Europ. d. Cancer Vol. 13, pp. 677-684. Pergamon Press 1977. Printed in Great Britain
Serum Prolactin Concentrations Throughout the Menstrual Cycle of Normal Women and Patients with Recent Breast Cancer* E. N. COLE, + P. C. ENGLAND,~" R. A. SELLWOOD + and K. GRIFFITHS~f "~Tenovns Institute for Cancer Research, Heath, Cardiff, CF4 4XX. $Department of Surgery, University Hospital of South Manchester, Withington Hospital, Manchester, M20 8LR, Great Britain Abstraet--Prolactin concentration has been estimated by radioimmunoassay of serum samples taken daily throughout the menstrual cycle of 11 patients who had undergone mastectomy for primary breast cancer and 32 normal women. Although there were no marked cyclical changes in prolactin level, concentrations were lowest in the follicular phase. Hence, comparison of normal and cancer subjects required detailed statistical analysis of results from comparable stages of the monthly cycle. Mid-cycle peaks of oestradiol and follicle stimulating hormone and the onset of menstrual bleeding were used as reference points. Prolactin concentrations were very similar in samples from normal and cancer groups, although at certain stages of the cycle some significant differences werefound: on the fifth day preceding the mid-cycle oestradiol peak; during the follicular and periovulatory phases; and among the highest mid-cycle levels. At these stages of the cycle, samples from the breast cancerpatients had a greater prolactin concentration than normal controls, although levels were within the normal range. The physiological relevance of these higher prolactin concentrations is uncertain and, in general, this detailed study clearly indicates little difference between serum levels of prolactin in normal women and in patients with breast cancer whose primary tumour had been removed :more than three months previously.
cycles of differing lengths are usually aligned for periovulatory events, prolactin concentrations at the time of menstruation have been given much less attention [9]. The effects of prolactin on experimental m a m m a r y tumours are well documented, and their relevance to h u m a n breast cancer has been reviewed recently [10]. Whereas basal plasma prolactin concentrations were correlated with differing susceptibility to chemical induction of m a m m a r y tumours in 3 strains of female rats [11], a finding which also appears true for male rats [12], our studies of h u m a n breast cancer patients have shown similar basal prolactin levels at various stages of the disease and in control subjects [13, 14]. These and other studies [15-17] indicate that if there are abnormal prolactin concentrations in breast cancer patients then the differences will be small. However, elevated prolactin concentrations in breast cancer patients have been found
INTRODUCTION ALTHOUOH the mammotrophic-lactogenic and luteolytic-luteotrophic effects of prolactin are well established in experimental animals [1, 2], the physiological role of prolactin during the h u m a n menstrual cycle is not so well understood. Apart from generally higher plasma levels in the luteal phase, and a possible increase near raid cycle, no consistent patterns of prolacfin concentration have been found from daily blood sampling [3-8]. Indeed, such studies have emphasized the marked variations in prolactin concentration which occur not only between individuals but also from day to day within subjects. Furthermore, because Accepted 22 November 1976. *We thank the Tenovus Organization, the Medical Research Council[and the Cancer Research Campaign for generous financial support. 677
678
E. N. Cole, P. C. England, R. A. Sellwood and K. Gri~ths
using a heterologous radioimmunoassay [18] and also using a bioassay [19]. In this detailed study, basal prolactin concentrations throughout the menstrual cycle have been compared in normal women and in breast cancer patients, to examine the possibility that there may be differences at some stages of the cycle only. As it would be unethical to delay mastectomy for more than a month while collecting the daily blood samples, patients were studied at least three months after removal of the primary tumour, when they had resumed regular cycles. Such patients may still be considered to have breast cancer, though occult, since breast carcinoma is regarded by many as a systemic disease [20, 21]. MATERIAL AND METHODS
Subjects and samples Samples of peripheral venous blood (10 ml) were obtained daily, or as often as possible, between 09.00 and 12.00 hr for at least one menstrual cycle. The blood was allowed to clot, centrifuged and serum removed to be stored at -20°C. The subjects were 32 normal women, and 11 women who had undergone mastectomy for primary carcinoma of the breast but had since resumed regular menstrual cycles. None of the subjects had a history of gynaecological disorders nor was taking drugs, hormone preparations or other agents known to affect prolactin, ovarian steroid or gonadotrophin levels. Of the normal women, 11 were selected to be matched controls for the group of cancer patients on the basis of cycle length, age and completeness of sampling. The normal women, including matched controls, were placed into 3 subgroups by age for data analysis.
Measurement of prolactin, follicle stimulating hormone (FSH), oestradiol-17fl and progesterone Prolactin was measured by the radioimmunoassay established in these laboratories by Cole and Boyns [22]. Results are given as milliunits/ ml M.R.C. Res. Std. A 71/222 where 1 mu = 50 ng prolactin by this assay system. Serum samples were assayed in duplicate, all those for an individual subject being together in one of the 8 assays that were required. Each cancer patient was paired with her matched control and their samples also included within the same assays. Displacement of 5% of bound iodinated prolactin was achieved by 0-04 mu/ ml prolactin.
Measurements of FSH were limited to women over 40 years old. The radioimmunoassay of Groom et al. [23] was used and the results served to check whether subjects had normal cycles with a marked mid-cycle peak. Oestradiol-17fl and progesterone were determined by radioimmunoassays and the results for these subjects have been reported [24, 25].
Analysis of results Prolactin concentrations were calculated from radioimmunoassay standard curves by the preferred equation of Taljedal and Wold [26]. Values of less than 0"01 were entered into subsequent calculations as 0.01 mu/ml. Menstrual cycles varied greatly in length and so two preference points were used: the day of the mid-cycle peak of oestradiol-17p was designated Day 0, taking gonadotrophin and progesterone profiles into consideration; the day of appearance of menstrual bleeding was labelled M. Prolactin concentrations were first examined to assess whether they met the assumptions inherent in parametric tests of statistical significance [27]. A logarithmic transformation was found necessary. Back-transformation yielded geometric means, but the logarithmic values were tested for significant differences. As Student's t-test should be reserved for planned comparisons [27], for which the experimental design allowed relatively few, the Student-Newman-Keuls (SNK) procedure was applied to transformed daily prolactin values from the cancer, matched control, and normal 40s groups. The range of values in the cancer group was not wide enough to hold significant differences, but prolactin levels on days - 5 and + 1 in the other two groups lay beyond the maximum non-significant ranges at the 5% level. Planned comparisons were therefore made in the remaining groups using data from days - 5 and + 1. The experimental design could support the use of Student's t-test for the following planned comparisons: (a) test for homogeneity of normal subgroups and whether the cancer group is different. (b) test highest periovulatory prolactin levels in cancer vs matched control groups. Additionally, planned comparisons were made using samples taken during defined 5-day intervals which were evenly spaced over 4 consecutive weeks:days - 1 , 0, +1, 2, 3 (periovulatory phase) ; days - 8 to - 4 (fol-
Serum Prolactin Concentrations Throughout the Menstrual Cycle licular phase); days +6 to + 10 (Iuteal phase), and the period 2 days before and after Day M. By pooling the data in this way, the resulting prolactin levels should be less sensitive to misalignment of cycles. Values for the follicular phase were then compared with the periovulatory phase, and also the 5 days around menstruation, within all groups; the follicular phase prolactin :in the cancer group was compared with matched controls and combined normals. RESULTS Mid-cycle peaks of oestradiol-17/~ or FSH could be located in 25 normal cycles and in all the 11 cycles fi:om the patients with breast cancer. Two of the normal subjects (aged 53 and 46) were menopausal, having generally elevated FSH levels; critical samples were unavailable for a further 6 subjects and these were therefore omitted from detailed data analysis. One other cycle was rejected because
679
of low FSH levels in an abnormally long luteal phase. The remaining 24 normal cycles were placed in 3 age groups (Table 1). Distribution of ages within each group was essentially random, and cycle lengths were variable with no age-related trend. Table 1 also shows median value with range of both age and cycle length for the breast cancer group, matched control group and the combined normal group. The 30-yearold in the matched control group was chosen for her 22 day cycle to pair with the 23 day cycle of a 45-year-old cancer patient. Prolactin concentrations did not follow a Gaussian distribution but approximated to a log-normal distribution. This is evident from Table 2 where for each group the median value from raw data is compared with the calculated arithmetic and geometric means. In a normal distribution, median and mean values coincide: in Table 2, the geometric mean is the closer to the median. Furthermore,
Table 1. Allocation of subjects to groups, their age and the duration of their menstrual cycle Age (years) Median Range
Group
Cycle length (days) Median Range
Number of cycles
Normal 20s Normal 30s Normal 40s
25 34 44
22-29 30-39 40--49
30 26 27
26-32 22-32 25-32
8 7 9
Breast cancer Matched control Combined normal
44 41 35
38-48 30--49 22-49
27 27 28
23-33 22-32 22-32
11 11 24
Menst~:ual cycles of 23 normal women have been grouped by age and additionally by matchi!ng to the 11 cancer patients on the basis of cycle length and age. Note that 2 cycles were s~:udied from one 22 year old subject.
Table2. C•mparis•n of serum pr•lactin c•ncentrati•ns between gr•ups •f subjects using statistics of l•cati•n and dispersi•n that are derivedfrom raw data (median with 2.5-97,5 percentiles) and by arithmetic (mean, S.D. ) or logarithmic (geometric mean___1.96 S.D. range) computation Number of samples
Median value
2.5-97.5 percentiles
Normal 20s Normal 30s Normal 40s
187 147 226
0.10 0"09 0-10
0.01-0.45 0.01-0.33 0"03-0.28
0.15 0.12 0-12
Breast cancer Matched control Combined normal
292 262 550
0.11 0.10 0.10
0.02-0-44 0"02-0.29 0.01-0.38
0.15 0.12 0.13
Group
Arithmetic Mean S.D.
Geometric Mean
_+ 1.96 S.D. range
0.133 0.090 0.101
0-085 0.083 0.101
0.01-0.81 0-01-0.53 0.03-0.31
0.112 0.069 0.Ill
0.114 0'097 0.091
0-02-0"53 0.03-0.35 0.01-0.53
Serum prolactin concentrations in mu/ml M R C Res. Std. A 71/222 are given for menstrual cycles from subjects grouped as in Table 1. The median and 2.5-97-5 percentiles were found by inspection; arithmetic mean and standard deviation (S.D.) were calculated in the usual way; geometric mean + 1.96 S.D. range are back transformations of the logarithmic mean + 1.96 S.D., where 1.96 = t (approximately) at the 5% probability level for the appropriate degrees of freedom.
680
E. N. Cole, P. C. England, R. A. Sellwood and I£. Griffths
95% of raw data lie within the 2"5-97.5 percentiles by definition, and w i t h i n _ 1.96 S.D. of the mean of a normal distribution. These limits can be compared in Table 2 for each group of women, and it is obvious that a logarithmic transformation ofprolactin values is reasonable. Prolactin concentrations were similar in all groups of normal subjects and in the group of cancer patients (Table 2). The range of geometric mean prolactin for those individuals who were rejected from the normal group was 0.02-0.19, and this is similar to the ranges for normal subjects in their 20s, 30s and 40s or the range for breast cancer patients: 0-02-0.25; 0"02-0.21; 0"06-0.14; and 0.05-0.23 mu/ml respectively. There was no evidence for any trend in serum prolaetin concentration with 0.4
~
E
0.3
c
o.2, o .~_ 0.1-
0
I0
2o--....k__ Age,
Fig. 1. Relationship between serum prolactin (geometric mean of daily samples over the menstrual cycle) and age. The ellipse was calculated to include 95% of data points from normal women. • Normal group; [] normal subjects whose prolactin concentrations were rejected from detailed analysis; • breast cancer patients. 0.5 ¸
~'-
Normal subjects
age (Fig. 1), and it can be seen that the geometric means for the cancer patients are within the range for the normal women. Daily prolactin concentrations for the follicular, periovulatory and luteal phases and the period of menstruation showed fluctuations of the geometric mean which were small compared with the range of the values for each day. Figure 2 illustrates this for the combined normal group. Nevertheless, prolactin levels were generally higher in the luteal phase than in the follicular phase, and an elevation at midcycle was present (2P < 0.025 for day + 1 vs - 5 relative to the oestradiol-17/~ peak). A decline in serum prolactin occurred during the 2 days before onset of menstrual bleeding in 21 women out of 27 who were sampled on these days (significant at 1% level by a sign test). When daily prolactin concentrations in breast cancer patients and matched controls were compared, a significant difference was found on day - 5 in the follicular phase (2P < 0.01) but not for day + 1. A mid-cycle elevation of serum prolactin in the cancer group could not be shown (2P > 0.10) whereas the significance level was 0"001 for day - 5 vs + 1 in the matched control group (Fig. 3). However, by using a paired comparison of the mean values shown in Fig. 3, Wilcoxon's matched-pairs signed-rank test indicated an overall difference between the 2 groups at the 2% probability level. This statistical method takes no account of the considerable range of prolactin concentrations associated with each mean (Fig. 2). As the differences in mean prolactin levels within 5-day intervals were generally non-
20-50
0.4,
¢; "*-
0.3'
o
0.2.
0.1-
ODays
from onset of menstruotion(M) or o e s t r d d i o l - 1 7 ~ peak(O)
Serum prolactin in menstrual cycles of normal women. Hatched area encloses the range of concentrations for each day, and the line denotes the geometric mean for 24 cycles.
Serum Prolactin Concentrations Throughout the Menstrual Cycle • 0.2,
68l
Cancer
o Controls
•
Q.
0.1'
if)
o,
-~
~
Days from
~'-~-~ onset
.-~-~ of
6
menstrual'ion
k
~
(M)or
g
~ ,'o'~'-~ , oestrodiol-17/~
~
t~
peak (0)
Fig. 3. Comparison of daily prolactin concentrations in breast cancer patients and matched controls. Values are geometric means for 11 cancer patients (0) and 11 controls (©).
0,2-
Norma I 30s Norma~ 20s
Norrna I 40s
1i
" 0''" 1t
.c_ o.
_E o.2-
Breast
Matched
Combined
o~
cancer
controls
normals
0''
o
M
F 0
L
Weeks
M
iI
0
of menstrual
M
F 0
L
week by week (Fig. 4). In each group, serum prolactin was lowest in the follicular phase and was significantly higher in the periovulatory phase (2P < 0.1, 20s; < 0.05, 30s; < 0.05, 40s; < 0-01, combined normal age groups; < 0.01, cancer group; < 0.01, matched controis). In all the groups of normal women, prolaetin was higher in the week of menstruation than in the following week, significant at 2P < 0.01 for the combined group. The weekly pattern of prolactin in serum from the cancer patients differs from normal (Fig. 4). Prolactin remained at the level of the menstrual phase during the follicular phase and was higher for the next 2 weeks. Consequently, there was a difference (2P < 0.05) in the follicular phase serum prolactin concentration between 55 samples from 11 breast cancer patients and either 100 samples from 23 normal women or 49 from the matched controls. Similarly, these cancer patients have higher prolactin concentrations than normal in the periovulatory phase. In addition, by taking the highest values in the periovulatory phase, the geometric mean prolactin for the cancer patients (0.29) is greater than for the matched controls (0.20; 2P < 0.025).
cycle
Fig. 4. Weekly patterns of serum prolactin in normal subjects, grouped according to age or selected as matched controls, and in breast cancer patients. Geometric mean with 95% confidence limits are shown for prolactin concentrations over 5-day intervals in the weeks of menstruation (M) and ovulation ( 0 ) or within the follicular (F) and luteal (L) phases.
significant, pooled values were calculated to represent the pattern ofprolactin concentration
DISCUSSION
Using samples taken throughout the menstrual cycle, only small differences could be found in serum prolactin concentrations between normal women and a group of patients that had undergone mastectomy for primary breast cancer. Indeed, to show any significant differences in prolaetin levels, both the design
682
E. N. Cole, P. C. England, R. A. Sellwood and If. Griffiths
of the radioimmunoassays and the statistical treatment of results required very careful consideration. The physiological relevance of the differences is obviously uncertain. The a priori selection of control subjects was intended to minimize possible sources of error due to interassay variation, age-related trends and bias arising from, for example, a prolonged follicular phase which would tend to lower an individual's mean prolactin level. In practice, however, the major variance components arose from within and between subjects. Hence, geometric mean prolactin values over the four 5-day intervals were within 2% of those given in Table 2 for which sets of results over the whole cycle were taken into consideration. Several statistical treatments of the data were possible. Thus, prolactin concentrations could be normalized to reduce inter-subject variation and then tested for consistent cyclical changes. Alternatively, areas under the prolactin-time curve could be taken for the comparison of cancer and normal groups. Instead, the familiar Student's t-test has been applied to data which have undergone transformation in order that the assumptions of parametric significance testing were better met. Prolactin concentrations approximated quite closely to a log-normal distribution, and a slight skewness may be accounted for by less than 0.01 mu/ml prolactin in the "prolactinfree" plasma that was used in constructing the radioimmunoassay standard curve. This skewness is a likely cause of the over-estimation of standard deviation terms which is indicated by the ___1.96 S.D. limits being wider than the 2" 5-97. 5 percentiles (Table 2). The significance levels for differences between means are likely to be conservative estimates in consequence. Daily serum prolactin concentrations were variable and there were no consistent patterns from one person to another during the menstrual cycle. This is in agreement with similar studies from other centres [3-7]. However, by pooling the results from the defined 5-day periods, a clear pattern of serum prolactin emerged. This pattern throughout the menstrual cycle was consistent insofar as follicular phase prolactin was lower than in the periovulatory or menstrual phases for the combined group of 24 normal cycles and in the subgroups of 8, 7 or 9 cycles by age, or the matched control cycles. However, the pattern for the breast cancer group differed because prolactin in the follicular phase remained at the level of the preceding
week before increasing in the periovulatory and luteal phases (Fig. 4). Comparison of patterns of serum prolactin week-by-week has advantages over the simple comparison of mean concentrations from one group of subjects with another. In addition to smoothing out daily fluctuations, assay and alignment errors should be minimal, and the errors due to the selection of patients and matched controls reduced. Thus, neither worry related to the mastectomy nor residual postoperative stress are likely to affect the pattern of prolactin levels over the month, whereas it could be argued that a single sample might well be affected. The breast cancer patients for this study had undergone mastectomy for primary breast cancer and had since resumed normal cyclical activity. It is, of course, virtually impossible to obtain daily blood samples for a month from a woman with known or suspected breast cancer; it would be unethical to withhold treatment for this time, and hormone concentrations would be of doubtful value in a woman giving informed consent under such distressing circumstances. The abnormal prolactin pattern in our patients may indicate some relationship between prolactin and carcinogenesis. The occurrence of significantly greater prolactin concentrations in the cancer patients during the periovulatory phase and the possible prolonged elevation at this stage (Fig. 3) certainly provide further evidence that the endocrinology of these women still differs if only minimally from the 23 normal subjects. Just as the disease persists in occult metastatic foci even after masteetomy [20, 21], so there m a y remain an abnormal prolactin balance in the breast cancer patients. However loss of breast tissue may itself affect prolactin secretion or utilization, although such a feedback mechanism from prolactin target tissue remains to be clarified. Mastectomy could result in neural stimulation of prolactin release, which might explain the generally elevated levels, but is unlikely to account for the observed difference in the weekly pattern. Although differences in serum prolactin concentration were found, their interpretation requires care as it is probably unwise to generalize from a sample of only 11 premenopausal breast cancer patients. The differences between cancer and control levels are very small and their relationship to the disease is still uncertain.
Serum Prolactin Concentrations Throughout the Menstrual Cycle REFERENCES
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5. 6. 7. 8. 9. 10. 11.
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19. 20. 21. 22. 23.
C.S. NICOLLand H. A. BERN,On the actions of prolactin among the vertebrates: is there a common denominator? In Lactogenic Hormones. (Edited by G. E. W. WOLSTEmtOLMEand J. KNIOHT) p. 299, Churchill-Livingstone, London (1972). J. MEITES, K. H. Lu, W. WUTTKE, C. W. WELSCH,H. NAOASAWAand S. K. QUADRI, Recent studies on functions and control of prolactin secretion in rats. Recent Prog. hormone Res. 28~ 471 (1972). H. FRIESEN, P. HWANO, H. GUYDA, G. TOLLS,J. TYsoN and R. MYERS, A radioimmunoassay for human prolactin. In Prolactin and Carcinogenesis. (Edited by A. R. BOYNS and K. GRIrrITHS) p. 64, Alpha Omega Alpha, Cardiff (1972). M. L'HERMITE,P. DELVOYE,J. NOKIN, M. VEKEMANSand C. ROBYN, Human prolactin secretion, as studied by radioimmunoassay: some aspects of its regulation. In Prolactin and Carcinogenesis. (Edited by A. R. Bo,zNs and K. GRIFFITHS) p. 64, Alpha Omega Alpha, Cardiff (1972). Y. EHARA, T. SILER, G. VANDENBERo, Y. N. SINHA and S. S. C. YEN, Circulating prolactin levels during the menstrual cycle: episodic release and diurnal variation. Amer. J. Obstet. Gynec. 117~ 962 (1973). A.S. McNEILLYand C. HAGEN, Prolactin, TSH, LH and FSH responses to a combined L H R H / T R H test at different stages of the menstrual cycle. Clin. Endocr. 3~ 435 (1974). M. SCHMIDT-GOLLWlTZERand B. B. SAXENA, Radioimmunoassay of human prolactin (PRL). Acta Endocr. 80, 262 (1975). N.A. SHETH,K. J. RANADIVE,J. N. SURAIYAand A. R. SHETH,Circulating levels of prolactin in human breast cancer. Brit. J. Cancer 32~ 160 (1975). A . S . McNEILLY and T. CHARD, Circulating levels of prolactin during the menstrual cycle. Clin. Endocr. 3~ 105 (1974). F. SMITHLINE,L. SHERMANand H. D. KOLODNY,Prolactin and breast carcinoma. New Engl. J. Med. 292~ 784, (1975). A . R . BOYNS,R. BUCHAN,E. N. COLE,A. P. 1VL FORRESTand K. GRIFFITHS, Basal prolactin blood levels in three strains of rat with differing incidence of 7,12-dimethylbenz(a)anthracene induced mammary tumours. Europ. or. Cancer 9, 169 (1973). C.W. WELSCH,G. LOUKS,D. FOX and C. BROOKS,Enhancement by prolactin of carcinogen induced mammary cancerigenesis in the male rat. Brit. J. Cancer 32, 427 (1975). A . R . BOYNS,E. N. COLE, K. GRIFFITHS,M. M. ROBERTS,R. BUCHAN,R. G. WILSON and A. P. M. FORP.~ST, Plasma prolactin in breast cancer. Europ. J. Cancer, 9~ 99 (1973). R . G . WILSON,R. BUCHAN,M. M. ROBERTS,A. P. M. FORm~ST,A. R. BOYNS, E. N. COLE and K. GRII~FITHS. Plasma prolactin and breast cancer. Cancer (Philad.) 33, 1325 (1974). I. MITTm% J. L. HAYWA~D and A. S. McNEILLY, Hypothalamic-pituitaryprolactin axis in breast cancer. Lancet i, 889 (1974). H . G . KWA, M. DEJONG-BAKKER,E. ENOLESMANand F. J. CLETON, Plasma prolactin in human breast cancer. Lancet i~ 433 (1974). A. GORINSand A. NETTER,La prolactine. Nouv. Presse med. 3~ 73 (1974). R . M . L . MURRAY, G. MOZAFFARIANand O. H. PEARSON, Prolactin levels with L-DOPA treatment in metastatic breast carcinoma. In Prolactin and Carcinogenesis. (Edited by A. R. BOYNSand K. GRIFrlTHS) p. 158, Alpha Omega Alpha, Cardiff (1972). P. BERLEand K. D. VOIGT, Evidence of prolactin levels in patients with breast cancer. Amer. J. Obstet. Gynec. 114~ 1101 (1972). W . H . BOND. In The Treatment of Carcinoma of the Breast. (Edited by A. S. JARRETT) p. 1, Excerpta Medica, Amsterdam (1968). M. BAUM,The curability of breast cancer. Brit. med. or. i~ 439 (1976). E . N . COLE and A. R. BOYNS, Radioimmunoassay for human pituitary prolactin, using antiserum against an extract of human amniotic fluid. Hormone Res. 4~ 261 (1973). G.V. GROOM,M. A. GROOM, I. D. COOKEand A. R. BOYNS,The secretion of immuno-reactive luteinizing hormone and follicle stimulating hormone by the human foetal pituitary in organ culture. J. Endocr. 49, 335 (1971).
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25. 26. 27.
P.C. ENOLAND,L. G. SKINNER,K. M. COTTRILLand R. A. SELLWOOD,Serum oestradiol-17~ in women with benign and malignant breast disease. Brit. J. Cancer ~ , 571 (1974). P.C. ENGLAND,L. G. SKINNER,K. M. COTTRILLand R. A. SELLWOOD,Sex hormones in breast disease. Brit. J. Surg. 62, 806 (1975). I.-B.TALJEDALand S. WOLD,Fit of some analyticalfunctionsto insulin radioimmunoassay standard curves. Bfochem. J. 119, 134 (1970). i~. R. SOKALand F. J. ROHLF.B{omet~, W. H. Freeman, San Francisco (1969).
Serum Prolactin Concentrations Throughout the Menstrual Cycle of Normal Women and Patients with Recent Breast Cancer* E. N. COLE, + P. C. ENGLAND,~" R. A. SELLWOOD + and K. GRIFFITHS~f "~Tenovns Institute for Cancer Research, Heath, Cardiff, CF4 4XX. $Department of Surgery, University Hospital of South Manchester, Withington Hospital, Manchester, M20 8LR, Great Britain Abstraet--Prolactin concentration has been estimated by radioimmunoassay of serum samples taken daily throughout the menstrual cycle of 11 patients who had undergone mastectomy for primary breast cancer and 32 normal women. Although there were no marked cyclical changes in prolactin level, concentrations were lowest in the follicular phase. Hence, comparison of normal and cancer subjects required detailed statistical analysis of results from comparable stages of the monthly cycle. Mid-cycle peaks of oestradiol and follicle stimulating hormone and the onset of menstrual bleeding were used as reference points. Prolactin concentrations were very similar in samples from normal and cancer groups, although at certain stages of the cycle some significant differences werefound: on the fifth day preceding the mid-cycle oestradiol peak; during the follicular and periovulatory phases; and among the highest mid-cycle levels. At these stages of the cycle, samples from the breast cancerpatients had a greater prolactin concentration than normal controls, although levels were within the normal range. The physiological relevance of these higher prolactin concentrations is uncertain and, in general, this detailed study clearly indicates little difference between serum levels of prolactin in normal women and in patients with breast cancer whose primary tumour had been removed :more than three months previously.
cycles of differing lengths are usually aligned for periovulatory events, prolactin concentrations at the time of menstruation have been given much less attention [9]. The effects of prolactin on experimental m a m m a r y tumours are well documented, and their relevance to h u m a n breast cancer has been reviewed recently [10]. Whereas basal plasma prolactin concentrations were correlated with differing susceptibility to chemical induction of m a m m a r y tumours in 3 strains of female rats [11], a finding which also appears true for male rats [12], our studies of h u m a n breast cancer patients have shown similar basal prolactin levels at various stages of the disease and in control subjects [13, 14]. These and other studies [15-17] indicate that if there are abnormal prolactin concentrations in breast cancer patients then the differences will be small. However, elevated prolactin concentrations in breast cancer patients have been found
INTRODUCTION ALTHOUOH the mammotrophic-lactogenic and luteolytic-luteotrophic effects of prolactin are well established in experimental animals [1, 2], the physiological role of prolactin during the h u m a n menstrual cycle is not so well understood. Apart from generally higher plasma levels in the luteal phase, and a possible increase near raid cycle, no consistent patterns of prolacfin concentration have been found from daily blood sampling [3-8]. Indeed, such studies have emphasized the marked variations in prolactin concentration which occur not only between individuals but also from day to day within subjects. Furthermore, because Accepted 22 November 1976. *We thank the Tenovus Organization, the Medical Research Council[and the Cancer Research Campaign for generous financial support. 677
678
E. N. Cole, P. C. England, R. A. Sellwood and K. Gri~ths
using a heterologous radioimmunoassay [18] and also using a bioassay [19]. In this detailed study, basal prolactin concentrations throughout the menstrual cycle have been compared in normal women and in breast cancer patients, to examine the possibility that there may be differences at some stages of the cycle only. As it would be unethical to delay mastectomy for more than a month while collecting the daily blood samples, patients were studied at least three months after removal of the primary tumour, when they had resumed regular cycles. Such patients may still be considered to have breast cancer, though occult, since breast carcinoma is regarded by many as a systemic disease [20, 21]. MATERIAL AND METHODS
Subjects and samples Samples of peripheral venous blood (10 ml) were obtained daily, or as often as possible, between 09.00 and 12.00 hr for at least one menstrual cycle. The blood was allowed to clot, centrifuged and serum removed to be stored at -20°C. The subjects were 32 normal women, and 11 women who had undergone mastectomy for primary carcinoma of the breast but had since resumed regular menstrual cycles. None of the subjects had a history of gynaecological disorders nor was taking drugs, hormone preparations or other agents known to affect prolactin, ovarian steroid or gonadotrophin levels. Of the normal women, 11 were selected to be matched controls for the group of cancer patients on the basis of cycle length, age and completeness of sampling. The normal women, including matched controls, were placed into 3 subgroups by age for data analysis.
Measurement of prolactin, follicle stimulating hormone (FSH), oestradiol-17fl and progesterone Prolactin was measured by the radioimmunoassay established in these laboratories by Cole and Boyns [22]. Results are given as milliunits/ ml M.R.C. Res. Std. A 71/222 where 1 mu = 50 ng prolactin by this assay system. Serum samples were assayed in duplicate, all those for an individual subject being together in one of the 8 assays that were required. Each cancer patient was paired with her matched control and their samples also included within the same assays. Displacement of 5% of bound iodinated prolactin was achieved by 0-04 mu/ ml prolactin.
Measurements of FSH were limited to women over 40 years old. The radioimmunoassay of Groom et al. [23] was used and the results served to check whether subjects had normal cycles with a marked mid-cycle peak. Oestradiol-17fl and progesterone were determined by radioimmunoassays and the results for these subjects have been reported [24, 25].
Analysis of results Prolactin concentrations were calculated from radioimmunoassay standard curves by the preferred equation of Taljedal and Wold [26]. Values of less than 0"01 were entered into subsequent calculations as 0.01 mu/ml. Menstrual cycles varied greatly in length and so two preference points were used: the day of the mid-cycle peak of oestradiol-17p was designated Day 0, taking gonadotrophin and progesterone profiles into consideration; the day of appearance of menstrual bleeding was labelled M. Prolactin concentrations were first examined to assess whether they met the assumptions inherent in parametric tests of statistical significance [27]. A logarithmic transformation was found necessary. Back-transformation yielded geometric means, but the logarithmic values were tested for significant differences. As Student's t-test should be reserved for planned comparisons [27], for which the experimental design allowed relatively few, the Student-Newman-Keuls (SNK) procedure was applied to transformed daily prolactin values from the cancer, matched control, and normal 40s groups. The range of values in the cancer group was not wide enough to hold significant differences, but prolactin levels on days - 5 and + 1 in the other two groups lay beyond the maximum non-significant ranges at the 5% level. Planned comparisons were therefore made in the remaining groups using data from days - 5 and + 1. The experimental design could support the use of Student's t-test for the following planned comparisons: (a) test for homogeneity of normal subgroups and whether the cancer group is different. (b) test highest periovulatory prolactin levels in cancer vs matched control groups. Additionally, planned comparisons were made using samples taken during defined 5-day intervals which were evenly spaced over 4 consecutive weeks:days - 1 , 0, +1, 2, 3 (periovulatory phase) ; days - 8 to - 4 (fol-
Serum Prolactin Concentrations Throughout the Menstrual Cycle licular phase); days +6 to + 10 (Iuteal phase), and the period 2 days before and after Day M. By pooling the data in this way, the resulting prolactin levels should be less sensitive to misalignment of cycles. Values for the follicular phase were then compared with the periovulatory phase, and also the 5 days around menstruation, within all groups; the follicular phase prolactin :in the cancer group was compared with matched controls and combined normals. RESULTS Mid-cycle peaks of oestradiol-17/~ or FSH could be located in 25 normal cycles and in all the 11 cycles fi:om the patients with breast cancer. Two of the normal subjects (aged 53 and 46) were menopausal, having generally elevated FSH levels; critical samples were unavailable for a further 6 subjects and these were therefore omitted from detailed data analysis. One other cycle was rejected because
679
of low FSH levels in an abnormally long luteal phase. The remaining 24 normal cycles were placed in 3 age groups (Table 1). Distribution of ages within each group was essentially random, and cycle lengths were variable with no age-related trend. Table 1 also shows median value with range of both age and cycle length for the breast cancer group, matched control group and the combined normal group. The 30-yearold in the matched control group was chosen for her 22 day cycle to pair with the 23 day cycle of a 45-year-old cancer patient. Prolactin concentrations did not follow a Gaussian distribution but approximated to a log-normal distribution. This is evident from Table 2 where for each group the median value from raw data is compared with the calculated arithmetic and geometric means. In a normal distribution, median and mean values coincide: in Table 2, the geometric mean is the closer to the median. Furthermore,
Table 1. Allocation of subjects to groups, their age and the duration of their menstrual cycle Age (years) Median Range
Group
Cycle length (days) Median Range
Number of cycles
Normal 20s Normal 30s Normal 40s
25 34 44
22-29 30-39 40--49
30 26 27
26-32 22-32 25-32
8 7 9
Breast cancer Matched control Combined normal
44 41 35
38-48 30--49 22-49
27 27 28
23-33 22-32 22-32
11 11 24
Menst~:ual cycles of 23 normal women have been grouped by age and additionally by matchi!ng to the 11 cancer patients on the basis of cycle length and age. Note that 2 cycles were s~:udied from one 22 year old subject.
Table2. C•mparis•n of serum pr•lactin c•ncentrati•ns between gr•ups •f subjects using statistics of l•cati•n and dispersi•n that are derivedfrom raw data (median with 2.5-97,5 percentiles) and by arithmetic (mean, S.D. ) or logarithmic (geometric mean___1.96 S.D. range) computation Number of samples
Median value
2.5-97.5 percentiles
Normal 20s Normal 30s Normal 40s
187 147 226
0.10 0"09 0-10
0.01-0.45 0.01-0.33 0"03-0.28
0.15 0.12 0-12
Breast cancer Matched control Combined normal
292 262 550
0.11 0.10 0.10
0.02-0-44 0"02-0.29 0.01-0.38
0.15 0.12 0.13
Group
Arithmetic Mean S.D.
Geometric Mean
_+ 1.96 S.D. range
0.133 0.090 0.101
0-085 0.083 0.101
0.01-0.81 0-01-0.53 0.03-0.31
0.112 0.069 0.Ill
0.114 0'097 0.091
0-02-0"53 0.03-0.35 0.01-0.53
Serum prolactin concentrations in mu/ml M R C Res. Std. A 71/222 are given for menstrual cycles from subjects grouped as in Table 1. The median and 2.5-97-5 percentiles were found by inspection; arithmetic mean and standard deviation (S.D.) were calculated in the usual way; geometric mean + 1.96 S.D. range are back transformations of the logarithmic mean + 1.96 S.D., where 1.96 = t (approximately) at the 5% probability level for the appropriate degrees of freedom.
680
E. N. Cole, P. C. England, R. A. Sellwood and I£. Griffths
95% of raw data lie within the 2"5-97.5 percentiles by definition, and w i t h i n _ 1.96 S.D. of the mean of a normal distribution. These limits can be compared in Table 2 for each group of women, and it is obvious that a logarithmic transformation ofprolactin values is reasonable. Prolactin concentrations were similar in all groups of normal subjects and in the group of cancer patients (Table 2). The range of geometric mean prolactin for those individuals who were rejected from the normal group was 0.02-0.19, and this is similar to the ranges for normal subjects in their 20s, 30s and 40s or the range for breast cancer patients: 0-02-0.25; 0"02-0.21; 0"06-0.14; and 0.05-0.23 mu/ml respectively. There was no evidence for any trend in serum prolaetin concentration with 0.4
~
E
0.3
c
o.2, o .~_ 0.1-
0
I0
2o--....k__ Age,
Fig. 1. Relationship between serum prolactin (geometric mean of daily samples over the menstrual cycle) and age. The ellipse was calculated to include 95% of data points from normal women. • Normal group; [] normal subjects whose prolactin concentrations were rejected from detailed analysis; • breast cancer patients. 0.5 ¸
~'-
Normal subjects
age (Fig. 1), and it can be seen that the geometric means for the cancer patients are within the range for the normal women. Daily prolactin concentrations for the follicular, periovulatory and luteal phases and the period of menstruation showed fluctuations of the geometric mean which were small compared with the range of the values for each day. Figure 2 illustrates this for the combined normal group. Nevertheless, prolactin levels were generally higher in the luteal phase than in the follicular phase, and an elevation at midcycle was present (2P < 0.025 for day + 1 vs - 5 relative to the oestradiol-17/~ peak). A decline in serum prolactin occurred during the 2 days before onset of menstrual bleeding in 21 women out of 27 who were sampled on these days (significant at 1% level by a sign test). When daily prolactin concentrations in breast cancer patients and matched controls were compared, a significant difference was found on day - 5 in the follicular phase (2P < 0.01) but not for day + 1. A mid-cycle elevation of serum prolactin in the cancer group could not be shown (2P > 0.10) whereas the significance level was 0"001 for day - 5 vs + 1 in the matched control group (Fig. 3). However, by using a paired comparison of the mean values shown in Fig. 3, Wilcoxon's matched-pairs signed-rank test indicated an overall difference between the 2 groups at the 2% probability level. This statistical method takes no account of the considerable range of prolactin concentrations associated with each mean (Fig. 2). As the differences in mean prolactin levels within 5-day intervals were generally non-
20-50
0.4,
¢; "*-
0.3'
o
0.2.
0.1-
ODays
from onset of menstruotion(M) or o e s t r d d i o l - 1 7 ~ peak(O)
Serum prolactin in menstrual cycles of normal women. Hatched area encloses the range of concentrations for each day, and the line denotes the geometric mean for 24 cycles.
Serum Prolactin Concentrations Throughout the Menstrual Cycle • 0.2,
68l
Cancer
o Controls
•
Q.
0.1'
if)
o,
-~
~
Days from
~'-~-~ onset
.-~-~ of
6
menstrual'ion
k
~
(M)or
g
~ ,'o'~'-~ , oestrodiol-17/~
~
t~
peak (0)
Fig. 3. Comparison of daily prolactin concentrations in breast cancer patients and matched controls. Values are geometric means for 11 cancer patients (0) and 11 controls (©).
0,2-
Norma I 30s Norma~ 20s
Norrna I 40s
1i
" 0''" 1t
.c_ o.
_E o.2-
Breast
Matched
Combined
o~
cancer
controls
normals
0''
o
M
F 0
L
Weeks
M
iI
0
of menstrual
M
F 0
L
week by week (Fig. 4). In each group, serum prolactin was lowest in the follicular phase and was significantly higher in the periovulatory phase (2P < 0.1, 20s; < 0.05, 30s; < 0.05, 40s; < 0-01, combined normal age groups; < 0.01, cancer group; < 0.01, matched controis). In all the groups of normal women, prolaetin was higher in the week of menstruation than in the following week, significant at 2P < 0.01 for the combined group. The weekly pattern of prolactin in serum from the cancer patients differs from normal (Fig. 4). Prolactin remained at the level of the menstrual phase during the follicular phase and was higher for the next 2 weeks. Consequently, there was a difference (2P < 0.05) in the follicular phase serum prolactin concentration between 55 samples from 11 breast cancer patients and either 100 samples from 23 normal women or 49 from the matched controls. Similarly, these cancer patients have higher prolactin concentrations than normal in the periovulatory phase. In addition, by taking the highest values in the periovulatory phase, the geometric mean prolactin for the cancer patients (0.29) is greater than for the matched controls (0.20; 2P < 0.025).
cycle
Fig. 4. Weekly patterns of serum prolactin in normal subjects, grouped according to age or selected as matched controls, and in breast cancer patients. Geometric mean with 95% confidence limits are shown for prolactin concentrations over 5-day intervals in the weeks of menstruation (M) and ovulation ( 0 ) or within the follicular (F) and luteal (L) phases.
significant, pooled values were calculated to represent the pattern ofprolactin concentration
DISCUSSION
Using samples taken throughout the menstrual cycle, only small differences could be found in serum prolactin concentrations between normal women and a group of patients that had undergone mastectomy for primary breast cancer. Indeed, to show any significant differences in prolaetin levels, both the design
682
E. N. Cole, P. C. England, R. A. Sellwood and If. Griffiths
of the radioimmunoassays and the statistical treatment of results required very careful consideration. The physiological relevance of the differences is obviously uncertain. The a priori selection of control subjects was intended to minimize possible sources of error due to interassay variation, age-related trends and bias arising from, for example, a prolonged follicular phase which would tend to lower an individual's mean prolactin level. In practice, however, the major variance components arose from within and between subjects. Hence, geometric mean prolactin values over the four 5-day intervals were within 2% of those given in Table 2 for which sets of results over the whole cycle were taken into consideration. Several statistical treatments of the data were possible. Thus, prolactin concentrations could be normalized to reduce inter-subject variation and then tested for consistent cyclical changes. Alternatively, areas under the prolactin-time curve could be taken for the comparison of cancer and normal groups. Instead, the familiar Student's t-test has been applied to data which have undergone transformation in order that the assumptions of parametric significance testing were better met. Prolactin concentrations approximated quite closely to a log-normal distribution, and a slight skewness may be accounted for by less than 0.01 mu/ml prolactin in the "prolactinfree" plasma that was used in constructing the radioimmunoassay standard curve. This skewness is a likely cause of the over-estimation of standard deviation terms which is indicated by the ___1.96 S.D. limits being wider than the 2" 5-97. 5 percentiles (Table 2). The significance levels for differences between means are likely to be conservative estimates in consequence. Daily serum prolactin concentrations were variable and there were no consistent patterns from one person to another during the menstrual cycle. This is in agreement with similar studies from other centres [3-7]. However, by pooling the results from the defined 5-day periods, a clear pattern of serum prolactin emerged. This pattern throughout the menstrual cycle was consistent insofar as follicular phase prolactin was lower than in the periovulatory or menstrual phases for the combined group of 24 normal cycles and in the subgroups of 8, 7 or 9 cycles by age, or the matched control cycles. However, the pattern for the breast cancer group differed because prolactin in the follicular phase remained at the level of the preceding
week before increasing in the periovulatory and luteal phases (Fig. 4). Comparison of patterns of serum prolactin week-by-week has advantages over the simple comparison of mean concentrations from one group of subjects with another. In addition to smoothing out daily fluctuations, assay and alignment errors should be minimal, and the errors due to the selection of patients and matched controls reduced. Thus, neither worry related to the mastectomy nor residual postoperative stress are likely to affect the pattern of prolactin levels over the month, whereas it could be argued that a single sample might well be affected. The breast cancer patients for this study had undergone mastectomy for primary breast cancer and had since resumed normal cyclical activity. It is, of course, virtually impossible to obtain daily blood samples for a month from a woman with known or suspected breast cancer; it would be unethical to withhold treatment for this time, and hormone concentrations would be of doubtful value in a woman giving informed consent under such distressing circumstances. The abnormal prolactin pattern in our patients may indicate some relationship between prolactin and carcinogenesis. The occurrence of significantly greater prolactin concentrations in the cancer patients during the periovulatory phase and the possible prolonged elevation at this stage (Fig. 3) certainly provide further evidence that the endocrinology of these women still differs if only minimally from the 23 normal subjects. Just as the disease persists in occult metastatic foci even after masteetomy [20, 21], so there m a y remain an abnormal prolactin balance in the breast cancer patients. However loss of breast tissue may itself affect prolactin secretion or utilization, although such a feedback mechanism from prolactin target tissue remains to be clarified. Mastectomy could result in neural stimulation of prolactin release, which might explain the generally elevated levels, but is unlikely to account for the observed difference in the weekly pattern. Although differences in serum prolactin concentration were found, their interpretation requires care as it is probably unwise to generalize from a sample of only 11 premenopausal breast cancer patients. The differences between cancer and control levels are very small and their relationship to the disease is still uncertain.
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