Europ. J. Cancer Vol. 9, pp. 553-556. Pergamon Press 1973. Printed in Great Britain

Plasma Oestradiol and Progesterone in Benign Breast Disease

MARGARET C. SWAIN, J. L. HAYWARD" and R. D. BULBROOK Imperial Cancer Research Fund, Lincoln's Inn Fields, London WC2A 3PX,

England A b s ~ - - T h e concentrations of plasma oestradiol and progesterone have been measured in women with benign breast disease and in a control group of ostensibly normal women.

There were no significant differences in the steroid levels of the two groups in the folli- cular, ovulatory and luteal phases of the menstrual cycle. The hormone levels were not related to any of the histological diagnoses made on tissue obtained at biopsy from the women with benign breast disease.

INTRODUCTION

THE MAJORITY of diseases of the breast classed as "benign" can be divided into two main cate- gories, those in which the lesion is confined to the lobules such as fibroadenoma and cystic disease, and those in which the ductal system is affected such as duct ectasia and epitheliosis. In many cases both types of lesion occur and it has been generally assumed that the proliferative nature of epitheliosis is due to hypersecretion of one or more of the hormones known to be in- volved in the development of the mammary gland.

Measurement of the endocrine environment associated with particular benign breast diseases is of interest since the results might give a further insight into the aetiology of the benign conditions, and also because there is consider- able evidence that women with benign breast disease have a greater risk of subsequent breast cancer [1-7]. Studies on benign disease might therefore provide information about the role of the endocrine system in the genesis of breast cancer.

There is little published work on the hormonal status of women with benign breast disease [8-12], and these studies are confined to assays of urinary metabolites. Recently, methods have been devised for the measurement of oestrogens and progesterone in blood and since these

Accepted 8 May 1973. *Breast Unit, Guy's Hospital, London, SE1 9RT.

553

estimations may reflect more closely the hor- monal stimuli to the tissues, a study of the plasma concentrations of these compounds in normal pre-menopausal women and in patients with benign breast disease was undertaken.

MATERIAL AND METHODS

The patients in this study had been admitted to the Guy's Hospital Breast Unit for the biopsy of lumps considered to be benign. The lesions seen in the histological study of this group included epitheliosis, adenosis, fibroade- homo, cystic disease, duct abscess and simple cysts. Blood samples were collected by vene- puncture on the day prior to operation and the plasma stored at -20°C. The control group consisted of women living in Guernsey, none of whom had any history of menstrual disorders or breast disease. Both groups were within the age range 20-44 years, and the women in the control group were matched for age and the day of the menstrual cycle with the hospital patients; blood was collected and the plasma stored at -20°C.

Tissue specimens obtained at biopsy were fixed in formalin and embedded in paraffin wax. Sections (5 #m thick) were cut and stained with haematoxylin and eosin.

Plasma oestradiol was measured by a modifi- cation of the method of Corker [13]. 3H- Oestradiol (2000 counts/min) was added to plasma (4 ml) which was extracted twice with dichloromethane (10 ml). The dried extract

554 Margaret C. Swain, J. L. Hayward and R. D. Bulbrook

w a s chromatographed on columns of Sephadex LH20 with benzene: methanol (85:15) as elution mixture. The oestradiol fraction was evaporated to dryness, dissolved in tris-hydro- chloric acid buffer (0.01 M, pH 7.4) an aliquot taken for estimation of procedural losses and duplicate aliquots for oestradiol assay in the system described by Corker.

Plasma progesterone was estimated by a com- petitive protein binding assay which used diluted guinea-pig serum as the specific binding agent [ 1 4 ] .

R E S U L T S

The plasma samples were collected at various times in the menstrual cycle. This makes exact comparisons between patients and controls difficult since there is considerable variability between individuals in cycle length and time of

250

20C

15C

o

5¢

¢0)339

0

o

o

o

o

** 8

o

| o

4 ~

o

o

I f

0 6 - - 1 2

0

I: o o

• 0

~ o

8o

o

o o oO

~ o

la3 - 17' 18 - 2 6

Days of cycle

Fig. 1. Plasma oestradiol concentrations during the follicular, ovulatory and luteal phases of the menstrual cycle.

represents control group. © representspatients with benign breast disease. The horizontal lines indicate the mean values for each group.

ovulation. The cycle has therefore been divided into three phases: the follicular phase (for plasma specimens collected between day 6 and day 12); the ovulatory period (day 13-day 17) and the lutea] phase (day 18-day 26).

The plasma oestradiol concentrations of the controls and patients with benign breast disease in these three phases are shown in Fig 1. The plasma oestradiol concentrations of women with benign breast disease who were in the ovulatory and luteal phases are marginally lower than those in the control groups but the differences are not significant. The mean values are shown in Table 1.

The plasma progesterone concentrations in the two groups of women are shown in Fig. 2.

2 0

g ¢- o

4 -

o cl .

o E u

E

1 5

I O

t t i

6 - - 12

0

!

o • o

o • o

o

o

o

o

o • o

Z o •

o

o . 8 o ! ~

• • o

13 ~ 17' i , 1 8 - 2 6

Days of cycle

Fig. 2. Plasma progesterone concentrations during the follicular, ovulatory and luteal phases of the menstrual cycle.

O represents control group. 0 represents patients with benign breast disease. The horizontal lines indicate the mean values for each group.

Table 1. Plasma oestradiol concentrations in normal pre-menopausal women and in patients with benign breast disease

Days of cycle 6-12 13-17 18-26

Control Benign Control Benign Control Benign

No. of subjects 23

Mean concentration of plasma oestradiol (pg/ml) _+ S.E.M. 87 _+ 11

18 12 8 21 17

89__+13 112+26 754-19 100_+12 78_+13 i

Plasma Oestradiol and Progesterone in Benign Breast Disease

Once again there are no significant differences between the plasma concentrations in the nor- mal women and those with benign breast disease, although the latter have marginally higher progesterone concentrations in the folli- cular and ovulatory phases and lower concen- trations in the luteal phase. The mean values are shown in Table 2.

555

A study of the histology of the benign lesions is in progress. Current results show that there were 15 women with marked epithelial prolifera- tion, the lesion considered to carry the greatest risk of subsequent malignancy [3, 6]. The oestradiol and progesterone concentrations in these women were distributed evenly through- out the normal range of values. While further

Table 2. Plasma progesterone concentrations in normal pre-menopausal women and in patients with benign breast disease

i

Days of cycle 6-12 13-17 18-26

Control Benign Control Benign Control Benign

No. of subjects 23 18

Mean concentration of plasma progesterone (ng/ml) + S.E.M. 1.17+0.1l 2.93+ 1.06

Assays of plasma oestradiol and progesterone in a single plasma sample taken in the luteal phase of the cycle do not provide conclusive evidence whether or not ovulation has taken place, and it is obvious that if the frequency of anovulatory cycles differed between the normal controls and the patients with benign breast disease, a comparison of mean steroid values in the two groups would lead to bias.

For the purpose of this paper, anovulatory cycles have been defined as those in which the luteal phase oestradiol concentration was less than 60 pg/ml coupled with a progesterone con- centration of less than 3 ng/ml. By this defini- tion 4 normal women and 5 patients with benign breast disease would be classed as having an anovulatory cycle.

When the results for such patients and controls are excluded the mean oestradiol and progesterone concentrations in the luteal phase of the normal women were l l4pg /ml and 8.93 ng/ml, respectively, compared with 91 pg/ ml and 6.46 ng/ml in the patients with benign breast disease. The differences are not signifi- cant.

DISCUSSION

There are obvious limitations in a study in which single plasma specimens are taken in the menstrual cycle. In ideal conditions, serial samples would be much more informative. Nevertheless, the present results indicate that there are no discernible differences between the plasma oestradiol and progesterone concentra- tions in patients with benign breast disease and normal women.

12 8 21 17

5.76+ 1.40 6.64+ 1.98 7.48+ 1.07 5.14+0.95 i i

work is required it seems highly unlikely that this condition is associated with abnormalities in the plasma concentrations of the female sex hormones.

In a study in which blood samples were collected from a large number of ostensibly normal women, 5 women have subsequently developed breast cancer. Plasma oestradiol was measured in the samples from these women and in controls with no evidence of breast disease, matched for age and day of the menstrual cycle. Preliminary results show no apparent abnorma- lities in plasma oestradiol concentrations in the pre-clinical phase of the disease [15].

It is generally believed that oestradiol is an important hormone in the genesis of human breast cancer [16]. However, in women with benign breast disease and in women who subse- quently developed breast cancer, normal con- centrations of plasma oestradiol and pro- gesterone were found.

One explanation for these findings is that raised levels of plasma oestradiol are not a necessary factor in the aetiology of either benign or malignant breast disease. Alternatively, an effective imbalance in oestrogenic and pro- gestational stimuli could be mediated by ab- normalities in target cell response to normal peripheral concentrations of the relevant hormones.

The only convincing endocrine abnormality in patients with breast disease is a subnormal plasma level of androgen sulphates and of urinary androgen metabolites [17]. It would be tempting to suppose that oestrogenic and pro- gestational stimuli to the breast are modified by androgen secretion and that deficiencies in androgen production lead to an abnormal

556 Margaret C. Swain, J. L. Hayward and R. D. Bulbrook

stimulus to breast tissue by plasma levels of oestradiol and progesterone that are within the normal range.

Whatever the final explanation of our results may be, the original concept of oestradiol as a putative breast carcinogen, which stems from

the clear demonstrations of its role as such in rodents, appears less tenable in man.

Aclmowledgeznents--The authors wish to thank Dr. Stretton Young and Dr. Lillian Pang for their help in the histological study of biopsy specimens from the patients with benign breast disease.

REFERENCES

1. S. WARREN, The relation of chronic mastiffs to carcinoma of the breast. Surg. G~c . Obstet. 71, 257 (1940).

2. C.D. HAAOENS~N, In Diseases oftl~ Breast W. B. Saunders, Philadelphia (1956). 3. L.J . HUMPHREY and M. SW~RDLOW, Relationship of benign breast disease to

carcinoma of the breast. Surgery 52, 841 (1962). 4. A. BEHgEND, Cystic disease of the breast: its relation to the development of

carcinoma. J. int. Coll. Surg. 40, 549 (1967). 5. H .H . DAvis, M. SIMONS and J. B. DAvis, Cystic disease of the breast: relation-

ship to carcinoma. Cancer (Philad.) 17, 957 (1964). 6. C .M. K~RP~, H. P. LEIS, A. OPPEmtZna and W. C. MERSHEI~mR, Relation-

ship offibrocystic disease to carcinoma of the breast. Ann. Surg. 162, 1 (1965). 7. M . M . BLACK, T. H. C. BARCLAY, S. S. CUTLER, B. F. HANKEY and A. J.

AsmE, Association of atypical characteristics of benign breast lesions with subsequent risk of breast cancer. Cancer (Philad.) 29, 338 (1972).

8. N . L . R . BUCHER and C. F. GESCHICgTER, Corpus luteum studies II: preg- nandiol and oestrogen output in the urine of patients with chronic cystic mastiffs, o r. din. Endocr. Metab. 1~ 58 (1941).

9. H. C. TAYLOR, The endocrine aspects of chronic mastiffs. Surg. Gyncc. Obstet. 74, 326 (1942).

10. I . T . NATHANSON, The relationship of hormones to diseases of the breast. Surgery 16~ 108 (1944).

11. J. M,tgMORSTON, L. G. CROWLEY, S. M. MYERS, E. STERN and C. E. HOPKINS, Urinary excretion of neutral 17-ketosteroids and pregnandiol by patients with breast cancer and benign breast disease. Am. J. Obstet. Gynec. 9"2, 447 (1965).

12. J. MARMORSTON, L. G. CROWLEY, S. M. MYERS, E. STERN and C. E. HOPKINS, Urinary excretion of estrone, estradiol and estriol by patients with breast cancer and benign breast disease. Am. or. Obstet. Gynec, 9'2, 460 (1965).

13. C.S. CORKER and D. EXLEY, The determination of plasma estradiol-17fl by competitive protein binding radioassay. Steroids, 15, 469 (1970).

14. M.C. SWAm, The assay of plasma progesterone in women by a simple com- petitive protein binding method using guinea-pig serum. Clin. chim. Aeta 39, 455 (1972).

15. D.Y. WANG, M. C. SWAIN, J. L. HAYWARD and R. D. BULBROOK, in Current Problems in the Epidemiology of Cancer and Lymphomas (Edited by E. GRUNDtaANN and H. TULINmS) p. 177. Springer-Verlag, Berlin, (1972).

16. B. McMAHoN, P. COLE and J. BROWN, Etiology of human breast cancer: a review, or. nat. Cancerlnst. 50, 21 (1973).

17. M.J . BRENNAN, R. D. BULBROOK, N. DESHPANDE, J. L. HAYWARD and D. Y. WANO, Urinary and plasma androgens in patients with benign breast disease. Possible relation to breast cancer. Lancet (In press).