t s e r u m sample w a s t a k e n a f t e r a d m i s s i o n t c t h e h o s p i t a l b u t b e f o r e o p e r a t i o n and was used for t h e d e t e r m i n a t i o n o f f o l l i c l e s t i m u l a t . i n g hormone (FSH) and f o r t h e a s s e s s m e n t of t h y r o i d f u n c t i o n . The second serum sample was drawn randomly be- tween 0 9 . 0 0 a m and 04.00 pm more t h a n o n e w e e k p o s t o p e r a t i v e l y , t h e median t i m e b e i n g t h r e e w e e k s and t h r e e q u a r t e r s o f t h e s a m p l e s t a k e n w i t h i n s i x w e e k s . The s e c o n d serum sample u a s u s e d f o r a n a l y s e s of t h e s t e r o i d hormones and SHBG. T a b l e 1 . S t a g i n g a c c o r d i n g t o t h e number TNM c l a s s i f i c a t i o n of women T N M - c l a s s i f i c a t i o n 301 C a t e g o r y Number % I 52 37 20- I1 6 8 48 I11 13 9 I V 8 6 1 4 1 100 45 55 65 1 1, 85 95 age F i g . 1 . Age d i s t r i b u t i o n o f 1 4 1 pa- t i e n t s and c o r r e s p o n d i n g c o n t r o l s C o n t r o l s The c o n t r o l g r o u p c o n s i s t e d o f age-matched women w i t h o u t b r e a s t c a n c e r t h i s s t u d y t h o s e 151 w i t h i n t h e t o t a l c o n t r o l g r o u p o f 1 7 9 women who were menopausal were i n c l u d e d . They were s e l e c t e d from t h e c o m p u t e r i z e d p o p u l a I n p o s t - i o n r e g i s t e r w i t h i n e a c h c o u n t y where t h e women closest i n a g e t o e a c h r e s p e c t i v e b r e a s t c a n c e r p a t i e n t was c h o s e n . C o n t r o l women who r e f u s e d t o p a r t i c i p a t e (25=14 % ) were r e p l a c e d by a n a l t e r n a t i v e c o n t r o l s e l e c t e d i n t h e same way a s t h e f i r s t o n e . A s a r e s u l t e a c h b r e a s t c a n c e r p a t i e n t had o n e age-matched con- t r o l w i t h n o t more t h a n 3 d a y s a g e d i f f e r e n c e t o t h e c o r r e s p o n d i n g p a t i e n t . A l l women i n t h e c o n t r o l g r o u p answered a q u e s t i o n n a i r e i d e n t i c a l to t h a t i n t h e p a t i e n t g r o u p . I n a d d i t i o n t h e y were a l l examined a t t h e o f f i c e o f t h e i r d i s t r i c t m e d i c a l n u r s e by o n e o f u s (HOA). A t t h e e x a m i n a t i o n blood s a m p l e s f o r a n a l y s i s o f hormones i n s e r u m were drawn and c e n t r i f u g e d . T h i s o c c u r r e d , a s i n 26 1 t h e p a t i e n t g r o u p , randomly between 0 9 . 0 0 am and 04.00 pm. The sera were d i r e c t - l y s e n t t o Uppsala i n t h e same way a s i n t h e p a t i e n t g r o u p and were f r o z e n and s t o r e d a t -90 C . The s u b s e q u e n t a n a l y s i s i n c l u d e d , a s i n t h e p a t i e n t g r o u p , FSH and SHBG i n a d d i t i o n t o t h e s t e r o i d hormones. 0 S e l e c t i o n of t h e p o s t m e n o p a u s a l g r o u p - When comparing t h e age-matched p a i r s b o t h t h e p a t i e n t and t h e c o r r e s p o n d i n g c o n t r o l were p o s t m e n o p a u s a l a c c o r d i n g t o h i s t o r y and a n i n c r e a s e d c o n c e n t r a t i o n of FSH i n t h e s e r u m ( 6 0 ) i n 141 i n s t a n c e s . The a g e d i s t r i b u t i o n of t h i s g r o u p i s shown i n F i g . l . , t h e a v e r a g e and t h e median a g e b e i n g 68 y e a r s . I n t h e s e g r o u p s two p a t i e n t s and o n e c o n t r o l had t o be e x c l u d e d due t o e s t r o g e n t r e a t - ment. I n t h e r e m a i n i n g g r o u p p o s t o p e r a t i v e l y t a k e n s e r u m s a m p l e s were a v a i l a b l e i n o n l y 1 2 2 p a t i e n t s . With r e s p e c t t o t h e f a c t o r s r e l e v a n t f o r t h e i n v e s t i g a t i o n , t h e loss c a n be c o n s i d e r e d random and t h e 1 2 2 p a i r s a n a l y z e d t h u s r e p r e s e n t a t i v e f o r t h e whole p o s t m e n o p a u s a l g r o u p s . Radioimmunoa s s a y s T e s t o s t e r o n e : An a n t i b o d y r a i s e d i n s h e e p a g a i n s t testosterone-3-0-carboxy- m e t h y l b o v i n e serum a l b u m i n was used ( g i f t from D r . L a r s - E r i k E d q v i s t , Royal V e t e r i n a r y High S c h o o l , U p p s a l a , Sweden). The a n t i b o d y c r o s s r e a c t s 56 % w i t h d e h y d r o t e s t o s t e r o n e and 7 % w i t h a n d r o s t e n e d i o n e b u t l e s s t h a n 1 % w i t h dehydro- e p i a n d r o s t e r o n e and e s t r a d i o l . I t was used i n a 1:15 000 d i l u t i o n w i t h e t h a n o l . A s t r a c e r 1 , 2 , 6 , 7 - H - t e s t o s t e r o n e w i t h a s p e c i f i c a c t i v i t y of 8 5 C i / m m o l p u r - c h a s e d from t h e N e w England N u c l e a r , Boston M a s s a c h u s e t t s , U S A w a s u s e d . I t was d i l u t e d t o g i v e a p p r o x i m a t e l y 85 pg p e r a s s a y t u b e . The p r o c e d u r e and r e a g e n t s of t h e a s s a y were t h e same a s d e s c r i b e d p r e v i o u s l y ( 1 4 ) . 0.4 m l a l i q u o t s of plasma were e x t r a c t e d w i t h 4 m l of d i e t h y l e t h e r f o r t h e a s s a y . A b l a n k of 55 pg (CV 2 1 % ) was found i n c h a r c o a l t r e a t e d plasma a t t h i s e x t r a c t i o n volume and r e s u l t s were c o r r e c t e d f o r t h i s plasma b l a n k . The w i t h i n and between a s s a y co- e f f i c i e n t s o f v a r i a t i o n were found t o be 1 4 and 18 % , r e s p e c t i v e l y . 3 A n d r o s t e n e d i o n e : A p r e v i o u s l y d e s c r i b e d a n t i b o d y r a i s e d i n a d u l t ewes a g a i n s t androstenedione-3-oxime human s e r u m albumin ( g i f t from Dr. Guy E . Abra- ham) was u s e d . The a n t i b o d y c r o s s r e a c t s 25 % w i t h d e h y d r o e p i a n d r o s t e r o n e ( 2 ) . -3 I t w a s u s e d i n a 1:500 d i l u t i o n . 1 , 2 , H - a n d r o s t e n e d i o n e w i t h a s p e c i f i c a c t i v - i t y of 4 0 C i / m m o l p u r c h a s e d from N e w England N u c l e a r was used a s t r a c e r . I t was d i l u t e d t o g i v e a p p r o x i m a t e l y 7 2 pg p e r a s s a y t u b e . The p r o c e d u r e and r e a g e n t s of t h e a s s a y were t h e same a s d e s c r i b e d p r e v i o u s l y ( 1 4 ) . 0 . 1 m l a l i q u o t s of s e r u m were e x t r a c t e d w i t h 1 . 5 m l of d i e t h y l e t h e r f o r t h e a s s a y . A plasma blank of 1.03 pg (CV 20 % ) was found i n c h a r c o a l t r e a t e d plasma a t t h i s e x t r a c t i o n volume and t h e r e s u l t s were c o r r e c t e d f o r t h i s b l a n k . The w i t h i n and between c o e f f i c i e n t s of v a r i a t i o n f o r t h e a s s a y were found t o be 10 and 20 % , r e s p e c t - i v e l y . 262 E s t r o n e was d e t e r m i n e d by radioimmunoassay a s d e s c r i b e d by Axelsson e t a l . (7). Sex-hormone-binding g l o b u l i n w a s a s s a y e d by t h e ammoniumsulphate p r e c i p i t a - t i o n t e c h n i q u e d e s c r i b e d by Rosner ( 5 0 ) and t h e r e s u l t s are e x p r e s s e d a s t h e dihydrotestosterone-binding c a p a c i t y of plasma i n nmol/l. c a l c u l a t i o n s and s t a t i s t i c a l methods A s a n i n d e x o f o b e s i t y i n d e p e n d e n t of h e i g h t w e u s e d t h e i n d e x o f Q u e t e l e t ( w e i g h t / h e i g h t ) which was shown t o f u l f i l l i m p o r t a n t c r i t e r i a f o r s u c h a n i n d e x ( 2 9 ) . A s t h e SHBG, El and T showed a skewed d i s t r i b u t i o n t h e i r l o g a r i t h m i c v a l u e s were c a l c u l a t e d . T h i s made t h e normal d i s t r i b u t i o n a s s u m p t i o n of t h e t- t e s t f o r matched g r o u p s more p l a u s i b l e . 2 The c o r r e c t i o n f o r t h e r e l a t i v e l y h i g h plasma b l a n k o f A by s u b t r a c t i o n o f i t s mean v a l u e g a v e some n e g a t i v e r e s u l t s . I f t h e s e v a l u e s had been d i s c a r d e d t h e mean e s t i m a t e o f A would have become p o s i t i v e l y b i a s e d . They were t h e r e f o r e i n c l u d e d i n t h e c a l c u l a t i o n which, however, made t h e u s e o f l o g a r i t h m e d v a l u e s i m p o s s i b l e . Due t o t h e s k e w e d d i s t r i b u t i o n of A Wilcoxon's n o n p a r a m e t r i c m a t c h e d - p a i r s s i g n e d - r a n k s test w a s u s e d a s a complement t o t h e t-test. I n t h e c a l c u l a t i o n of t h e c o n f i d e n c e i n t e r v a l s of t h e p o p u l a t i o n means of A t h e s t a n - d a r d d e v i a t i o n o f t h e b l a n k had t o be i n c l u d e d . The p r o d u c t moment c o r r e l a t i o n c o e f f i c i e n t w i t h t h e c o r r e s p o n d i n g p-value o f t h e i n d e p e n d e n c e h y p o t h e s i s was u s e d a s a n measure o f c o r r e l a t i o n . A m u l t i p l e r e g r e s s i o n a n a l y s i s was a l s o a p p l i e d t o e v a l u a t e how much of t h e v a r i a n c e i n E l c o u l d be e x p l a i n e d by v a r i a n c e i n o t h e r " i n d e p e n d e n t " v a r i a b l e s . A l l v a l u e s r e f e r t o t w o - t a i l e d tests. When v a l u e s were m i s s i n g i n t h e p a t i e n t or t h e c o r r e s p o n d i n g c o n t r o l , b o t h were e x c l u d e d from t h e c a l c u l a t i o n s v a r i a b l e . E s t r o n e ( E l ) . c a n t l y h i g h e r (p ( T a b l e 2 ) . T h e r e v a l u e s e x c e e d i n g RESULTS The mean v a l u e i n t h e p a t i e n t g r o u p (132 p m o l / l ) w a s < 0.01) t h a n t h e mean v a l u e i n t h e c o n t r o l g r o u p ( 1 0 8 was a wide r a n g e o f v a r i a t i o n i n b o t h g r o u p s ( F i g . 2) 200 pmol/l were found i n o n l y 15 c o n t r o l s compared t o f o r t h a t s i g n i f i- pmol/l) b u t 29 pa- t i e n t s . The means c a l c u l a t e d on a r i t h m e t i c v a l u e s were 152 pmol/l ( 4 1 . 2 pg/ml) i n t h e p a t i e n t g r o u p and 1 2 4 pmol/1 (33.5 pg/ml) i n t h e c o n t r o l g r o u p . A n d r o s t e n e d i o n e ( A ) . The d i s t r i b u t i o n was s l i g h t l y s h i f t e d t o h i g h e r v a l u e s i n t h e p a t i e n t g r o u p ( F i g . 3 ) whose mean v a l u e a f t e r c o r r e c t i o n f o r t h e b l a n k was 2 . 5 nmol/l (0.71 ng/ml) which i s s i g n i f i c a n t l y h i g h e r (p=0.009, t - t e s t ; p0.006, Wilcoxon's t e s t ) t h a n t h e mean v a l u e i n t h e c o n t r o l g r o u p of 1.6 n m o l / l (0.46 ng/ml) ( T a b l e 2 ) . V a l u e s lower t h a n t h e mean v a l u e of t h e blank (3.7 nmol/l) were found i n 16 p a t i e n t s and 2 2 c o n t r o l s . T e s t o s t e r o n e ( T ) . Even t h e d i s t r i b u t i o n of testosterone was s h i f t e d t o 263 - a J 4 u - m u m m c a o u ' 4 a m u c z m u m ar -4 0 m 4 w 3 [ r Q U c 0 -4 -4 4 E c cnc aJ 3 d m I a c m .rl 5 aJ z aJ 0 aJ 9 w c C r n O a J U E oh0 a, s m u m w a 0 m m u c -4 m E aJ .A E d -4 CI (I) aJ u I u ffl d z CI c U (I) u C aJ .rl u m a m rl z u c 0 m u C ar .4 u m a Tr 0 0 0 c c c 0 m c m c c I 00 m W 0 c Lo -3 ?- I N c ?- N m c d d \ 2 CI W c m 0 0 0 m c 0 N c N I c c W c c m I m c m N ? 5 4 0 4 N v 0 0 m m c 0 W c 0 m c I 00 N c W m c m W c I m -r c q m c d \ 4 0 5 €+ W 0 0 0 m 0 m c 0 0 m 0 In I 0 q -3 W P -J m m q I W W W N 0 xr d \ rl 0 5 u z m m 264 number of women 7 / / / / L patients controls 30 20 i a r 121 number Of women 30. 20- 1 ESTRONE pmolll patients 0 controls 2 3 ANOROSTENEDIONE nrnolll Fig. 2. Distribution of serum levels Fig. 3. Distribution of serum levels of estrone in patient and control of androstenedione in patient and group control group number of women 0 patients 0 controls l,25 150 175 2pO 2.25 2,50 2.75 "3,OO TESTOSTERONE nmolll number of women patients controls n 50 60 70 80 290 SHBG nmolll Fig. 4. Distribution of serum levels Fig. 5. Distribution of serum levels of testosterone in patient and control group patient and control group of sex-hormone-binding globulin in 265 higher values in the patient group (Fig. 4 ) but the difference in mean values between the groups was very small albeit significant (p < 0.05)(Table 2). The skewness of the distributions (Fig. 4 ) was less pronounced than for El (Fig. 2) and A (Fig. 3 ) and the arithmetic means - 1.65 nmol/l ( 0 . 4 8 ng/ml) and 1.52 nmol/l ( 0 . 4 4 ng/ml) - close to the logarithmic. Sex-hormone-binding globulin (SHBG). In contrast to E l , A and T this dis- tribution was shifted towards lower values in the patient group (Fig, 5 ) . The difference is highly significant (p < 0 . 0 1 ) . Due to some very high values in the patient group (Fig. 5 ) the mean values - 4 0 . 2 nmol/l and 4 7 . 3 nmol/l - differ less than the median values - 4 0 . 1 and 5 0 . 9 , respectively (Table 2). The arith- metic means were 4 5 . 2 and 5 0 . 8 nmol in the patient and control groups. Relations to age, weight and stage of disease. Subgrouping of the material according to age revealed no obvious trend in the differences between patients and controls for any of the variables. Thus in all decades from 4 5 to 7 5 years or more the patients, on the average, had a slightly higher A and T concentra- tion in the serum. E l concentration did not differ between patients and controls aged 4 5 - 5 4 years but the patients had higher mean values in all subsequent age- groups. The patients had a lower mean SHBG value in all groups except in that including women over 7 5 years of age. Not any of the variables was significantly correlated to age, either in the control or in the patient group (Table 3 ) . Table 3 . Correlation coefficients to age, weight, Quetelet’s index and stage of disease according to the TNM-classification Correlated to Test Group age Quetelet’s TNM weight index classification E l patient 0;Ol 0.05 0 . 0 7 0 . 0 4 control 0.07 0 . 1 4 0. 22L’ - A patient 0.00 0.07 0.09 - 0 . 0 8 control - 0 . 0 1 0.03 0.00 - T patient -0.11 -0.04 -0.03 -0.01 control -0.09 0.01 - 0 . 0 5 - 0 . 0 1 4 / 4 / SHBG patient 0.13 - 0 . 2 8 3 / - 0 . 3 1 control 0.12 -0.29 3 / 0.34 2 I / Quetelet’s index = weight/height 2 / p < 0 . 0 5 3 / p < 0.01 4 / p < 0 . 0 0 1 266 350 300 250 _i . . . . . . - . . . . . . . - . . . - .. . . . . . . . - -. . ? ... .. I . - . . . . . . . . . . . . . . . . . . . 250 2w- 150 I00 50. I 0 2 L 6 8 1 0 350. 300. 2507 A n d l l 200. 150 100. 5 0 F i g . 6 . C o r r e l a t i o n b e t w e e n s e r u m l e v e l s of e s t r o n e a n d a n d r o s t e n e - d i o n e i n t h e c o n t r o l g r o u p 150. loo. 0 p W l LOO! .. . . . . . . . . . . . . .. . . . . . . . . " . . . . . . .I .. . . . . . . . . . . . . . " . ... I = m o pzOD5 50. . . . . . . . . . . . . . . . " . . . . . . r = Oh5 pcMOl F i g . 7 . C o r r e l a t i o n b e t w e e n s e r u m l e v e l s of e s t r o n e a n d t e s t o s t e r o n e i n t h e c o n t r o l g r o u p E l pmdll mi r 5 0.25 p 4 0 1 . . . . . / . ." . . . . . . . .... . . .. . . . . . . . . . . . . . . - " * u : .: _. . . . . . . I 015 (0 1:5 2b 25 3'0 15 T nrnolll F i g . 9. C o r r e l a t i o n b e t w e e n s e r u m l e v e l s of e s t r o n e a n d t e s t o s t e r o n e i n t h e p a t i e n t g r o u p 267 Weight varied from 41 to 110 kg with a close accordance between the distribu- tions and a mean weight of 65.55 kg in the patient group and 65.75 kg in the control group. The same accordance was found concerning height with a range from 140 to 178 and mean values of 161.7 and 161.4 cm, respectively. The differences were, as reported elsewhere (4), not statistically significant in either weight, height or the Quetelet’s index. When weight and the index for overweight (Quetelet’s index) were correlated to the steroid hormone levels the correlation coefficients were very low (Table 3). Except for a low degree of significance (p < 0.05) for the correlation be- tween El and Quetelet’s index in the control group (Table 3) they were all in- significant (p > 0.05). The same was true in the patient group in the correla- tion to the stage of the disease according to the TNM classification (Table 3). A slight but significant negative correlation was found between Quetelet’s index and the SHBG, in both groups (Table 3). Interrelations between El, A and T were finally analyzed with calculation of correlation coefficients and with a multiple regression analysis using El as a dependent variable and A and T as independent variables in addition to age and the weight index. In the control group there was a highly significant correlation between El and A (r=0.48) (Fig. 6) and El and T (r=0.45) (Fig. 7). The situation was quite different in the patient group where no significant correlation (r=0.10) was found between El and A (Fig. 8) and a low correlation (~0.25) between El and T (Fig. 9). According to the multiple regression analysis the variances in the serum level of El could be explained to 14 % and 58 % in the patient and control group, respectively, by variations primarily in A and T with very little contri- bution to the determination coefficient from age and the weight index. SHBG was not significantly correlated to El, A or T in any of the groups (r=0.17). DISCUSSION The estrogen and androgen metabolism and the serum concentration after the menopaus differ in many aspects from the premenopausal state. The serum concen- trations of estradiol and estriol were thus generally found to be low (8, 15, 16, 17, 27, 35, 46) while estrone is the dominant estrogen in the postmenopause with normal values in most reports which were in agreement with the mean value in our control group (17, 25, 27, 35, 42, 46) and in some reports even higher (3, 9, 58). A l s o concerning testosterone our study revealed a mean value in accordance with previous authors (3, 10, 24, 30, 58) although lower (25, 42) as well as higher (16) normal values have been reported. Our mean value for andro- stenedione in the control group when corrected for the blank, is in the range found by some other investigators using the same antibody for the radioimmuno- a s s a y ( 2 , 3 , 4 2 ) b u t somewhat lower t h a n t h a t r e p o r t e d by o t h e r s who u s u a l l y found v a l u e s i n t h e i n t e r v a l 0.75-1.09 ng/ml ( 1 6 , 1 7 , 2 5 , 5 8 ) . The u s e of n o n - h o s p i t a l i z e d c o n t r o l s made a c o m p a r a b i l i t y w i t h t h e p a t i e n t g r o u p w i t h r e g a r d t o a r e c e n t o p e r a t i o n i m p o s s i b l e . S u r g i c a l trauma h a s been shown t o i n d u c e a s m a l l and t r a n s i t o r y f a l l of A and T b u t no change o f E l i n normal women ( 2 8 ) . The f a c t t h a t t h e s e c h a n g e s were n o r m a l i z e d w i t h i n one w e e k ( 2 8 ) and t h a t o u r p a t i e n t s had h i g h e r mean v a l u e s t h a n t h e c o n t r o l s seems t o e x c l u d e p o s t o p e r a t i v e c h a n g e s a s a cause of t h e d i f f e r e n c e s found i n t h i s s t u d y . D i u r n a l v a r i a t i o n s o f A and T i n women were shown by Vermeulen ( 5 8 ) whereas r e s u l t s c o n c e r n i n g E l have been c o n t r a d i c t o r y ( 8 , 5 8 ) . A s i g n i f i c a n t d i f f e r e n c e i n t h e a v e r a g e s a m p l i n g t i m e between p a t i e n t s and c o n t r o l s was, however, con- s i d e r e d u n l i k e l y and t h e c h a n g e s d u r i n g t h e day too s m a l l ( 5 8 ) t o a c c o u n t f o r t h e d i f f e r e n c e s f o u n d between t h e g r o u p s c o n c e r n i n g A and T. Accumulated e v i d e n c e now s u p p o r t t h e view t h a t n e g l i g i b l e amounts of estro- g e n s a r e s e c r e t e d by t h e p o s t m e n o p a u s a l o v a r i e s or a d r e n a l s ( 1 2 , 1 6 , 2 5 , 3 6 - 3 8 , 4 3 ) . I n s t e a d most e s t r o g e n s a f t e r t h e menopause a r e d e r i v e d from t h e p e r i p h e r a l c o n v e r s i o n of t h e a n d r o g e n s ( 1 7 , 3 4 , 3 7 ) s e c r e t e d by t h e o v a r i e s and a d r e n a l s ( 1 6 , 1 7 , 2 5 , 2 6 , 3 6 ) . Thus E l c a n be a c c o u n t e d f o r n e a r l y e x c l u s i v e l y by t h e p e r i p h e r a l c o n v e r s i o n o f A w h e r e a s t h e p r o d u c t i o n of E 2 i n t h e blood is m a i n l y due t o t h e c o n v e r s i o n of T b o t h d i r e c t l y and i n d i r e c t l y v i a A and E l ( 3 4 , 3 8 ) . T h e r e f o r e t h e h i g h e r mean c o n c e n t r a t i o n of E l found i n women w i t h b r e a s t c a n c e r i n t h i s s t u d y h a s t o be d i s c u s s e d i n terms of a lowered m e t a b o l i c c l e a r a n c e r a t e or a n i n c r e a s e d p r o d u c t i o n a s a consequence of e i t h e r i n c r e a s e d a v a i l a b i l i t y o f plasma p r e c u r s o r s or o f a n i n c r e a s e d e x t e n t of c o n v e r s i o n of t h e s e p r e c u r s o r s t o t h e p r o d u c t hormone ( 3 9 ) . The m e t a b o l i c c l e a r a n c e r a t e o f e s t r o g e n s ( E l , E 2 , E 3 ) h a s been shown t o be d e c r e a s e d i n p o s t m e n o p a u s a l women compared t o premenopausal ( 1 5 , 3 5 ) b u t i n b r e a s t c a n c e r p a t i e n t s t h e r e h a s r a t h e r been a t e n d e n c y t o h i g h e r m e t a b o l i c c l e a r a n c e o f E l ( 3 1 ) and even A ( 3 1 , 4 5 ) t h a n i n n o r m a l s . The c o n v e r s i o n r a t e o f a n d r o s t e n e d i o n e t o e s t r o n e i n b r e a s t c a n c e r p a t i e n t s d i d n o t d i f f e r from t h a t i n normal c o n t r o l s ( 3 1 , 4 5 ) . B u t i n women w i t h endo- m e t r i a l c a r c i n o m a ( 1 8 , 5 3 ) and e n d o m e t r i a l h y p e r p l a s i a ( 5 3 ) a n i n c r e a s e d con- v e r s i o n was found and s u g g e s t e d an i m p o r t a n t e t i o l o g i c f a c t o r ( 3 9 ) . These f i n d - i n g s m i g h t imply t h a t t h e p r i n c i p a l r e a s o n f o r t h e h i g h e r e s t r o n e l e v e l i n t h e b r e a s t c a n c e r g r o u p i s a n i n c r e a s e d a v a i l a b i l i t y of t h e p r e c u r s o r s , m a i n l y A and T . T h i s c o n c e p t was s u p p o r t e d by t h e s i g n i f i c a n t l y h i g h e r v a l u e s found i n t h e p a t i e n t g r o u p i n o u r s t u d y ( T a b l e 2 ) . A s l i g h t d i f f e r e n c e i n t h e same d i r e c - t i o n w a s a l s o s u g g e s t e d f o r a n d r o s t e n e d i o n e i n a r e c e n t r e p o r t by Rose e t a l . ( 4 8 ) . I n c r e a s e d t e s t o s t e r o n e l e v e l s were a l s o r e c e n t l y f o u n d i n b r e a s t c a n c e r p a t i e n t s by McFadyen e t a l . ( 4 4 ) and S a r f a t y e t a l . ( 5 1 ) a l t h o u g h t w o p r e v i o u s r e p o r t s a r e i n d i s a g r e e m e n t w i t h t h i s f i n d i n g ( 2 2 , 5 6 ) . Whereas i n t h e c o n t r o l g r o u p a c o n s i d e r a b l e p a r t o f t h e e s t r o n e v a r i a t i o n s 269 were r e l a t e d t o t h e a v a i l a b i l i t y o f p r e c u r s o r s ( F i g s 6 and 7 ) , t h e l a c k o f c o r - r e l a t i o n between E l and A and t h e l o w c o r r e l a t i o n between E l and T i n t h e p a t i e n t g r o u p a r e c o n f u s i n g and s u g g e s t t h a t o t h e r f a c t o r s have a n i n f l u e n c e on t h e s e r u m e s t r o n e l e v e l . The r e s u l t s of Poortman e t a l . ( 4 5 ) and K i r s c h n e r e t a l . (31) a r e a l s o i n c o n s i s t e n t w i t h t h e c o n c e p t o f a n i n c r e a s e d a v a i l a b i l i t y of p r e - cursor hormones i n b r e a s t c a n c e r p a t i e n t s a s t h e y found no d i f f e r e n c e i n t h e a n d r o s t e n e d i o n e p r o d u c t i o n r a t e compared t o age-matched c o n t r o l s . Although t h e p r o d u c t i o n of A and T c a n be a c c o u n t e d f o r e x c l u s i v e l y by a d r e - n a l and o v a r i a n s e c r e t i o n and i n t e r c o n v e r s i o n between t h e s e hormones ( 5 8 ) a n a d d i t i o n a l source of e s t r o g e n was s u g g e s t e d by H i l l e t a l . ( 2 1 ) . They demon- s t r a t e d t h e p r o d u c t i o n of e s t r o g e n s by b a c t e r i a from t h e b i l i a r y s t e r o i d s re- l a t e d t o t h e f a t c o n t e n t i n t h e d i e t . T h i s c o u l d t h e n l i n k t h e o b s e r v e d g r e a t i n t e r n a t i o n a l v a r i a t i o n s i n b r e a s t c a n c e r i n c i d e n c e - o b v i o u s l y r e l a t e d t o e n v i r o n m e n t a l f a c t o r s ( 4 ) - t o an e n d o c r i n e h y p o t h e s i s . The d i s t r i b u t i o n and mean v a l u e of SHBG i n t h i s m a t e r i a l is i n t h e r a n g e p r e - v i o u s l y r e p o r t e d i n normal women ( 4 9 ) . V a l u e s i n b r e a s t c a n c e r p a t i e n t s a r e n o t a v a i l a b l e f o r c o m p a r i s o n . Serum SHBG l e v e l s a r e known to be r e g u l a t e d by t h e b i o l o g i c a l a c t i v e , unbound hormone f r a c t i o n w i t h t h e a n d r o g e n s h a v i n g a n i n h i b i - t o r y and e s t r o g e n s a s t i m u l a t o r y i n f l u e n c e ( 6 , 5 7 ) . T h y r o i d f u n c t i o n , a s s e s s e d i n t h i s m a t e r i a l by d e t e r m i n a t i o n of TSH, T3, rT3, T4 and T 3 - r e s i n u p t a k e and r e p o r t e d e l s e w h e r e ( 5 ) , is a l s o known t o i n f l u e n c e t h e SHBG l e v e l ( 6 ) and t h e s t e r o i d hormone m e t a b o l i s m ( 5 4 ) . The r e s u l t s were, however, m a i n l y w i t h i n t h e l i m i t s o f t h e r e f e r e n c e r a n g e and no s i g n i f i c a n t c o r r e l a t i o n s t o SHBG, E l , A or T were f o u n d . The lower c o n c e n t r a t i o n of SHBG i n t h e p a t i e n t g r o u p s p e a k s a g a i n s t i n c r e a s e d e s t r o g e n i c i t y i n b r e a s t c a n c e r p a t i e n t s and might r a t h e r i n d i c a t e a predominance of a n d r o g e n i c i n f l u e n c e ( 6 ) c o n s i s t e n t w i t h t h e c o n c e p t o f i n - c r e a s e d p r o d u c t i o n of A and T. T h e r e i s a well-known r e l a t i o n between t h e b i o l o g i c a l a c t i v i t y o f s t e r o i d h o r - mones and t h e i r b i n d i n g t o SHBG. C o n s e q u e n t l y t h e b i n d i n g o f t e s t o s t e r o n e is h i g h and t h a t o f a n d r o s t e n e d i o n e and e s t r o n e v e r y l o w or n e g l i g i b l e ( 6 , 4 9 ) . A lower SHBG l e v e l i n t h e p a t i e n t g r o u p w i l l t h e r e f o r e p r i m a r i l y a f f e c t testo- s t e r o n e and cause a n i n c r e a s e d f r a c t i o n o f t h e unbound hormone. An a d d i t i o n a l consequence would b e a n i n c r e a s e d m e t a b o l i c c l e a r a n c e r a t e o f t e s t o s t e r o n e ( 1 1 , 57) which c o u l d d i m i n i s h t h e i n f l u e n c e of a n i n c r e a s e d p r o d u c t i o n r a t e on t h e p e r i p h e r a l c o n c e n t r a t i o n ( 1 ) . The a b i l i t y o f a d c p o s e t i s s u e t o m e t a b o l i z e C 1 9 s t e r o i d s t o e s t r o g e n s h a s been d e m o n s t r a t e d i n v i t r o ( 5 2 ) . A p o s i t i v e c o r r e l a t i o n was a l s o found between w e i g h t and c o n v e r s i o n r a t e of A t o E l i n normal p o s t m e n o p a u s a l women ( 5 3 ) and i n women w i t h e n d o m e t r i a l c a n c e r ( 4 7 ) . The l a c k of s i g n i f i c a n t c o r r e l a t i o n between w e i g h t and s e r u m e s t r o n e l e v e l s i n normal p o s t m e n o p a u s a l women found i n t h e p r e s e n t s t u d y , i s i n a c c o r d a n c e w i t h t h e f i n d i n g s o f J u d d e t a l . ( 2 5 , 2 7 ) b u t a 270 positive correlation was found in women with endometrial carcinoma ( 2 7 ) . Our study did not reveal any difference in weight or in different weight indices between patients and controls ( 4 ) and no correlations to E l , A or T except for a low correlation between E l and Quetelet's index in the control group. If the amount of fat tissue is positively correlated to the conversion rate of A to E l , an influence on the serum levels might be obscured if the body weight is nega- tively correlated to the percentage of E l derived from plasma A which enters the circulation, as noticed by Grodin et al. ( 1 7 ) . But if obesity was a risk factor for breast cancer and the amount of fat tissue quantitatively influenced the aromatization of estrogen precursors, then the conversion rate of A to E l ought to be increased in breast cancer patients. This was, as pointed out above, not confirmed by Poortman et al. ( 4 5 ) or by Kirschner et al. ( 3 1 ) who found a con- version rate in close agreement with other studies in normal postmenopausal women ( 3 7 , 5 3 ) . A negative correlation between SHBG and obesity, confirmed in this study, was previously noticed by Vermeulen et al. ( 5 7 ) . The conversion rate of A to E l has repeatedly been found to be 1 . 2 - 1 . 3 % in premenopausal women ( 3 7 , 5 3 ) and at least two times higher in postmenopausal women ( 1 7 , 3 7 , 3 8 , 4 5 , 5 3 ) . The concept of a progressive increase with advancing age, as suggested by Hemsell et al. ( 2 0 ) has less support and was not confirmed in women with endometrial carcinoma ( 4 7 ) . It is also in disagreement with the lack of correlation between age and excretion of urinary estrogens found by Thijssen et al. ( 5 5 ) in normal postmenopausal women. Neither was there any cor- relation between age and serum levels of E l or A in the present study nor in those by Judd et al. ( 2 5 , 2 7 ) . In conclusion, serum concentrations of E l and SHBG have, to our knowledge, not been studied before in breast cancer patients and thus the increased E l and decreased SHBG never observed, whereas the increased A and T levels have some support in a few recent studies. Although an increased production of estrogen precursors seems to be the most reasonable explanation for these findings, they are difficult to fit into a definite hypothesis and uniform support for this view was not perceivable in earlier studies. The significance of these findings needs therefore further conformation, primarily by studies of steroid hormone kinetics including production, conversion and metabolic clearance in breast cancer patients and comparable controls. ACK€?OWLEDGMENT This study was supported by contract No. 1-CB 5 3 9 6 8 from the National Cancer Institute, USA and by grants from the Swedish Medical Research Council No. 0 3 4 9 5 and the Ford Foundation. 27 1 REFERENCES 1 . Abraham, G . E . , L o b o t s k y , J . & L l o y d , C.W.: M e t a b o l i s m o f t e s t o s t e r o n e a n d a n d r o s t e n e d i o n e i n n o r m a l and o v a r i e c t o m i z e d women. J C l i n I n v e s t 48: 696-703, 1969. c y c l e i n a b i l a t e r a l l y a d r e n a l e c t o m i z e d woman. J C l i n E n d o c r i n o l Metab 37: 58 1-587 , 1973. 3. Abraham, G.E. & M a r o u l i s , G.B.: E f f e c t o f e x o g e n o u s e s t r o g e n o n s e r u m p r e g - 2 . Abraham, G . E . & C h a k m a k j i a n , Z . H . : Serum s t e r o i d l e v e l s d u r i n g t h e m e n s t r u a l n e n o l o n e , c o r t i s o l , a n d a n d r o g e n s i n p o s t m e n o p a u s a l women. J O b s t e t G y n e c o l 45:271-275, 1975. w e i g h t a n d o b e s i t y on risk o f b r e a s t c a n c e r i n a n u n s e l e c t e d S w e d i s h popu- l a t i o n . B r J C a n c e r 36:787-792, 1977. 5. Adami, H . O . , R i m s t e n , A . , ThorCn, L . , V e g e l i u s , J . & Wide, L.: T h y r o i d d i s - ease a n d f u n c t i o n i n b r e a s t c a n c e r p a t i e n t s a n d n o n - h o s p i t a l i z e d c o n t r o l s e v a l u a t e d b y d e t e r m i n a t i o n o f TSH, T 3 , r T 3 a n d T4 l e v e l s i n s e r u m . Acta C h i r S c a n d 144:89-97, 1978. 69-96, 1974. 4. Adami, H.O., R i m s t e n , A . , S t e n k v i s t , B. & V e g e l i u s , J.: I n f l u e n c e Of h e i g h t , 6 . A n d e r s o n , D.C.: S e x - h o r m o n e - b i n d i n g g l o b u l i n . C l i n E n d o c r i n o l ( O x f ) 3: 7 . A x e l s s o n , O., L i n d b e r g , B.S., N i l s s o n , B . A . & J o h a n s s o n , E . D . B . : A r a d i o - i m m u n o l o g i c a l method f o r o e s t r o n e . P l a s m a l e v e l s o f n o n c o n j u g a t e d o e s t r o n e d u r i n g u n c o m p l i c a t e d p r e g n a n c y . Acta O b s t e t G y n e c o l S c a n d 56:49-54, 1977. 8 . B a i r d , D.T. & G u e v a r a , A . : C o n c e n t r a t i o n o f u n c o n j u g a t e d e s t r o n e a n d e s t r a - d i o l i n p e r i p h e r a l p l a s m a i n n o n p r e g n a n t women t h r o u g h o u t t h e m e n s t r u a l c y c l e , c a s t r a t e a n d p o s t m e n o p a u s a l women a n d i n men. J C l i n E n d o c r i n o l M e t a b 29: 149-159, 1 9 6 9 . 9. B a i r d , D.T. S t e r o i d s i n b l o o d r e f l e c t i n g o v a r i a n f u n c t i o n . In: C o n t r o l o f G o n a d a l S t e r o i d S e c r e t i o n ( e d . D.T. B a i r d ) , p p . 176-191, U n i v . P r e s s , E d i n b u r g h , 197 1 . 1 0 . B a r b e r i a , J . M . & T h r o n e y c r a f t , I . H . : S i m u l t a n e o u s r a d i o i m m u n o a s s a y of t e s t o - s t e r o n e a n d d i h y d r o t e s t o s t e r o n e . S t e r o i d s 23:757-766, 1974. 1 1 . B a r d i n , C.W. & L i p s e t t , M.B.: T e s t o s t e r o n e a n d a n d r o s t e n e d i o n e b l o o d p r o d u c - t i o n r a t e s i n n o r m a l women a n d women w i t h i d i o p a t h i c h i r s u t i s m or p o l y c y s t i c o v a r i e s . J C l i n I n v e s t 46:891-902, 1967. 12. Barlow, J.J., Emerson, K . & S a x e n a , B . N . : E s t r a d i o l p r o d u c t i o n a f t e r o v a r i - e c t o m y f o r c a r c i n o m a of t h e b r e a s t . N E n g l J Med 280:633-637, 1969. 13. D i c k i n s o n , L . , MacMahon, B . , Cole, P . & Brown, J.: E s t r o g e n p r o f i l e s of o r i e n t a l a n d C a u c a s i a n women i n H a w a i i . N E n g l J Med 291:1211-1213, 1974. 1 4 . E d q v i s t , L.E. & J o h a n s s o n , E . D . B . : Radioimmunoassay o f o e s t r o n e a n d oestra- d i o l i n human a n d b o v i n e p e r i p h e r a l p l a s m a . Acta E n d o c r i n o l (Kbh) 71: 716-730, 1972. 1 5 . F l o o d , C . , P r a t t , J . H . & L o n g c o p e , C . : The m e t a b o l i c c l e a r a n c e a n d b l o o d p r o d u c t i o n r a t e s of e s t r i o l i n n o r m a l , n o n - p r e g n a n t women. J C l i n E n d o c r i n o l M e t a b 4 2 : l - 8 , 1976. 16. G r e e n b l a t t , R.B., C o l l e , M.L. & Mahesh, V . B . : O v a r i a n a n d a d r e n a l s t e r o i d p r o d u c t i o n i n t h e p o s t m e n o p a u s a l women. J O b s t e t G y n a e c o l 47:383-387, 1976. 17. G r o d i n , J . H . , S i i t e r i , P.K. & MacDonald, P.C.: S o u r c e o f e s t r o g e n p r o d u c t i o n i n p o s t m e n o p a u s a l women. J C l i n E n d o c r i n o l Metab 36:207-214, 1973. 1 8 . H a u s k n e c k t , R.U. & G e s b e r g , S.B.: E s t r o g e n m e t a b o l i s m i n p a t i e n t s a t h i g h r i s k f o r e n d o m e t r i a l c a r c i n o m a . Am J O b s t e t G y n e c o l 116:981-984, 1973. 19. Hayward, J.: Hormones a n d Human B r e a s t C a n c e r . S p r i n g e r - V e r l a g , B e r l i n - Heidelberg-New Y o r k , 1 9 7 0 . 20. H e m s e l l , D . L . , G r o d i n , J . M . , B r e n n e r , P . F . , S i i t e r i , P.K. & MacDonald, P.C.: P l a s m a p r e c u r s o r s o f e s t r o g e n . 11. C o r r e l a t i o n o f t h e e x t e n t o f c o n v e r s i o n o f p l a s m a a n d r o s t e n e d i o n e t o e s t r o n e w i t h a g e . J C l i n E n d o c r i n o l Metab 38: 476-479, 1974. 21. H i l l , M . J . , G o d d a r d , P . & W i l l i a m s , R.E.O.: G u t b a c t e r i a a n d a e t i o l o g y o f 22. H o r n , H . & G o r d a n , G.S.: P l a s m a t e s t o s t e r o n e i n a d v a n c e d b r e a s t c a n c e r . c a n c e r o f t h e b r e a s t . L a n c e t I I : 4 7 2 - 4 7 3 , 1971. O n c o l o g y 30:147-151, 1 9 7 4 . 272 23. I n t e r n a t i o n a l Union A g a i n s t Cancer ( U I C C ) , TNM C l a s s i f i c a t i o n of M a l i g n a n t Tumors. Second e d i t i o n . Geneva, 1974. 24. I t o , T. & H o r t o n , R.: The source Of plasma d i h y d r o t e s t o s t e r o n e i n man. J C l i n I n v e s t 50:1621-1627, 1971. 25. J u d d , H.L., J u d d , E . G . , L u c a s , W.E. & Yen, S.S.C.: E n d o c r i n e f u n c t i o n o f t h e p o s t m e n o p a u s a l o v a r y : C o n c e n t r a t i o n of a n d r o g e n s and e s t r o g e n s i n o v a r i a n and p e r i p h e r a l v e i n b l o o d . J C l i n E n d o c r i n o l Metab 39:1020-1024, 1974. 26. J u d d , H . L . , L u c a s , W.E. & Yen, S.S.C.: E f f e c t of oophorectomy o n c i r c u l a t i n g t e s t o s t e r o n e and a n d r o s t e n e d i o n e l e v e l s i n p a t i e n t s w i t h e n d o m e t r i a l c a n c e r . Am J O b s t e t Gynecol 118:793-798, 1974. 27. J u d d , H.L., L u c a s , W.E. & Yen, S.S.C.: Serum 1 7 B - e s t r a d i o l and e s t r o n e l e v e l s i n p o s t m e n o p a u s a l women w i t h and w i t h o u t e n d o m e t r i a l c a n c e r . J C l i n Endo- c r i n o l Metab 43:272-278, 1976. 28. J u d d , H . L . , R i g g , L . A . , Anderson, D . C . & Y e n , S.S.C.: The e f f e c t s of o v a r i a n wedge r e s e c t i o n on c i r c u l a t i n g g o n a d o t r o p i n and o v a r i a n s t e r o i d l e v e l s i n p a t i e n t s w i t h p o l y c y s t i c o v a r y syndrome. J C l i n E n d o c r i n o l Metab 43:347- 355, 1976. h e i g h t . B r J P r e v SOC Med 21:122-128, 1967. androgen p r o d u c t i o n and m e t a b o l i s m i n normal and v i r i l i z e d women. Metabolism 21:667-688, 1972. 31. K i r s c h n e r , M . A . , Cohen, F.B., Ryan, C . & Adrews, S.: I n c r e a s e d c l e a r a n c e of a n d r o s t e n e d i o n e and oestrone i n women w i t h b r e a s t c a n c e r . Program of X I I n t e r n a t i o n a l Cancer C o n g r e s s , 249. F l o r e n c e , I t a l y , 1974. Cancer 39:2716-2726, 1977. t i o n i n p a t i e n t s w i t h b r e a s t c a n c e r p r i o r t o e n d o c r i n e t h e r a p y . JAMA 196: 29. K h o s l a , T . & L o w e , C.R.: I n d i c e s o f o b e s i t y d e r i v e d from body w e i g h t and 30. K i r s c h n e r , M.A. & B a r d i n , W.C.: P r o g r e s s i n e n d o c r i n o l o g y and metabolism: 32. K i r s c h n e r , M.A.: The role o f hormones i n t h e e t i o l o g y of human b r e a s t c a n c e r . 33. Lemon, H . M . , W o t i z , H . H . , P a r s o n s , L . & Mozden, P . J . : Reduced e s t r i o l excre- 112-120, 1966. g e n s i n normal a d u l t men and women. J C l i n I n v e s t 48:2191-2201, 1969. p o s t m e n o p a u s a l women. Am J O b s t e t Gynecol 111:778-781, 1971. I n t Cong E n d o c r i n o l 403:484-485, 1977. a n d r o s t e n e d i o n e for e s t r o n e p r o d u c t i o n i n women. P r o c I n t Cong E n d o c r i n o l 34. Longcope, C . , K a t o , T . & H o r t o n , R . : C o n v e r s i o n o f blood a n d r o g e n s t o estro- 35. Longcope, C.: M e t a b o l i c c l e a r a n c e and blood p r o d u c t i o n r a t e s o f e s t r o g e n s i n 36. Luukainen, T., Lahteenmaki, P. & S i p i n e n , S.: Postmenopausal o v a r y . Proc 37. MacDonald, P . C . , G r o d i n , J . M . & S i i t e r i , P.K.: The u t i l i z a t i o n o f plasma 184:770-776 , 1969. 38. MacDonald, P.C., G r o d i n , J . M . & S i i t e r i , P.K.: Dynamics o f a n d r o g e n and o e s t r o g e n s e c r e t i o n . I n : C o n t r o l of Gonadal S t e r o i d S e c r e t i o n ( e d . D.T. B a i r d & J . A . S t r o n g ) , pp. 158-174, Univ. P r e s s , E d i n b u r g h , 1971. p r o d u c t i o n of e s t r o n e and t h e o c c u r r e n c e o f e n d o m e t r i a l n e o p l a s i a . Gynecol Oncol 2:259-263, 1974. view. J N a t l Cancer I n s t 5O:Zl-42, 1973. Woo, H.C.: U r i n e o e s t r o g e n p r o f i l e s of Asian and N o r t h American women. I n t J Cancer 14:161-167, 1974. 42. Maroulis, G.B. & Abraham, G.E.: O v a r i a n and a d r e n a l c o n t r i b u t i o n s t o p e r i - p h e r a l s t e r o i d l e v e l s i n p o s t m e n o p a u s a l women. O b s t e t Gynecol 48:150-154, 1976. 43. M a t t i n g l y , R.F. & Huang, W.Y.: S t e r o i d g e n e s i s of t h e menopausal and p o s t - menopausal o v a r y . Am J O b s t e t Gynecol 103:679-693, 1969. 44. McFadyen, I . J . , P r e s c o t t , R . J . , G r o o m , G . V . , F o r r e s t , A.P.M., G o l d e r , M.P. & Fahmy, D . R . : C i r c u l a t i n g hormone c o n c e n t r a t i o n s i n women w i t h b r e a s t c a n c e r . L a n c e t 1:llOO-1102, 1976. s i o n t o e s t r o g e n s i n normal p o s t m e n o p a u s a l women and i n s e l e c t e d b r e a s t cancer p a t i e n t s . J C l i n E n d o c r i n o l Metab 37:lOl-109, 1973. 39. MacDonald, P.C. & S i i t e r i , P.K.: The r e l a t i o n s h i p between t h e e x t r a g l a n d u l a r 40. MacMahon, B . , Cole, P. & BKown, J.: E t i o l o g y of human b r e a s t c a n c e r : a re- 41. MacMahon, B . , Cole, P . , B r o w n , J . B . , A o k i , K . , L i n , T.M., Morgan, R.W. & 45. Poortman, J., T h i j s s e n , H . H . & Schwarz, F.: Androgen p r o d u c t i o n and c o n v e r - 273 46. R a d e r , M . D . , F l i c k i n g e r , C . L . , D e v i l l a , G . O . , M i k u t a , J.J. & M i k h a i l , G . : Plasma e s t r o g e n s i n p o s t m e n o p a u s a l women. Am J O b s t e t Gynecol 116:1069-,1073, 1973. 47. R i z k a l l a h , T . H . , T o v e l l , H . M . M . & K e l l y , W . G . : P r o d u c t i o n o f e s t r o n e and f r a c t i o n a l c o n v e r s i o n of c i r c u l a t i n g a n d r o s t e n e d i o n e t o e s t r o n e i n women w i t h e n d o m e t r i a l c a r c i n o m a . J C l i n E n d o c r i n o l Metab 40:1045-1056, 1975. 48. Rose, D.P., S t a u b e r , P . , T h i e l , A . , Crowley, J.J. & M i l b r a t h , J . R . : Plasma d e h y d r o e p i a n d r o s t e r o n e s u l f a t e , a n d r o s t e n e d i o n e and c o r t i s o l , and u r i n a r y f r e e cortisol e x c r e t i o n i n b r e a s t c a n c e r . E u r J C a n c e r 13:43-47, 1917. 49. R o s n e r , W . : I n t e r a c t i o n o f a d r e n a l and g o n a d a l s t e r o i d s w i t h p r o t e i n s i n human plasma. N Engl J Med 281:658-665, 1969. 50. R o s n e r , W.: A s i m p l i f i e d method f o r t h e q u a n t i t a t i v e d e t e r m i n a t i o n o f testo- s t e r o n e - e s t r a d i o l b i n d i n g g l o b u l i n a c t i v i t y i n plasma. J C l i n E n d o c r i n o l Metab 34:983-988, 1972. P . : Hormonal c o n c o m i t a n t s o f b r e a s t c a n c e r : u r i n a r y a n d r o g e n m e t a b o l i t e s , plasma t e s t o s t e r o n e and p r o l a c t i n . P r o c I n t Cong E n d o c r i n o l 403:399-404, 1977. 52. S c h i n d l e r , A.E.: C o n v e r s i o n of a n d r o s t e n e d i o n e t o e s t r o n e by human f a t t i s s u e . J C l i n E n d o c r i n o l Metab 35:627-630, 1972. 53. S i i t e r i , P.K. & MacDonald, P.C.: Role o f e x t r a g l a n d u l a r e s t r o g e n . I n : Human E n d o c r i n o l o g y : Handbook o f P h y s i o l o g y . S e c t i o n 7,2, pp. 615-629. The Ameri- c a n P h y s i o l o g i c S o c i e t y , N e w Y o r k , 1973. The c o n v e r s i o n of a n d r o g e n s t o e s t r o g e n s i n h y p e r t h y r o i d i s m . J C l i n Endo- c r i n o l Metab 38:207-214, 1974. 55. T h i j s s e n , J . H . H . , Poortman, J . , Schwarz, F. & d e Waard, F.: Post-menopausal e s t r o g e n p r o d u c t i o n , w i t h s p e c i a l r e f e r e n c e t o p a t i e n t s w i t h mammary c a r c i - noma. I n : F r o n t Hormone R e s , v o l . 3, pp. 45-62, K a r g e r , B a s e l , 1975. 56. Wang, D . Y . , Hayward, J . L . & Bulbrook, R.D.: T e s t o s t e r o n e l e v e l s i n t h e plasma of normal women and p a t i e n t s w i t h b e n i g n b r e a s t d i s e a s e or w i t h b r e a s t c a n c e r . E u r J Cancer 2:373-376, 1966. t h e t e s t o s t e r o n e - b i n d i n g g l o b u l i n i n human plasma and i n f l u e n c e o f s p e c i f i c b i n d i n g o f t e s t o s t e r o n e o n i t s m e t a b o l i c c l e a r a n c e r a t e . J C l i n E n d o c r i n o l Metab 29: 1470-1480, 1969. E n d o c r i n o l Metab 42:247-253, 1976. b i n d i n g g l o b u l i n and D - n o r g e s t r e l l e v e l s i n plasma. Acta E n d o c r i n o l (Kbh) 86:430-436, 1977. 60. Wide, L . , N i l l i u s , S . J . , G e m z e l l , C . & ROOS, P . : Radioimmunosorbent a s s a y o f f o l l i c l e - s t i m u l a t i n g hormone and l u t e i n i z i n g hormone i n s e r u m and u r i n e from men and women. Acta E n d o c r i n o l [ S u p p l l 174:l-58, 1973. s i o n o f a n d r o g e n s t o normal 1 7 - k e t o s t e r o i d m e t a b o l i t e s : a n o n s p e c i f i c con- s e q u e n c e of i l l n e s s . J C l i n E n d o c r i n o l Metab 32:824-832, 1971. hormone-dependent c a n c e r . Cancer R e s 35:3365-3373, 1975. 51. S a r f a t y , G . , T a l l i s , G . M . , Murray, R.M.L., P i t t , P . , G a r d n e r , H . & Leppard, 54. S o u t h r e n , A . L . , O l i v o , J., Gordon, G . G . , V i t t e k , J., B r e n e r , J . & R a f f i , F.: 57. Vermeulen, A . , Verdonah, L . , van d e r S t r a e t e n , M. & O r i e , N . : C a p a c i t y o f 58. V e r m e u l e n , A.: The hormonal a c t i v i t y of t h e p o s t m e n o p a u s a l o v a r y . J C l i n 59. V i c t o r , A , , Weiner, E . & J o h a n s s o n , E.D.B.: R e l a t i o n between s e x hormone 61. Zumoff, B . , Bradlow, H . L . , G a l l a g h e r , T.F. & Hellman, L.: D e c r e a s e d c o n v e r - 62. Zumoff, B., Fishman, J., Bradlow, H.L. & Hellman, L.: Hormone p r o f i l e s i n Accepted J a n u a r y 25, 1979 Address f o r r e p r i n t s : Hans-Olov Adami, M.D. Department o f S u r g e r y U n i v e r s i t y H o s p i t a l 5-750 14 Uppsala Sweden 274