Hormone Therapy, Estrogen Metabolism, and Risk of Breast Cancer in the Women’s Health Initiative Hormone Therapy Trial

Abstract

Background

In the Women’s Health Initiative Hormone Trials (WHI-HT), breast cancer risk was increased with estrogen plus progestin (E+P) but not with unopposed estrogen (E-alone). We hypothesized that E+P would preferentially metabolize to 16α-hydroxyestrone (16α-OHE1) rather than 2-hydroxyestrone (2-OHE1), and that breast cancer risk would be associated with baseline and 1 year changes in estrogen metabolites: positively for 16α-OHE1 levels and negatively for levels of 2-OHE-1 and the 2:16 ratio.

Methods

In a prospective case-control study nested in the WHI-HT, 845 confirmed breast cancer cases were matched to 1690 controls by age and ethnicity. Using stored serum, 2-OHE1 and 16α-OHE1 levels were measured by EIA at baseline, and for those randomized to active treatment (n=1259), at 1 year.

Results

The 1 year increase in 16α-OHE1 was greater with E+P than E-alone (median 55.5 vs. 43.5 pg/ml, p<0.001), but both increased 2-OHE1 by ~300 pg/ml. Breast cancer risk was modestly associated with higher baseline levels of 2-OHE1 and the 2:16 ratio, and. for estrogen receptor+/progesterone+ cases only, higher baseline 16α-OHE1 levels. For those randomized to active treatment, breast cancer risk was associated with greater increase in 2-OHE-1 and the 2:16 ratio, but associations were not significant.

Conclusions

Although E+P modestly increased 16α-OHE1 more than E-alone, increase in 16α-OHE1 was not associated with breast cancer.

Impact

Study results do not explain differences between the WHI E+P and WHI E-alone breast cancer results but metabolism of oral HT which may explain smaller than expected increase in breast cancer compared with endogenous estrogens.

Introduction

Endogenous estrogen levels are positively associated with postmenopausal breast cancer for non-hormone users. (1;2) In the WHI randomized placebo-controlled clinical trials combined estrogen+progestin (E+P) was associated with increased breast cancer risk (relative risk (RR) of 1.26 (1.00-1.59)), (3;4) but unopposed estrogen (E-alone) was not (RR= 0.77 (0.59-1.01). (5) Breast cancer risk increases less with E+P postmenopausal hormone therapy (HT) than expected based on the increase in circulating estrogens. (6) Furthermore, a recent case-cohort study in the WHI-HT trial showed that 1-year increases in estrogens and sex hormone binding globulin (SHBG) were similar for E+P compared with E-alone, and that progesterone decreased slightly with E+P. (6) However, with both E+P and E-alone, bioavailable and free estradiol increased less than total estradiol, related to the 2.5-fold increase in SHBG with either HT regimen. (6) A case-cohort study of non-hormone users in the WHI Observational Study (WHI-OS) recently reported that median levels of bioavailable estradiol were significantly higher in invasive breast cancer cases than cohort controls, especially for estrogen receptor (ER)+ breast cancer, (adjusted HR 1.86(1.00,3.45), quartiles 4 versus 1.) (7) Therefore, the difference in the risk of breast cancer between E-alone (decreased risk) and in E+P (increased risk) in the WHI-HT do not appear to be explained by similar changes in hormone levels for E+P compared with E-alone.(6)

Therefore, we hypothesized that differences in estrogen metabolism might explain differences in breast cancer risk for E+P and E-alone. Estrogens (both estradiol and estrone) metabolize along several competing pathways including of 2-hydroxy estrone (2-OHE1), 16α- hydroxy estrone (16α-OHE1), and 4-hydroxy estrone (4-OHE1) that may vary in estrogenic and genotoxic potential. (8-10) Most studies of estrogen metabolites and breast cancer have focused on 2-OHE1, 16α-OHE1 and their ratio (2:16). 16α-OHE1 has strong estrogenic effects related to cell proliferation, and has a higher binding affinity for the estrogen receptor than 2- OHE1, which has limited proliferation effects and possibly even anti-estrogenic effects. (8;9;11) Several studies have reported that breast cancer risk was associated with higher levels of 16α- OHE1(12-16), and lower levels of the 2:16 ratio. (16-21) Other studies have found no associations, or a positive association of 2-OHE-1 or the 2:16 ratio with breast cancer risk.(22-25) Most studies have excluded hormone users. In a previous case-cohort study nested in the WHI Observational Study, HT users had substantially higher levels of levels of 2-OHE1 and higher levels of 16α-OHE1 than non-users, and combined E+P users had slightly higher 16α- OHE1 levels than E-alone users. (26) However, no prospective studies have assessed the effect of HT regimen on changes in estrogen metabolism and subsequent breast cancer risk within a randomized clinical trial of HT.

Therefore, we conducted a prospective case control study of 2,535 postmenopausal women nested within the randomized WHI-HTs to test three hypotheses: 1) incident breast cancer is associated with higher baseline levels of 16α-OHE1 and lower levels of the 2:16 ratio, 2) compared to E-alone, E+P is associated with a greater increase in 16α-OHE1 levels, and a smaller increase in levels of 2-OHE1 and the 2:16 ratio 1 year after randomization, and 3) for women randomized to active HT, incident breast cancer is associated with a larger 1 year increase in 16α-OHE1 levels, and a smaller 1 year increase in the 2:16 ratio. Finally, we hypothesized that associations are stronger for cases that are positive for estrogen receptor (ER+) and progesterone receptor (PR+).

Methods

Overview of the WHI Hormone Trials (WHI-HT)

Between 1993 and 1998, the WHI recruited 27,347 postmenopausal women aged 50 to 79 at 40 centers in the United States into 2 randomized clinical trials: E+P and E-alone. Details of the methods and results of these trials have been published. (27;28) Briefly, the E+P trial randomized 16,608 postmenopausal women to conjugated equine estrogen (CEE) (0.625 mg per day) plus medroxy-progesterone acetate (MPA) (2.5 mg/day) or placebo. The E-alone trial randomized 10,738 women with a prior hysterectomy to CEE (0.625 mg/day) or placebo. Women using hormones at the screening visit underwent a 3 month washout period prior to study randomization. The protocol and consent forms were approved by the institutional review boards of the participating institutions, and all women provided written informed consent. Follow-up contacts by telephone or clinic visit occurred every 6 months with clinic visits required annually. Covariates were collected via questionnaires or examinations and included age, ethnicity, BMI, waist circumference, reproductive history, alcohol intake, smoking status, physical activity, prior HT use, family history of breast cancer, and oophorectomy status. BMI and waist circumference were measured at clinic visits.

Outcomes

Baseline and annual mammograms were required for women in the WHI-HT to identify potential breast cancer cases. Details of breast cancer endpoints were abstracted and confirmed by review of medical records and pathology reports (available on 98.2% of participants) by physician adjudicators at local clinics masked to treatment assignments. Estrogen receptor/progesterone receptor (ER/PR) tumor status was obtained by pathology reports. Only ER+, PR+ and ER-, PR- status was specifically evaluated because of limited sample size in other groups.

Study Design and Participants

The current study selected breast cancer cases and controls from both the E+P and E- alone WHI-HT, to compare changes in metabolites and their associations with subsequent breast cancer risk according to HT regimen (E+P trial vs. E-alone trial). Estrogen metabolites were measured on breast cancer cases and controls at baseline, and for participants randomized to active treatment, metabolites were also measured at 1 year post-randomization. Cases (N=845) include all women diagnosed with breast cancer after study enrollment through August 2006 who met the following study eligibility criteria: diagnosis of breast cancer during WHI follow-up, breast cancer diagnosis that occurred at least 5 years after menopause, no diagnosis of other cancers during follow-up, no use of medications that could affect estrogen levels (i.e. anti-mycobacterial, imidazole-related anti-fungals, androgen-anabolic steroids, adrenal steroid inhibitors, hormone receptor modulators, histamine H₂ receptor antagonists, cyclosporine analogs, and herbal estrogens), self-reported race or ethnicity other than “unknown,” and adequate serum available at baseline and year 1. To preserve the temporal relationship between exposure and incident breast cancer, the study design called for the exclusion of breast cancer cases with diagnosis ≤ 1 year post-randomization (n=48 at baseline, reducing the total number of cases to n=797 with baseline estrogen metabolites (n=627 invasive).

Potential controls were selected from participants who were free of breast cancer diagnosis through August 2006 and met the following criteria: postmenopausal for at least 5 years, no use of medications that could affect estrogen levels, self-reported race or ethnicity other than “unknown,” and with adequate serum available. For each case, 2 controls (N=1,690) were selected, frequency-matched for age and ethnicity. Approximately half of the controls were selected from existing WHI case-control studies of breast cancer and hip fracture that include measurements of both baseline and 1 year follow up sex steroid hormone levels, with the remainder (n=827) selected from the remaining risk set as of August, 2006. Among women randomized to active treatment (n=1259), 21 breast cancer cases were excluded for occurring ≤1 year follow-up, and 24 participants did not have 1 year samples available. Therefore 1 year change in estrogen metabolites were available on n =1214, including n=401 breast cancer cases.

Laboratory analyses

At baseline and 1 year post-randomization blood was drawn after a minimum 10 hours of fasting. Serum and plasma samples were shipped to a central repository and stored at −70°C and not previously thawed. (29) For the current study, stored serum from baseline (all participants) and 1 year (participants randomized to active HT) was shipped on dry ice to Immuna Care, Inc., (Tampa, FL) for measurement of estrogen metabolites under the direction of Dr. Thomas Klug. Using serum (150 μl per assay) 16α-OHE1 and 2-OHE1 were measured by immunoassay (ESTRAMET 2/16 kits, Immuna Care). The limits of detection are ~10 pg/ml for the 16αOHE1 assay and ~20 pg/ml for the 2-OHE1 assay. Postmenopausal women screened for WHI provided 210 additional samples that were split into duplicates for quality control (QC) testing. Vials were coded to provide no participant information to study investigators or laboratory staff. Cases, controls, intervention groups and QC samples were randomly distributed throughout each batch of specimens, and to minimize potential differences between cases and controls that might arise from batching on a plate, each plate batch contained a ratio of one breast cancer case to two controls (masked). Coefficients of variation (CV) and intraclass correlation coefficients (ICC) were calculated using the 210 pairs of split QC samples.

Statistical analysis

The characteristics of study participants according to HT arm and case/control status were compared using T-tests and Wilcoxon tests for normally and non-normally distributed continuous variables, respectively, and Chi-square and Fisher’s exact tests for categorical variables. (Table 1) Associations between the estrogen metabolites and breast cancer risk were assessed using conditional logistic regression models to account for the primary matching variables (age, ethnicity). Quartiles were defined based on the controls’ estrogen metabolite levels. For continuous models, metabolite values were log-transformed, and 1 year changes in metabolites were increased by a constant to create all positive values and then log transformed. Differences in by trial and active vs. placebo assignment were tested by including an interaction term in the logistic regression models. Sensitivity analyses included adjusting for BMI and treatment assignment, and for baseline levels in models assessing change in metabolites, and also stratifying by ER+/PR+ status, and restricting to invasive cancer only (n=697). All p-values are two sided. SAS 9.2 (SAS Institute) was used for analyses.

Table 1.

Baseline demographics of study participants by WHI Hormone Trials

	E-alone Trial			E+P Trial
Variable	Breast Cancer n=269	Control n=639	p	Breast Cancer n=528	Control n=1051	p
Oophorectomy status - a part, one, or both ovaries removed, N, %	145, 54.31	375, 58.6	0.06c	21, 4.0	41, 3.6	0.73d
Age, years	63.9 (0.43)	65.3 (0.29)	0.01 a	64.1 (0.29)	64.6 (0.22)	0.16a
BMI, kg/m2	31.3 (0.37)	29.2 (0.22)	<0.0 001a	29.3 (0.25)	28.4 (0.18)	0.001a
Waist circumference, cm	95.1 (0.93)	90.2 (0.51)	<0.0 001a	90.3 (0.57)	87.8 (0.41)	0.0005a
Family History breast cancer, N, %	64, 46.7	101, 37.8	0.09c	110, 45.3	167, 36.0	0.02c
Active treatment assignment, N, %	115, 42.8	323, 50.6	0.03c	310, 58.7	490, 46.6	<.0001c
Age at first birth, N, %
No births	8,3.7	11,2.0		14,3.3	33,3.8
<20 years old	59,27.4	128,23.8		14,3.3	33,3.8
20-<30 years old	139,64.7	321,67.1		286,68.3	640,72.9
30 or more years old	9, 4.2	38, 7.1	0.19c	53, 12.7	85, 9.7	0.24c
Ethnicity, N, %
Amer. Indian/Alaskan	2, 0.7	3, 0.5		1, 0.2	2, 0.2
Native
Asian/Pacific Islander	5, 1.9	10, 1.6		17, 3.2	23, 2.2
Black/African American	39, 14.5	77,12.1		38, 7.2	23, 2.2
Hispanic/Latino	10, 3.7	23,3.6		15, 2.8	26, 2.5
White	213, 79.2	525, 82.2	0.85 d	457, 86.6	940, 89.4
Other	0, 0	1, 0.2		0, 0	0, 0	0.45d
Alcohol intake, N, %
Non-drinker	46, 17.4	81, 12.8		56, 10.8	112, 10.8
Past-drinker	63, 23.9	132, 20.8		91, 17.5	168, 16.2
<1 drink/month	33, 12.5	98, 15.5		72, 13.9	164, 15.8
<1 drink/week
1-6 drinks/ week	46, 17.4	122, 19.2		128, 24.7	297, 28.6
7 or more drinks/ week	29, 11.0	50, 7.9	0.07c	69, 13.3	107, 10.3	0.28c
Current smoker, N, %	20, 7.5	55, 8.7	0.79c	44, 8.5	93, 8.9	0.04c
Parity, children, N, %
Never pregnant	21, 7.8	52, 8.2		44, 8.4	75, 7.2
Never had term	8, 3.0	11, 1.7		14, 2.7	33, 3.2
Pregnancy
1	16, 6.0	39, 6.2		48, 9.2	68, 6.5
2	51, 19.0	114, 18.0		115, 22.0	210, 20.1
3	62, 23.1	143, 22.6		131, 25.1	252, 24.1
4	59, 22.0	123, 19.4		95, 18.2	203, 19.4
5 or more	51, 19.0	152, 24.0	0.64c	76, 14.5	206, 19.7	0.10c
Recreational physical activity, minutes/week (SEM)	132.9 (9.6)	160.3 (7.30)	0.07 b	154.6 (7.56)	171.6 (5.56)	0.08b
Prior E+P use, years (SEM)	2.7 (0.69)	5.2 (1.06)	0.57 b	4.3 (0.55)	3.9 (0.35)	0.70b
Previous hormone use at baseline, N, %
Never used	144, 53.5	331, 519		401, 76.0	782, 74.4
<5 years ago	24, 8.9	73, 11.4		38, 7.2	72, 6.9
5-<10 years ago	17,6.3	29, 4.6		324, 4.6	41, 3.9
10 or more years ago	49, 18.2	125, 19.6		41, 7.8	94, 8.9
Current-user	35, 13.0	80, 12.5	0.62c	24, 4.6	62, 5.9	0.68c

^at-test

^bWilcoxon test

^cChi-square test

^dFisher’s exact test

SEM=standard error of the mean P-values reflect comparisons between cases and controls within each HT trial arm.

Results

The overall CVs for 2-OHE1,16α-OHE1,and the 2:16 ratio were 26.4%, 15.2%, and 28.0% respectively, with intra-class correlations (ICCs) of 0.94, 0.86 and 0.90, respectively. Spearman correlations between 2-OHE-1 and 16αOHE-1 were ρ=0.18, p<0.0001 at baseline, not on HT, and ρ=0.24, p<0.0001 at 1 year for women randomized to HT.

Table 1 presents baseline characteristics of the study population, stratified by E+P versus E-alone trials and breast cancer case-control status. Study participants from the E-alone arm differed from those from the E+P arm, (e.g., ~60% of study participants had partial or total oophorectomy in addition to their eligibility-requirement of hysterectomy, compared with ~4% in the E+P arm). Women in the E-alone arm were also more likely to be non-white. These characteristics are similar to the overall sample of women in the HT clinical trials. For both trials, breast cancer cases were heavier, had larger waist circumference, and were more likely to have a family history of breast cancer than controls (p<0.05). Mean (SD) time to breast cancer was 4.6 (2.4) years, with similar overall follow-up time for cases (9.1(1.6)) and controls (9.0(1.5)) years, respectively.

Relationship of baseline estrogen metabolite levels to incident breast cancer

The median levels of serum 16α-OHE1, 2-OHE1 and the 2:16 ratio at baseline were not significantly different between women who developed breast cancer during follow-up compared with controls in both E-alone and E+P arms. (Table 2) In logistic models that accounted for age and ethnicity-matching, breast cancer risk was significantly higher for quartile 4 versus 1 of baseline levels of both 2-OHE1 and the 2:16 ratio, but not associated with baseline levels of 16α-OHE1. (Table 3) Higher baseline levels of 2-OHE1 and the 2:16 ratio (but not 16α-OHE1) were associated with breast cancer risk in models with the estrogen metabolites as continuous variables. These associations persisted after further adjustment for BMI, and for treatment assignment. The association of higher baseline 16αOHE-1 with increased odds of breast cancer was slightly stronger 1) if restricted to invasive cancer only, or 2) for women randomized to active treatment in the E-alone trial, but associations and interactions by treatment assignment, were not statistically significant (data not shown).

Table 2.

Median baseline levels of 16α-OHE1, 2-OHE1, and the 2:16 ratio by treatment assignment and breast cancer case/control status among study participants from the WHI Hormone Trials

E-alone			E-alone Placebo		E+P		E+P Placebo
N cases/ controls	115/323		154/316		310/490		218/561
16α-OHE1 (pg/ml)
	Median	p	Median	p	Median	p	Median	p
Cases	349.8	0.71	345.3	0.62	338.5	0.96	345.5	0.38
Controls	353.5		351.4		335.8		339.8
2-OHE1 (pg/ml)
	Median	p	Median	p	Median	p	Median	p
Cases	99.0	0.22	104.3	0.52	102.9	0.48	106.0	0.26
Controls	93.5		102.5		99.8		100.8
2:16 ratio
	Median	p	Median	p	Median	p	Median	p
Cases	0.30	0.38	0.32	0.28	0.31	0.51	0.31	0.77
Controls	0.29		0.30		0.31		0.30

P value from Wilcoxon test

Table 3.

Association of baseline estrogen metabolite levels with breast cancer risk among study participants in the WHI Hormone Trials

	N Cases/Controls	Median (IQR)	OR (95% CI)	p
16α-OHE1 (pg/ml)
Quartile 1a	177/423	239.0 (212.1, 257.3)		0.21d
Quartile 2a	219/420	309.5 (291.5, 327)
Quartile 3a	217/425	377.5 (359, 397.5)
Quartile 4a	184/422	489.5 (450, 564.5)
Continuousa			0.96 (0.73-1.25)	0.76
Continuousb			0.89 (0.68-1.17)	0.40
Continuousc			0.91 (0.70-1.20)	0.52
2-OHE1 (pg/ml)
Quartile 1a	179/428	60 (49.5, 69)		0.11d
Quartile 2a	194/420	88 (81.7, 93.5)
Quartile 3a	195/419	116 (107.5, 124.5)
Quartile 4a	229/423	173.3 (151.6, 227.8)
Continuousa			1.23 (1.04-1.45)	0.01
Continuousb			1.20 (1.02-1.42)	0.03
Continuousc			1.18 (0.99-1.40)	0.05
2:16 ratio
Quartile 1a	184/422	0.17 (0.13, 0.19)		0.20d
Quartile 2a	186/421	0.26 (0.24, 0.28)
Quartile 3a	203/422	0.35 (0.32, 0.38)
Quartile 4a	220/420	0.53 (0.47, 0.68)
Continuousa			1.21 (1.04-1.41)	0.02
Continuousb			1.21 (1.04-1.42)	0.02
Continuousc			1.19 (1.01-1.39)	0.03

All models include matching factors: age ethnicity. Estrogen metabolite levels are logtransformed for continuous models.

^aUnadjusted

^bAdjusted for BMI

^cAdjusted for BMI and treatment arm

^dp-value for Wald Χ2 test for differences among quartiles

IQR=interquartile range

OR=odds ratio

CI=confidence interval

Baseline Estrogen metabolite-breast cancer risk stratified by ER+/PR+ vs. ER-PR- status

Associations between baseline estrogen metabolites and breast cancer risk were further evaluated after stratifying cases by ER and PR tumor status (ER+/PR+ vs. ER-/PR-)(Table 4). Associations were somewhat stronger for (ER+/PR+) cases and weaker for ER-/PR- cases. For ER+/PR+ cases, 16αOHE-1 was positively associated with breast cancer risk. Stratified by trial and treatment group, positive associations of 16αOHE-1 with breast cancer were stronger for women randomized to placebo in both trials, although interactions with treatment assignment were not statistically significant (data not shown). Among ER+/PR+ cases, higher baseline 2- OHE1 was also associated with increased breast cancer risk (p<.05) for the highest vs. lowest quartile, and also in continuous models adjusted for BMI and treatment assignment. Compared with the overall results, associations with the 2:16 ratio were slightly attenuated for ER+/PR+ cases and seemed absent for ER-/PR- cases. Overall results were similar when restricted to invasive cancer only, or when cases were stratified by median follow-up time (data not shown).

Table 4.

Association of baseline estrogen metabolite levels with breast cancer risk by ER/PR status among women in the WHI Hormone Trials

Estrogen receptor (ER) Progesterone Receptor (PR) Status
ER+/PR+					ER-/PRN
	N case/ control	Median (IQR)	OR (95% CI)	p		N Case/ Control	Median (IQR)	OR (95% CI)	p
16α-OHE1(pg/ml)					16α-OHE1(pg/ml)
Quartile 1a	78/423	239.5 (212.3-257.5)		0.09d	Quartile 1a	18/423	238.8 (211.0, 257.0)		0.86d
Quartile 2a	109/420	309.5 (291.0-327.0)			Quartile 2a	23/420	309.5 (291.0, 326.5)
Quartile 3a	115/425	378.0 (358.0-398.0)			Quartile 3a	20/425	377.5 (359.0, 398.0)
Quartile 4a	97/422	492.0 (450.5-570.0)			Quartile 4a	17/422	491.5 (450.5, 569.0)
Continuousa			1.23 (0.87-1.74)	0.24	Continuousa			0.75 (0.36-1.59)	0.46
Continuousb			1.12 (0.79-1.59)	0.53	Continuousb			0.75 (0.35-1.58)	0.44
Continuousc			1.17 (0.82-1.67)	0.40	Continuousc			0.77 (0.36-1.64)	0.50
2-OHE1(pg/ml)					2-OHE1(pg/ml)
Quartile 1a	86/428	60.0 (49.0, 69.0)		0.12d	Quartile 1a	17/428	59.5 (48.5, 69.0)		0.35d
Quartile 2a	94/420	88.5 (82.0, 93.5)			Quartile 2a	25/420	88.5 (82.0, 93.5)
Quartile 3a	105/419	116.0 (107.5, 125.0)			Quartile 3a	15/419	115.5 (107.3, 125.0)
Quartile 4a	114/423	174.5 (151.5, 227.0)			Quartile 4a	21/423	174.0 (153.0, 228.5)
Continuousa			1.35 (1.09-1.68)	0.006	Continuousa			1.15 (0.75-1.76)	0.54
Continuousb			1.34 (1.08-1.66)	0.008	Continuousb			1.12 (0.73-1.73)	0.60
Continuousc			1.31 (1.05-1.63)	0.02	Continuousc			1.13 (0.72-1.75)	0.60
2:16 ratio					2:16 ratio
Quartile 1a	87/420	0.17 (0.13-0.19)		0.48d	Quartile 1a	24/420	0.17 (0.13-0.19)		0.31d
Quartile 2a	99/422	0.26 (0.24-0.28)			Quartile 2a	11/422	0.26 (0.24-0.28)
Quartile 3a	107/422	0.35 (0.32-0.38)			Quartile 3a	19/422	0.35 (0.33-0.38)
Quartile 4a	102/421	0.53 (0.47-0.67)			Quartile 4a	24/421	0.53 (0.47-0.67)
Continuousa			1.21 (0.99-1.49)	0.06	Continuousa			1.22 (0.82-1.82)	0.33
Continuousb			1.24 (1.01-1.53)e	0.04	Continuousb			1.20 (0.81-1.79)	0.37
Continuousc			1.20 (0.98-1.49)	0.08	Continuousc			1.20 (0.79-1.81)	0.39

All models account for age and ethnicity matching. Estrogen metabolite levels are log-transformed for continuous models.

^aUnadjusted

^bAdjusted for BMI

^cAdjusted for BMI and treatment arm

^dp-value for Wald Χ2 test for differences among quartiles

IQR=interquartile range

OR=odds ratio

CI=confidence interval

Change in estrogen metabolites following 1 year active HT treatment

Among those randomized to active hormone treatment (n=1259), estrogen metabolites were also measured in serum from the 1 year follow-up after starting HT. (Table 5) As hypothesized, the increase in 16α-OHE1 was greater with E+P vs. E-alone (median increase 58 vs. 44 pg/ml, p =0.0002). However, the increase in 16α-OHE1 was dwarfed by the approximately 4-fold increase in 2-OHE1 (median increase= ~ 300 pg/ml) that occurred with both E-alone and E+P. This is consistent with previous studies. Following 1 year of HT, levels of 2-OHE1 were similar to levels of 16α-OHE1, resulting in a 2:16 ratio = 1.0 for both treatment arms.

Table 5.

Median(IQR) baseline, 1-year, and 1-year change in 16α-OHE1, 2-OHE1, and the 2:16 ratio among study participants randomized to active treatment in the E-alone and E+P WHI Hormone Trials

	E-alone	E+P	pa
N	431	783
16α-OHE1 (pg/ml)
Baseline	352.0 (277.5, 434.5)	337.5 (272.0, 412.0)	0.045
1-year	393.5 (322.5, 489.5)	398.0 (327.5, 495.5)	0.49
Change	43.8 (−4.5, 93.5)	58.0 (12.0, 122.5)	0.0002
2-OHE1 (pg/ml)
Baseline	96.0 (74.5, 134.7)	101.0 (77.0, 137.5)	0.17
1-year	390.5 (245.0, 537.5)	410.8 (247.3, 573.0)	0.14
Change	273.3 (142.0, 423.0)	305.0 (151.5, 457.5)	0.17
2:16 Ratio
Baseline	0.30 (0.21, 0.40)	0.31 (0.23, 0.43)	0.02
1-year	1.00 (0.60, 1.40)	1.00 (0.57, 1.42)	0.53
Change	0.66 (0.28, 1.06)	0.66 (0.27, 1.08)	0.94

^aIQR=interquartile range Wilcoxon test

Associations of 1- year change in estrogen metabolites to incident breast cancer

Overall, none of the 1-year changes in estrogen metabolites showed a clear pattern of association with breast cancer risk, evaluated in quartiles or per (log-transformed) unit change (Table 6). For E-alone, breast cancer risk was associated with smaller increase in 16αOHE-1, 2-OHE-1 and the 2:16 ratio, although ORs were generally not significant. ORs for a 1 ln-unit increase in estrogen metabolites generally showed a similar pattern that was relatively unaffected by adjusting for BMI. However, greater 1 year increase in both metabolites and the 2:16 ratio was associated with lower odds of breast cancer in models that adjusted for baseline levels, particularly for the E-alone arm. However, confidence intervals were wide and results were not significant. Results were similar if restricted to ER+/PR+ total or ER+/PR+ invasive cancer only (not shown). However, when stratified by median follow-up time (online Table 1), unadjusted ORs for quartiles of 1 year change in both 2-OHE-1 and the 2:16 ratio showed a stronger protective association with breast cancer that occurred before median follow-time compared with cases that occurred post-median follow-up time, or overall.

Table 6.

Association between 1-year HT-induced change in estrogen metabolites and breast cancer risk among study participants randomized to active treatment, stratified by WHI Hormone Trials (E-alone vs. E+P)

		N Cases/Controlsa	Median (IQR)	OR (95% CI)	p
Change in 16α-OHE1 (pg/ml)
E-alone	Quartile 1b	32/80	−36.0 (−64.5, −14.0)		0.77e
	Quartile 2b	29/82	28.0 (8.0, 36.0)
	Quartile 3b	22/81	66.0 (55.0, 81.0)
	Quartile 4b	24/80	152.8 (117.8, 204.3)
	Continuousb			0.34 (0.008-13.8)	0.57
	Continuousc			0.42 (0.01-17.66)	0.65
	Continuousd			0.06 (0.00, 7.46)	0.26
E+P	Quartile 1b	74/122	−20.0(−45.5, −2.5)		0.45e
	Quartile 2b	69/123	32.8 (23.0, 45.1)
	Quartile 3b	85/125	87.0 (73.0, 104.4)
	Quartile 4b	65/120	185.0 (149.5, 240.0)
	Continuousb			0.39 (0.07-2.08)	0.27
	Continuousc			0.48 (0.09-2.40)	0.37
	Continuousd			0.38 (0.06, 2.27)	0.29
Change in 2-OHE1 (pg/ml)
E-alone	Quartile 1b	32/80	18.5 (−8.6, 93.8)		0.52e
	Quartile 2b	29/81	220.1 (181.5, 252.0)
	Quartile 3b	27/81	344.8 (313.7, 389.5)
	Quartile 4b	20/81	606.0 (503.5, 725.5)
	Continuousb			0.69 (0.18-2.64)	0.58
	Continuousc			0.70 (0.18-2.77)	0.62
	Continuousd			0.10 (0.00, 3.03)	0.26
E+P	Quartile 1b	69/122	17.0 (−10.7, 85.5)		0.71e
	Quartile 2b	85/123	228.0 (182.3, 272.0)
	Quartile 3b	71/121	372.0 (340.0, 415.8)
	Quartile 4b	68/123f	582.5 (506.0, 738.7)
	Continuousb			0.88 (0.50-1.54)	0.65
	Continuousc			1.02 (0.57-1.84)	0.94
	Continuousd			0.72 (0.35, 1.49)	0.37
Change in 2:16 ratio
E-alone	Quartile 1b	32/81	0.08 (−0.02, 0.17)		0.07e
	Quartile 2b	34/80	0.49 (0.40, 0.59)
	Quartile 3b	18/81	0.87 (0.77, 0.96)
	Quartile 4b	23/81	1.38 (1.20,1.70)
	Continuousb			0.57 (0.08-3.90)	0.57
	Continuousc			0.59 (0.08-4.17)	0.59
	Continuousd			0.07 (0.00, 6.56)	0.25
E+P	Quartile 1b	71/123	0.04 (−0.03-0.14)		0.99e
	Quartile 2b	73/122	0.46 (0.37-0.54)
	Quartile 3b	73/122	0.83 (0.75-0.95)
	Quartile 4b	75/122	1.38 (1.21-1.74)
	Continuousb			1.01 (0.36-2.86)	0.99
	Continuousc			1.41 (0.47-4.23)	0.54
	Continuousd			0.60 (0.11, 3.38)	0.56

1-year changes assessed only among those assigned to active treatment, not those assigned to placebo. All models account for age and ethnicity-matching. For continuous models, change in estrogen metabolite levels are log-transformed (after adding a constant).

^an’s differ from total due to missing values: 16-OHE-1 (1 case and 1 control), 2-OHE-1 (1 control, and 2:16 ratio (2 cases, 1 control)

^bUnadjusted

^cAdjusted for BMI

^dAdjusted for ln-transformed baseline levels

^ep-value for Wald Χ2 test for differences among quartiles. HT= hormone therapy

IQR=interquartile range

OR=odds ratio

CI=confidence interval

Discussion

This is the first study to evaluate estrogen metabolites in relation to incident breast cancer within the context of a randomized clinical trial of HT. Incident breast cancer risk was modestly associated with higher baseline levels of 16-αOHE-1 among ER+/PR+ cases, but was also associated with higher baseline levels of 2-OHE-1 and the 2:16 ratio. For those randomized to active treatment, the 1-year increase in 16α-OHE1 was greater for E+P than for E-alone, but incident breast cancer was not associated with greater 1-year increases in 16α- OHE1 for either the E+P or the E-alone trial. Finally, greater 1-year increases in 2-OHE-1 or the 2:16 ratio were associated with lower odds of breast cancer but were generally not significant, although associations were stronger in the E-alone trial or for cases occurring before median follow-up time.

Although E+P treatment increased serum levels of 16α-OHE1 more than E-alone, this difference does not appear to explain the increased breast cancer risk with active treatment in the WHI E+P (E+P) trial vs. the decreased breast cancer risk in the WHI E-alone (E-alone) clinical trial. A noteworthy finding is that randomization to active treatment in either trial (E+P or E-alone) similarly increased 2-OHE1 ~4-fold, raising 2-OHE-1 to levels similar to 16α-OHE1, and changing the 2:16 ratio from ~0.35 at baseline to 1.0 at 1 year. The substantial increase in 2- compared to 16- is consistent with our previous reports from non-randomized HT (26;30) and the Woman On the Move through Activity and Nutrition (WOMAN) study, a randomized trial of weight loss among women on HT. (31) At baseline, compared with women not on HT, women on HT had 2-OHE1 levels five times higher but similar 16α-OHE1 levels. The WHI HT trial results were published shortly after randomization in the WOMAN study, and most of the participants stopped their HT, mostly E+P. There was a very substantial decline in levels of 2- OHE1 between baseline and the 18-month follow up visit for women who had stopped their HT. In contrast, for women not using HT at either visit, correlations between 2-OHE1 and 16α-OHE1 levels measured ~18 months apart were 0.54 and 0.85, respectively.

In the current study, the association of metabolites and risk of breast cancer were somewhat stronger among ER+/PR+ cases than ER-/PR- cases. For example, breast cancer risk was associated with higher levels of 16α-OHE1 among ER+/PR+ cases (not significant.) ER+/PR+ breast cancer also showed stronger associations with higher baseline 2-OHE1 levels, but there were n=423 ER+/PR+ cases and only n=67 ER-/PR- cases, so we had reduced power to detect a significant risk for ER-/PR- cases.

Several studies have reported results similar to our baseline estrogen metabolite associations. A large nested case-control study of postmenopausal breast cancer (n=426 cases and 426 controls) with urinary 2-OHE and 16α-OHE1 also found a positive association of 2-OHE with ER+ breast cancer, but among HT users only, with no associations among non-HT users or ER- cases.(24) Among postmenopausal non-HT users, the Nurses’ Health Study plasma levels of 2-OHE1 and the 2:16 ratio were positively associated with breast cancer, but only for ER-/PR- cases. (32) Studies that did not stratify by ER status have also reported a positive association of the 2:16 ratio with incident breast cancer for premenopausal women(25) and postmenopausal women. (18) Among postmenopausal non-HT users, Cauley et al. found a non-significant increased risk with increasing quartile of both 2-OHE1 and 16α-OHE1 but not with the 2:16 ratio. (23) The analyses did not stratify by ER status.

Our results are also in agreement with those of the Prostate, Lung, Colon and Ovary (PLCO) screening trial, which recently used new technologies to evaluate multiple estrogen metabolites in blood and urine. (10) The PLCO longitudinal study included 277 postmenopausal incident breast cancers and 433 controls, none of whom were using HT, and also found that incident breast cancer was associated with higher levels of serum 2-OHE1, and 16αOHE-1 (not statistically significant) as well as higher levels of total estrogens and unconjugated estradiol. (33) However, in the PLCO study, the ratio of the 2-OHE1 pathway to total estrogens was inversely related to breast cancer risk, as was the ratio of the entire 2-hydroxylation pathway to the 16 hydroxylation pathway, consistent with the current study’s original hypothesis that preferential metabolism to the 2-hydroxylation pathway is associated with lower breast cancer risk. In addition, both 4-OHE-1 and the ratio of 4-hydroxy pathway catechols to methylated catechols were positively related to breast cancer risk. (33) These results from the PLCO Study suggest that the evaluation of estrogen metabolites may need to be done in the context of total estrogen or unconjugated estrogen levels, and may explain, in part, the negative results in this study and previous studies.

Our results showing that in among women randomized to HT, 2-OHE1 increases much more than 16α-OHE1 suggests that differences in metabolism of exogenous estrogens may be a partial explanation for the apparent smaller increased risk of breast cancer with oral HT, compared with the more substantial increase in breast cancer associated with higher levels of endogenous estrone and estradiol. (1) It is possible that other estrogen metabolites, i.e. 4-OH pathway, not measured in this study could contribute to an increased or decreased risk of breast cancer. Some of the metabolites resulting from HT could be acting as a selective estrogen receptor modulator (SERM), decreasing both breast cancer risk and osteoporotic fracture with small effect on CHD. Another possible contributor to smaller than expected HT-related increased breast cancer risk is that although total estradiol increases substantially with both E- alone and E+P, free or bioavailable estradiol increases only slightly because of the concomitant increase in SHBG. Oral estrogen therapy clearly has estrogenic effects on bone, uterus and other tissues. It is very possible that the balance of the effects of the specific metabolites of oral E-alone and E+P, both estrogenic and anti-estrogenic effects, the effects of oral estrogens by their first pass from gut to liver, i.e. increased SHBG and other proteins, and host susceptibility (genetics), play a key role in the specific health effects of oral E-alone or E+P therapy. The WHI is currently measuring unconjugated estradiol, estrone, progesterone, testosterone and SHBG in the HT arm at baseline and 1 year. Further evaluation of estrogen metabolites, especially in relation to tumor characteristics, may provide a better rationale for effects of oral E-alone or E+P on breast cancer risks. Most important, it may be possible to individualize therapy based on both risk and metabolism of hormones.

A possible limitation of the study is the measurement of the metabolites in serum, whereas many previous studies have used plasma or urine specimens. The correlation between plasma and urine estrogen metabolites has been studied and found to be only fair (0.52 for the 2:16 ratio, 0.60 for 2:OHE1 and only 0.22 for 16α-OHE1) and to differ according to baseline urinary 2:16 ratio and other covariates. (34) The relationship of serum, plasma or urinary metabolites to metabolite levels in tissue is relatively unknown, although a recent study showed a fair correlation between urinary 2:16 ratio and breast tissue ratio levels. (35) Reproducibility of the estrogen metabolites was very good, judging by high intraclass correlations (ICCs)(86-94%), which were higher than in a previous study,(32) but %CVs (15-28%), while in the acceptable range, were lower than previous reports. A possible explanation for both is probable HT use by some of the women providing QC samples, particularly for the 2:16 ratio, for which paired samples would cluster around 1.0 for HT users and around 0.3 for non-users. Another reason for higher ICCs in our study is that our ICCs include only inter-assay variation of a single sample, whereas the previous study evaluated paired samples collected up to 3 years apart.(32)

In summary, to our knowledge, this study of over 2,500 women is the largest of its kind exploring breast cancer and its relationship to serum estrogen metabolites in relation to HT. This sample from the WHI study population represents a very large, geographically diverse cohort of U.S. women, with valuable incident and prevalent epidemiological and clinical information. Furthermore, this is the only study which evaluates the change in estrogen metabolites with randomization to E+P vs. E-alone. These results demonstrate that although 16α-OHE1 was increased more by E+P than by E-alone, the increase in the 2:16 ratio was similar for both HT regimens, due to ~4-fold greater increase in 2OHE-1 than 16α-OHE1 for both HT regimens. Second, baseline and 1 year change in 16α-OHE1 showed little relationship to incident breast cancer. In contrast, higher baseline 2-OHE1 and the 2:16 ratio were modestly associated with higher odds of incident breast cancer, but larger 1 year increase in 2-OHE-1 and the 2:16 ratio were also weakly associated with lower odds of breast cancer, even if adjusted for baseline metabolite levels. It is possible that differences in metabolism of oral estrogens, (i.e., as demonstrated in this study, the increase primarily in the 2-OHE1, a less potent estrogen with regard to breast cancer, and also by the previously reported increase in SHBG), may be important determinants of risk of breast cancer with oral E-alone or E+P.