Reproductive and Hormonal Risk Factors for Ductal Carcinoma In situ of the Breast

Carcinoma in situ (CIS) of the breast, a classification for malignant cells that have not moved beyond the epithelium to invade the basal membrane, is further categorized as either lobular CIS or ductal CIS (DCIS) depending on its location (1). In addition, DCIS can be classified into comedo (high-grade) and non-comedo (medium or low-grade) subtypes based on histopathologic characteristics such as pattern of necrosis and maximum nuclear diameter. Both biological and epidemiologic evidence suggest that some DCIS develops into invasive disease, whereas other forms of DCIS may not progress to invasive breast cancer (IBC) (2-7).

Age-adjusted incidence rates for DCIS increased from 2.3 per 100,000 females in 1973 (8) to 15.8 per 100,000 females in 1992 (9). The most dramatic increases have occurred since 1983, with a 17.5% annual increase in incidence rates between 1983 and 1992 compared with increases of 3.9% annually from 1973 to 1983 (9). Separate studies in Detroit (10), Connecticut (11), Vaud, Switzerland (12), and Florence, Italy (13) have shown that most of this increase was due to the introduction of screening mammography in the early 1980s and subsequent increases in its use in women age ≥40 years. However, since 1992, the proportional change in incidence rates for DCIS has slowed, especially for comedo DCIS (14). In addition, 80% of all DCIS diagnosed in the United States since 1980 were non-comedo type.

Whether DCIS lesions found through increased detection will progress to invasive disease is unknown. It is generally believed that comedo-type DCIS is more similar histologically to invasive disease than is the non-comedo-type. Studies of women diagnosed with concomitant DCIS and IBC or with IBC following a DCIS diagnosis have reported that higher-grade DCIS is associated with higher-grade IBC (15-20). Estimated DCIS prevalence rates based on autopsy studies of women who died from causes other than breast cancer range from 0.2% to 14.7% compared with 0% to 1.8% for IBC (21). Therefore, some in situ lesions may take much longer to develop invasive characteristics or may never become invasive during a woman's lifespan. Because of the uncertainties regarding DCIS progression, most lesions are treated aggressively. Understanding the differences in risk factor profiles, if any, between DCIS subtypes is a first step toward identifying the lesions that may be more likely to progress to invasive disease.

Many of the accepted risk factors for IBC involve hormonal exposures, particularly to estrogen, whether directly through exogenous use [oral contraceptives and hormone replacement therapy (HRT)] or indirectly through reproductive events such as timing of menarche and menopause, pregnancy, and lactation. Previous studies have found nulliparity, late age at first pregnancy, early menarche, late menopause, no lactation, and exogenous hormone use associated with IBC (22). The connection between estrogen and in situ breast cancer is less clear.

We examined known hormonal and reproductive risk factors for IBC to determine whether they are risk factors for DCIS and whether risk factors differ for comedo and non-comedo DCIS subtypes. Odds ratios (OR) for DCIS as well as for DCIS subtypes (comedo and non-comedo) were compared directly with those of IBC in the same North Carolina study population.

The Carolina Breast Cancer Study is a population-based case-control study of in situ breast cancer and IBC in African American and Caucasian women (23). Eligible study participants were residents of 24 contiguous counties of eastern and central North Carolina who were ages 20 to 74 years at the time of diagnosis (cases) or selection (controls). Women with first breast cancer diagnoses (in situ or invasive) were identified through a rapid ascertainment system in conjunction with the North Carolina Central Cancer Registry (24), and controls were located via computerized lists from the Department of Motor Vehicles (age <65 years) and the Health Care Finance Administration (age ≥65 years). Controls were frequency-matched to cases on race and 5-year age intervals.

Invasive cases were enrolled in two phases, between 1993 and 1996 (phase I) and from 1996 to 2001 (phase II), and were oversampled for African Americans and younger age (20-49 years). Specifically, a process of randomized recruitment using predetermined probabilities (25) was used to balance four groups based on age and race: younger African Americans, older African Americans, younger non-African Americans, and older non-African Americans, described in detail elsewhere (23).

In situ case enrollment occurred between 1996 and 2001 and included pure DCIS, DCIS with microinvasion to a depth of 2 mm, and lobular CIS. All in situ cases matching the age and geographic constraints mentioned above were eligible for the study, with no oversampling on race or age.

A total of 705 CIS cases were identified during the enrollment period. Of these, 50 were ineligible or deceased, leaving 655 eligible cases. Five cases could not be contacted, physicians refused participation for 51 cases, and 58 declined to participate. Therefore, 541 CIS cases participated resulting in an overall response rate (participants/eligible cases) of 82.6%. Thirty-eight participants were excluded from the current analysis because they completed only a mini questionnaire that did not include hormonal or reproductive questions along with 28 cases of pure lobular CIS and 29 cases of DCIS with microinvasion, leaving 446 pure DCIS cases. Of the 940 DCIS controls sampled, 122 were ineligible or deceased, 88 could not be located, and 197 refused participation. A total of 458 DCIS controls completed the full questionnaire, resulting in an overall response rate among eligible controls of 65.2%.

Risk factor distributions were similar for IBC cases enrolled in both phases of data collection, so data for all IBC cases from phases I and II were combined to maximize statistical power for a total of 2,704 identified cases. Of those, 201 were ineligible or deceased, 64 could not be contacted, physicians refused participation for 175, and 361 declined to participate, resulting in an overall response rate among eligible cases of 76.0%. In addition, 95 IBC cases were excluded because they completed only the mini questionnaire, leaving 1,808 IBC cases for the current analysis. A total of 3,600 controls for IBC cases were identified, of which 427 were ineligible or deceased, 689 could not be located, and 739 declined participation, resulting in an overall response rate for eligible controls of 55.0%. Controls who did not complete the full questionnaire were excluded (n = 175), leaving 1,564 controls for the IBC study analyses.

Initial DCIS diagnosis was assigned by the referring physician and verified for 446 cases by a pathologist employed by the Carolina Breast Cancer Study based on a review of pathology reports and H&E-stained slides. Less than 2% of the cases reviewed were classified as something other than DCIS based on the Carolina Breast Cancer Study pathologist's evaluation. DCIS subtype classification was based on a detailed microscopic examination of a H&E-stained slide for each case. Cases classified as comedo DCIS had a comedo-type pattern of necrosis and two of the following three characteristics: large or very large nuclear diameter (>2 times the diameter of a RBC), vesicular nuclear pleiomorphism, or prominent nucleoli. All other DCIS cases were classified as non-comedo. Fifty-six DCIS cases were not subtyped, leaving a total of 393 DCIS cases (163 comedo and 230 non-comedo) for DCIS subtype analyses.

Trained female nurses conducted in-person interviews at the woman's home or another agreed on location using a structured questionnaire. Topics covered by the questionnaire include sociodemographic factors, menstrual and pregnancy history, medical history, hormone use, family history of cancer, physical activity, and occupational history. The nurse measured height and weight at the time of the interview; all other questions were answered via self-report. Participants were given visual aids to assist with recall, such as pictures of common prescription and nonprescription drugs and calendars to pinpoint dates. Average time between diagnosis and interview was 198 days for DCIS cases and 145 days for IBC cases.

Main hormonal and reproductive risk variables included parity (categorized for analyses as no full-term pregnancies, 1, 2, ≥3), age at first full-term pregnancy (<26, ≥26 years), lactation (never, ever), oral contraceptive use (never, ever) and duration of oral contraceptive use (<5, 5-10, >10 years), use of HRT (never, ever), duration of HRT use (<5, 5-10, >10 years) and recency of HRT use (never, current, former ≥5 years since last use), age at menopause (<40, 40-49, ≥50 years), and age at menarche (≤11, 12, 13, ≥14 years). Participants who classified their race as American Indian/Eskimo, Asian or Pacific Islander, or Other (n = 13 for DCIS and n = 53 for invasive) were combined with Whites, resulting in two race categories: African American and non-African American. Age at the time of interview was computed from self-reported birth date and included in all analyses as a continuous variable. Women age <50 years were considered postmenopausal if they had undergone natural menopause (menstruation cessation), bilateral oophorectomy, or irradiation of the ovaries. In women ages ≥50 years, menopausal status was assigned based on menstruation cessation. Natural and surgical menopause were combined for analysis, because duration of estrogen exposure was the main focus.

The main outcome variable was DCIS, which included all cases of pure DCIS. Differences in exposure and outcome variable distributions by case-control status and histologic type were evaluated using χ² tests generating two-sided P values. ORs and 95% confidence intervals (95% CI) were computed using unconditional logistic regression. Tests for trend were conducted by evaluating the P value for the β coefficient where exposure was coded as an ordinal variable. Case-control analyses were conducted for all data to estimate main effects for the risk factors, comparing each case group (DCIS or invasive) with frequency-matched controls. In addition to analyses for all DCIS cases combined, univariate and multivariate analyses were conducted for DCIS cases stratified on histopathologic subtype (comedo versus non-comedo) and case-case analyses were used to identify factors with different relationships between comedo and non-comedo DCIS (26). All regression models contained an offset term to account for the sampling probabilities used to identify eligible cases and controls (25). If removal of the covariate from the model resulted in ≥10% change in stratum-specific regression coefficients, that variable was considered a confounder and remained in the final model.

All evaluations of potential effect measure modification and confounding were conducted on the in situ and invasive data sets separately. The resulting model included covariates that met modeling criteria for either data set to facilitate direct comparisons between in situ and invasive model estimates. All statistical analyses were conducted using SAS version 8.2 (SAS Institute).

A total of 904 women participated in the DCIS study, of which 18.1% were African American (n = 164). The mean ± SD age of all DCIS cases combined (55.16 ± 11.07 years) and of comedo and non-comedo cases (Table 1) was slightly higher than that of controls (54.46 ± 10.26 years), and a higher percentage of DCIS cases than controls were African American (21.1% of all DCIS combined versus 15.3% of controls). Comedo DCIS cases were slightly more likely to have a first-degree family member with breast cancer and a college education and were more often in the highest income bracket than non-comedo cases.

IBC cases and controls were slightly younger than their DCIS counterparts as reflected in both mean and median ages. IBC cases and controls included larger proportions of African Americans than corresponding DCIS cases and controls because African Americans were oversampled for the IBC study. Phase II IBC cases had a higher percentage of African American participants than phase I IBC cases.

Age and race were included in all multivariate models along with the offset terms to account for probability sampling by age and race age. No other covariates met our criteria for inclusion in models as a confounder, and there was no significant OR modification by any of the evaluated covariates at a 0.05 α level.

A first full-term birth at age <26 years was inversely associated with both DCIS and IBC. An inverse association with parity strengthened with number of full-term pregnancies in the DCIS group but remained relatively constant for IBC regardless of number of pregnancies (Table 2). Ever having breast-fed was not associated with DCIS but was inversely associated with invasive cancer.

Increasing duration of oral contraceptive use was positively associated with IBC but not DCIS. Any HRT use was not associated with DCIS in this study but was inversely associated with invasive disease, with some evidence of a stronger association for >10 years of HRT use. ORs were also more strongly inverse for IBC compared with DCIS for current but not former HRT use (data not shown). Younger age at menopause (<40 years) was inversely associated with both invasive disease and DCIS, although the strength and consistency of the relationship was more apparent for IBC. Similarly, older ages at menarche showed inverse associations with both DCIS and IBC but were more consistent for IBC. Each increase in age at menarche was associated with a decrease in OR for IBC.

Experiencing at least one full-term pregnancy was inversely associated with comedo DCIS, with stronger inverse associations for increasing numbers of full-term births (Table 3). Non-comedo DCIS were inversely associated with three or more full-term births, but the association was weaker than the corresponding OR for comedo DCIS. Ever breast-feeding was not significantly associated with either comedo or non-comedo DCIS, although the OR was <1.0 for comedo but not non-comedo DCIS. Similarly, ORs for ever use of HRT were not statistically significant for either DCIS subtype, although the association was inverse for comedo but not non-comedo DCIS. ORs for recent and former HRT were more strongly inverse for comedo compared with non-comedo DCIS (data not shown). When comedo and non-comedo DCIS cases were compared in a case-case analysis, ORs were statistically significant only for duration of oral contraceptive use; however, these associations were based on small numbers of cases.

Results did not differ substantially when we adjusted for history of screening mammography (data not shown).

Using a large, population-based study of CIS of the breast and IBC, we evaluated known hormonal and reproductive risk factors for IBC to determine whether they are also risk factors for DCIS. Parity and younger age at first full-term pregnancy and younger age at menopause (<40 years) were inversely associated with both DCIS and IBC, whereas older age at menopause was positively associated with IBC only and older age at menarche was inversely associated with IBC only. Ten or more years of oral contraceptive use showed a positive association with comedo-type DCIS and IBC but an inverse association for non-comedo DCIS.

When DCIS cases in our study were separated into the two main histologic subtypes, comedo and non-comedo, comedo-type DCIS associations paralleled invasive results more frequently than non-comedo DCIS. Specifically, parity, lactation, and HRT use were inversely associated with both comedo DCIS and IBC. These results support the theory that DCIS is not a uniform disease, and similarities in risk factors between comedo DCIS and IBC are in agreement with data showing that comedo DCIS is more closely related to IBC (27).

Many studies examined reproductive risk factors for IBC and DCIS, but differences in study designs, methods, and populations make comparisons of results difficult. Including both DCIS and invasive cases from the same population circumvents many of those issues, allowing for direct comparison between ORs. Six previous studies of reproductive or hormonal risk factors for DCIS have included invasive cases as well (28-33), and as with the current study, these studies found few differences in risk factors between DCIS and invasive disease. Two other studies examined reproductive risk factors in CIS cases only (34, 35). Parity (28-31, 33-35), young age at first full-term pregnancy (28, 29, 31-34), and older age at menarche (28, 33) were inversely associated with both outcomes, whereas older age at menopause and postmenopausal HRT use (31, 33) have been positively associated with both forms of cancer.

Only one other published study examined reproductive risk factors for comedo and non-comedo DCIS specifically, which were limited to parity and age at first full-term birth (29). In that study, the authors found no association with parity and either DCIS subtype and a positive association between comedo DCIS and age at first birth of ≥25 years (OR, 1.38; 95% CI, 1.02-1.88 for ages 25-29 years and OR, 1.63; 95% CI, 1.05-2.52 for ages ≥30 years). In contrast, parity showed a stronger inverse association with comedo DCIS than with non-comedo or all DCIS combined in our study, especially for two or more full-term pregnancies.

Evidence suggests that a woman's breasts reach full maturity after a full-term pregnancy, making the cells less vulnerable to neoplastic changes (36). In the current study, ever having a full-term pregnancy was inversely associated with IBC. For DCIS, the protective association was limited to those with a first full-term pregnancy at age <26 years. Nine previous DCIS risk factor studies included parity and age at first full-term pregnancy, all of which found results similar to ours (29-31, 33-35, 37, 38).

In the current study, lactation was inversely associated with IBC but showed no overall association with DCIS. Only three other studies assessed associations with lactation and DCIS (30, 31, 35). Two found no association between breast-feeding and either DCIS or IBC (30, 31), but in the third study, lactating for ≥24 months was associated with DCIS (OR, 2.00; 95% CI, 1.11-3.60; ref. 35). These varied findings may be due to differences in lactation practices in the underlying populations. For instance, breast-feeding is more prevalent and done for longer periods in China, where a significant inverse association with lactation for >24 months was found for IBC (OR, 0.46; 95% CI, 0.27-0.78; ref. 39).

Estrogen levels play an important role in reproductive events. Increase in estrogen leads to menarche, and decreasing levels precipitate menopause. In addition, estrogen augmented by progesterone has been shown to promote cell division, which increases the chance of mutant cell growth (40). The current study results support this theory for both IBC and DCIS. Other studies that examined age at menarche and menopause found mixed results. Three found no association between age at menarche and DCIS or invasive disease (30, 34, 38). Of the two others reporting an association with age at menarche, Longnecker et al. used the youngest age group as the reference and found an inverse association with the oldest age group (≥14 years) for both DCIS and invasive disease (OR, 0.36; 95% CI, 0.15-0.87 for DCIS and OR, 0.61; 95% CI, 0.43-0.86 for invasive; ref. 33), whereas Kerlikowske et al. found a positive association for the youngest age at menarche group (≤12 years) compared with those ages >12 years at menarche for IBC only (OR, 1.9; 95% CI, 1.4-2.7; ref. 28). Menopause at age ≥55 years was associated with DCIS (OR, 1.53; 95% CI, 1.07-2.18) and IBC (OR, 2.85; 95% CI, 1.37-6.35) in the Longnecker et al. study (33) but was associated with DCIS only in the Claus et al. (34) study (OR, 1.71; 95% CI, 1.05-2.77). Age ≥45 years at menopause showed an increased association with IBC only in the Trentham-Dietz et al. (31) study (OR, 1.03; 95% CI, 1.02-1.04 continuous per year). One other study reported no association between age at menopause and either DCIS or invasive disease (38).

The link between oral contraceptive use and breast cancer risk is less clear than with other hormonal risk factors, especially among earlier-stage cancer. Oral contraceptive use showed no association with DCIS or invasive disease in our study. All other studies that included both invasive and DCIS cases found that oral contraceptive use was positively associated with IBC but not associated with DCIS (31, 32, 41-43).

Although most other studies found that postmenopausal HRT was positively associated with either DCIS or IBC (31, 33, 34, 38, 44, 45), HRT was inversely associated with IBC in our study, especially among those using HRT for >10 years. Although this difference is puzzling, one explanation may be that we did not differentiate between estrogen-only and estrogen-plus-progestin regimens. Two studies that did examine HRT (estrogen and progesterone) and estrogen-only regimen separately found HRT associated with DCIS (OR, 1.75; 95% CI, 1.10-2.80 for Longnecker et al. and OR, 2.3; 95% CI, 1.3-3.9 for Schairer et al.) but not with IBC (OR, 1.14; 95% CI, 0.91-1.43 and OR, 1.1; 95% CI, 0.9-1.4, respectively), whereas estrogen-only regimen was not associated with either outcome (33, 45). A third study found estrogen-only regimen associated with IBC (OR, 2.22; 95% CI, 1.18-4.17) but not with DCIS and no association between HRT and either outcome (44). Another reason for the difference between our results and those of other studies for HRT could be that a higher percentage of our controls reported ever using HRT (50.3% for invasive and 63.9% for DCIS) than the rate of HRT use in the general population (44%; ref. 46).

A strength of the Carolina Breast Cancer Study is that the population base in North Carolina includes African Americans, who have been underrepresented in previous epidemiologic studies of DCIS. Differences between our results and other studies could reflect the underlying study populations. For example, in our study population, African American participants were statistically significantly less likely to use postmenopausal HRT than Caucasians (47). Unlike the invasive portion of the Carolina Breast Cancer Study where African Americans were oversampled, all cases of DCIS were eligible the in situ portion of the study. The number of minority participants with DCIS was not sufficient to conduct separate analyses by race, so it is difficult to draw specific conclusions about risk factors for DCIS among African Americans. In addition, overall response rates in our study were lowest for African American controls, suggesting that future studies should focus recruitment efforts on increasing participation among minorities and controls in particular.

Selection bias was a potential issue for this study, because case participants could have had better and more frequent access to healthcare and therefore mammography screening. However, the data were analyzed stratified on frequency of doctor's visits and having had a mammogram in the 2 years previous to participation in the study, and neither affected the ORs (data not shown). Finally, because there is no universal classification system for DCIS pathology, comedo and non-comedo cases could have been misclassified. Unpublished data by the authors of the current study on DCIS subtype classification errors indicate that pathologist errors are predominantly in favor of the more severe (comedo) category. Therefore, a sensitivity analysis was conducted by increasing the number of comedo cases using the methods described by Rothman and Greenland (48), which determined that over one-third would have to be incorrectly classified to have had any effect on the results.

It has already been established that women with non-comedo-type DCIS should be evaluated and treated using criteria different from those of the more aggressive types of DCIS. Our results support this conclusion, suggesting that comedo-type DCIS may be more similar to IBC with regard to underlying etiology. However, future studies will need to include larger numbers of both DCIS subtypes to clarify associations between each subtype and potential risk factors. With more women being diagnosed at earlier stages of breast cancer, large epidemiologic studies of DCIS with sufficient power to stratify on comedo versus non-comedo histology are feasible and likely to be highly informative.

No potential conflicts of interest were disclosed.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Chiu-Kit Tse (University of North Carolina at Chapel Hill) for analytic assistance and Dr. Dale Sandler (National Institute of Environmental Health Sciences) for guidance and expertise throughout the study process.