Background
Terminal duct lobular units (TDLUs), the structures within the breast where most cancers arise [
1], involute (shrink in number and size) with age. Involution is a complex process that is not observable; however, standardized, reproducible measures to quantify involution have been determined including TDLU count, TDLU span, and number of acini (milk producing substructures) per TDLU. Reduced involution (i.e., higher TDLU count, TDLU span, and acini/TDLU) has been associated with an increased risk of breast cancer among women with benign breast disease [
2‐
4]. Certain established reproductive breast cancer risk factors have been related to TDLU count; later age (≥ 30) at first term birth and lack of breastfeeding have been associated with higher TDLU count in premenopausal women, and earlier age at menarche has been associated with higher TDLU count in postmenopausal women [
5]. Circulating sex hormones and estrogen metabolites have also been associated with reduced TDLU involution as measured by TDLU count [
6,
7]. Thus, reduced TDLU involution is an intermediate marker of breast cancer risk.
Studies have suggested that outdoor air pollution exposure is related to an increased risk of breast cancer [
8]. The most consistent associations have been found for nitrogen dioxide (NO
2) and nitrogen oxides (NO
x) [
8‐
12], traffic-derived air pollutants. Although less evidence has been found for fine particulate matter < 2.5 μm in diameter (PM
2.5) in relation to breast cancer [
9,
11,
13,
14], two recent, large epidemiologic studies observed increased risks associated with higher levels [
10,
15]. White et al. reported that PM
2.5 was associated with a higher breast cancer risk overall, for ductal carcinoma in situ, and that there was variability by geographic region and PM
2.5 component profiles [
10]; Villeneuve et al. reported an increased risk of premenopausal breast cancer with higher PM
2.5 exposure [
15]. Additionally, studies have reported associations between multiple different air pollutants (PM
2.5, airborne metals, polycyclic aromatic hydrocarbons) and increased mammographic density [
16,
17], a strong risk factor for breast cancer [
18]. Lack of TDLU involution has been associated with higher mammographic density [
19‐
21], and it has been suggested that associations between mammographic density and breast cancer may partially reflect the amount of at-risk epithelium [
19,
20]. Therefore, there is support for examining the association between air pollution and TDLU involution. Addressing this question may help inform mechanisms underlying associations between outdoor air pollution and breast cancer risk; however, no epidemiologic studies to date have examined this question.
The objective of this study was to determine whether air pollution was associated with measures of TDLU involution (TDLU count, TDLU span, and acini/TDLU) in normal breast tissue samples from healthy volunteers. We considered PM2.5 total mass as the air pollutant of primary interest, but also examined associations for individual components of PM2.5, clusters of participants classified by PM2.5 component profiles, and gaseous pollutants, including NO2 and NO.
Discussion
In this study of women who volunteered to donate healthy breast tissue to the Komen Tissue Bank, we found that living in areas of higher exposure to PM2.5 was associated with reduced involution of the breast as measured by higher TDLU count. Consistent with this, most of the assessed individual components of PM2.5 were non-linearly associated with higher TDLU count and clusters of individuals with levels of all PM2.5 components above or at study population mean levels had higher TDLU counts compared to the cluster with levels below the population mean. Reduced TDLU involution has been associated with an increased risk of breast cancer and may reflect higher amounts of at-risk epithelium. Therefore, our results suggest PM2.5 could impact the histologic characteristics of breast tissue and inform early carcinogenic mechanisms relating air pollution to breast cancer risk.
To our knowledge, this was the first study to examine the association between air pollution and TDLU involution measures. While there is little prior epidemiologic work with which to compare our results, biologic and indirectly related results support our overall finding that PM
2.5 is associated with reduced involution of the breast. Reproductive factors, estrogen metabolites, and circulating sex hormones have all been associated with TDLU count, which suggests that involution of the breast may partially occur through a hormone-related pathway [
5‐
7]. Estrogenic and antiestrogenic effects of airborne particles were reported in a study of human T47D-KBluc breast cancer cells [
29], particulate matter < 1 μm in diameter is composed of compounds that affect estrogen-regulated pathways in vivo [
30], and genotoxic effects of PM
2.5 have been reported in mice [
31]. Polycyclic aromatic hydrocarbons and metals, components of PM
2.5 that we were unable to evaluate here but may have contributed to our PM
2.5 total mass finding, have been shown to be estrogenic and induce mammary tumors in animal models [
32,
33]. Additionally, among breast cancer patients, higher levels of multiple pro-inflammatory markers were associated with reduced involution of healthy breast tissue [
34]. PM
2.5 has been shown to increase systemic inflammation, as measured by C-reactive protein [
35,
36].
Although earlier epidemiologic studies of PM
2.5 and breast cancer did not report evidence of elevated risk [
9,
11,
13,
14], two recent large cohort studies found PM
2.5 was associated with an increased risk of breast cancer [
15], and that composition of PM
2.5 and geographic variability were important [
10]. Further, in a Breast Cancer Surveillance Consortium study of 279,967 women, PM
2.5 was positively associated with mammographic breast density (i.e., heterogeneously dense compared to scattered fibroglandular breasts) [
16], although a smaller study reported no significant association [
37]. Mammographic density is one of the strongest known breast cancer risk factors [
18], and reduced TDLU involution has been associated with higher breast density [
19,
21]. One hypothesis for the relationship between mammographic density and breast cancer risk is that it may be due in part to the amount of at-risk epithelium such as that measured by TDLU involution [
19]. Therefore, our results suggest exposure to PM
2.5 may impact characteristics of healthy breast tissue, which could influence future breast cancer risk.
In addition to PM
2.5 and its components, we found that the gaseous pollutants, CO, NO, NO
2, and SO
2, were non-linearly associated with elevated TDLU count. CO, NO, and NO
2 are common traffic-related air pollutants, and NO and NO
2 are the pollutants that have been most consistently associated with an increased risk of breast cancer [
8]. While biological mechanisms linking NO and NO
2 to TDLU involution are not established, these pollutants may be proxies for other traffic-related air pollutants such as polycyclic aromatic hydrocarbons, which have demonstrated both antiestrogenic and estrogenic activity [
38].
PM
2.5 total mass was not consistently associated with TDLU span or acini count/TDLU. However, individual components of PM
2.5, except for SO
4, and gaseous pollutants were inversely associated with TDLU span but not associated with acini count/TDLU. Differences in the associations across measures may be because TDLU span and acini count/TDLU were only assessed among women who had > 0 TDLUs, so there was reduced statistical power for these analyses. The underlying biologic mechanisms and significance of the inverse associations generally observed in relation to TDLU span, in contrast to the positive associations in relation to TDLU count, are unclear. However, as suggested previously, there is a weak correlation between TDLU count and acini count/TDLU or TDLU span and associations for certain other factors also vary by TDLU measure [
5]. For example, parity and higher levels of circulating sex hormones were associated with TDLU counts but not TDLU span or acini/TDLU [
5,
6]. Compared to never use of hormone replacement therapy in postmenopausal women, current use was associated with TDLU span but not TDLU count or acini/TDLU while former use was associated with TDLU count and span but not acini/TDLU [
5]. Therefore, the different markers of involution may represent distinct processes or stages of involution (i.e., complete disappearance vs reduction in size) [
5,
7,
39] and more research is needed to understand how they may contribute to breast cancer etiology.
Our results are based on a large sample size of women who were demographically diverse and donated healthy breast tissue. Given the dearth of research on air pollutants in relation to breast tissue characteristics, a strength of this study was the range of pollutants considered. However, it is important to note that the correlations across certain pollutants are high and independent associations may be difficult to disentangle. Each of the gaseous pollutants, NO
2, CO, and SO
2, is distinct criteria pollutants with varying relative contribution from different sources and is a regulatory priority of the Environmental Protection Agency, so understanding their individual health effects is important. For the PM
2.5 components, we were able to leverage these high correlations in our
K-means approach to identify subgroups of women who had similar patterns of exposure. This was an important consideration because PM
2.5 is a heterogeneous mixture and studies have noted that health effects may vary by the composition of the PM
2.5 [
10,
40,
41]. Another strength of this study was the consideration of clusters of individuals based on distinct PM
2.5 profiles that had differential associations with TDLU involution. While our clusters generally separated individuals based on high, average, or low levels of all PM
2.5 components rather than varying combinations of the components, these findings supported our overall PM
2.5 results.
A small breast tissue core has been shown to be generally representative of involution throughout the breast [
42], but we cannot exclude the possibility of non-representative sampling of breast tissue. We adjusted for percent of fat on the slide, which is inversely related to TDLU count [
5], to partially account for this possibility. A strength of this study was the inclusion of multiple measures of TDLU involution in addition to a range of different air pollutants. However, this resulted in many statistical comparisons, and thus, it is possible that some findings may be due to chance. Given this, we focused the interpretation of our results on the magnitude of point estimates, precision of confidence intervals, and trends observed in the data. Women provided their residential address at the time of tissue donation, but we did not have additional information about residential history, such as how long they had lived there or information on past residences. Therefore, we could not evaluate past air pollution exposure. Further, concentrations at a 12 × 12km grid level linked to residences do not fully account for variations in an individual’s daily activities, such as where they work, that could impact their exposure. PM
2.5 total mass data came from the EPA’s Downscaler Model that combines monitoring data (direct ambient measurements, but sparse geographic coverage) with CMAQ data (modeled estimates at all 12 × 12km grids across the USA, but subject to calibration/accuracy of the modeling parameters) [
22]. While this hinges on both sources’ strengths to increase the accuracy of the exposure assessment, non-differential exposure measurement error is still likely. Further, the components of PM
2.5 and gaseous pollutants were available from CMAQ-modeled data alone and are less accurate than the PM
2.5 total mass estimates from the Downscaler Model. One study reported that while fused models outperformed raw CMAQ data, the CMAQ predictions of PM
2.5 and certain components were within recommended model performance criteria [
43]. While we examined five components of PM
2.5, information on other components of PM
2.5 that may be carcinogenic, such as trace metals and polycyclic aromatic hydrocarbons, was not available from CMAQ. Finally, women in the Komen Tissue Bank volunteered to donate healthy breast tissue and a majority lived in Indiana, which may limit the generalizability of our results.
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