Detection of crown-like structures in breast adipose tissue and clinical outcomes among African-American and White women with breast cancer

Obesity is an established risk factor for postmenopausal breast cancer, notably hormone receptor-positive cancers [1, 2], and has been linked to a higher risk of recurrence and mortality regardless of menopausal and hormone receptor status [1, 3, 4]. Both systemic and local effects of obesity interact and contribute to the development and progression of breast cancer although mechanisms are not fully understood [1, 5]. Among postmenopausal women, adipose tissue is the main source of circulating estrogens, accounting for the link between obesity and increased breast cancer risk [6]. Obesity also results in chronic, subclinical inflammation characterized by elevated circulating pro-inflammatory mediators [7, 8] that have been linked to progression [9] and may increase risk via altered adipokine levels and insulin resistance [10]. Locally, adipocyte hypertrophy occurs in the breast of overweight/obese women that can lead to adipocyte death, resulting in macrophage recruitment and inflamed white adipose tissue (WAT) [11]. This adipocyte-macrophage interaction is characterized by the encirclement of necrotic adipocytes by macrophages in a crown-like pattern [11]. These crown-like structures in the breast adipose tissue (CLS-B) create a unique microenvironment rich in pro-inflammatory cytokines. Among women with breast cancer, CLS-B are associated with NF-κB activation, increased aromatase activity, and elevation of pro-inflammatory mediators, which may enhance invasiveness and metastatic potential [12‐16].

Previous epidemiologic studies have consistently demonstrated a strong, positive association between obesity, typically measured using body mass index (BMI), and CLS-B [16‐22], although CLS-B are also reportedly present in up to one third of lean women (BMI < 25 kg/m²). These data suggest that factors other than obesity contribute to the formation of CLS-B [23], but few studies have examined associations for other characteristics such as race/ethnicity, smoking status, and reproductive history. One previous study of 150 Black, non-Black Latinas, and Caucasian women with breast cancer found the highest numbers of CLS in the tumor tissue of Black patients and subsequently found CLS to be associated with lower survival; however, BMI was not available in this study [24]. Another analysis of CLS-B with survival included a low proportion of women with CLS-B and few clinical outcomes [25]. A case-only analysis of 127 patients who all developed metastatic breast cancer found that CLS-B was associated with shorter time to recurrence [19]. Thus, large epidemiologic studies with detailed clinical information in multi-racial populations are needed to assess determinants of CLS-B and their association with clinical outcomes.

Accordingly, we examined whether other patient factors, independent of BMI, are associated with detection of CLS-B in normal adjacent breast WAT tissue of cancer patients and whether CLS-B are associated with progression-free and overall survival in a large multi-racial study population. Given that African-American women have a higher prevalence of obesity than White women and higher rates of aggressive breast cancer subtypes and mortality, we were particularly focused on comparing the prevalence and clinical impact of CLS-B among African-American versus White women. Thus, we examined the detection of CLS-B in a study population with approximately equal numbers of African-American (n = 174) and White patients (n = 168) with stage I–III breast cancer diagnosed and treated at Emory University hospitals.

The present study provides additional evidence that higher BMI is strongly associated with the detection and density of CLS-B [16‐22] and provides new evidence that this association is similar in both White and African-American breast cancer patients. Race was not observed to be independently associated with detection or density of CLS-B. When we examined clinical outcomes over a median follow-up of 8 years, we found no difference in progression-free survival or overall survival by detection of CLS-B in unadjusted or adjusted models, contrary to two previous studies [19, 24]. However, estimates for these associations were imprecise, necessitating further research in larger study populations.

BMI is positively associated with CLS-B detection and density in numerous independent populations of breast cancer patients [16, 18, 20, 21, 24] and patients with benign breast disease [17]. The hypothesized biological mechanism relates higher BMI with adipocyte hypertrophy and the subsequent formation of CLS-B [5, 13]. Previous studies have found positive correlations between BMI, adipocyte size, and CLS-B that support this hypothesis [13, 18, 20]. Similarly, we found a positive correlation between BMI and CLS-B detection and density, and our finding of an inverse association between BMI and adipocytes per unit area is compatible with prior data linking obesity to adipocyte enlargement [18].

The association between BMI and CLS-B appears consistent across racial/ethnic groups as evidenced by the present study comparing African-American and White patients as well as previous studies in Taiwanese [20] and Hispanic/Latina [22] breast cancer patient populations. We did not find evidence that race is associated with the detection or density of CLS-B. The similar proportion of CLS-B+ cases among African-American (32%) and White women (29%) despite the higher prevalence of obesity among African-American women (52% vs 24%) was driven by the lower proportion of CLS-B+ cases at every BMI level compared to White women (Fig. 2e and Supplementary Table 1). For example, 43% of obese African-American women were CLS-B+ while 50% were CLS-B+ among obese White women. This may be a reflection of race-dependent differences in body composition since African-Americans are known to have lower overall adiposity and percent body fat than Whites with the same BMI [30]. However, BMI was still strongly associated with CLS-B among African-Americans and when adjusted for in analyses, any positive association between race and CLS-B attenuated considerably. Differences in BMI or other patient factors could explain the previous finding of an association between Black race and CLS-B [24].

Data on the association between other risk factors and detection or density of CLS-B are sparse. In the present study, age ≥ 60 years at diagnosis was related to increased detection of CLS-B. Previous studies have found that patients with CLS-B tend to be older than those without CLS-B [16, 21, 22, 25] although all but one study estimate included the null [25]. Menopausal status has also been identified as related to CLS-B [16]. Current smoking was also associated with increased detection of CLS-B in our study although estimates were imprecise and depended on the definition used for detection of CLS-B (i.e., ≥ 50%, ≥ 75%, or ≥ 90% adipocyte encirclement). One previous study in Hispanic/Latina breast cancer patients also observed a positive association with current smoking; however, this study and the present study had few current smokers (n = 8 in the study by Greenlee et al. [22] and n = 24 in the present study). We also observed an inverse association between parity and CLS-B detection, which has not been previously reported. Future studies are needed to confirm whether the risk factors identified in the present study persist in other, larger study populations—ideally with pre-malignant breast tissue.

We did not observe an association between CLS-B detection or density with overall or progression-free survival, contrary to two previous studies that found CLS-B to be associated with worse clinical outcomes among breast cancer patients [19, 24]. There are several differences in the methods for CLS-B evaluation and outcome assessment between our study and the previous ones that could account for the discrepant results. In the study by Koru-Sengul et al., CLS was assessed using three different macrophage markers for detection (CD206, CD40, and CD163), and the observed association with worse overall survival was limited to CLS detected by CD40, an M1 macrophage marker that has been linked to pro-inflammatory conditions [24]. It is possible that different types of macrophages (M1 vs M2) have different effects on clinical outcomes that could be obscured by using a pan-macrophage marker like CD68, which was employed in the present study. In further contrast to our study, CLS was analyzed using banked tumor tissue in the study by Koru-Sengul et al., and it was unclear how far the evaluated adipose tissue was from the tumor. We evaluated adipose tissue remote from the tumor to avoid local effects of biopsy or inflammatory responses directed against the cancer cells as opposed to localized adipose tissue specifically. In the study by Iyengar et al., CLS-B was detected by CD68 staining, which was the same as the present study, but assessed time to distant recurrence in a group of patients that all developed metastatic disease, limiting generalizability of their study findings [19]. In contrast, we evaluated patients unrestricted to outcome. Another key difference was that the previous study by Iyengar et al. used five tissue sections per case to determine the presence of CLS-B (41% CLS-B+) while the present study used one (30% CLS-B+), which could have resulted in more frequent misclassification of CLS-B presence in our study. We attempted to adjust for this potential misclassification using a probabilistic bias analysis with the results suggesting a potential bias towards the null in our main analysis but with a large amount of uncertainty—as demonstrated by the wide simulation interval (PFS: HR = 1.17, 95% SI 0.61–2.26). This estimate was still closer to the null than the estimate found in the study by Iyengar et al. (HR = 1.83, 95% CI = 1.07–3.13), so the discrepant results are likely due to other differences between the two studies [19]. For instance, our study population included equal proportions of African-American and White women while the Iyengar et al. study included predominantly White women. Heterogeneity in associations by race such that CLS-B increases risk in one group but decreases risk in the other (potentially driven by differences in tumor subtype by race) could result in an overall null association. Larger studies with enough statistical power to stratify by both race and tumor subtype are needed to examine these potential differences and further understand how CLS-B might influence breast cancer prognosis. Finally, it seems possible that if BMI and CLS-B are etiologically related to breast cancer development, then index event bias—a type of collider bias commonly referred to as the “obesity paradox”—could lead to artificial associations between CLS-B and prognosis [31]. Given the limitations of the previous studies and the uncertainty surrounding the estimates in the present study (i.e., wide 95% CIs), it remains unclear whether CLS-B negatively influences breast cancer prognosis and further research is needed.

The present study has several strengths and limitations. Strengths include the large number of well-characterized breast cancer patients with detailed follow-up information. Additionally, sampling equal numbers of African-American and White breast cancer patients allowed for comparisons by race. However, because our study population only consisted of breast cancer patients, our results do not provide evidence for how CLS-B may affect the risk and development of breast cancer. To date, there has only been one study that has examined the association between CLS-B and the risk of breast cancer that was among patients with benign breast disease and found a higher risk for patients with abundant CLS-B [17]. Further, our study was limited to mastectomy specimens to enable sampling of breast fat remote from the cancers, which may have resulted in some differences in patients versus those treated with lumpectomy. Additionally, the use of only one tissue specimen per case to assess CLS-B could have resulted in lower detection. This potential misclassification is expected to be non-differential since pathologists were blinded to patient characteristics and clinical outcomes at the time of CLS-B assessment. This non-differential misclassification is less likely to influence the analyses examining risk factors for CLS-B, supported by our simple bias analysis and the consistency of the strong association with BMI in our study with those from previous studies [18, 21]. Under-detection of CLS-B may have biased our results with OS and PFS towards the null; however, accounting for this potential misclassification using probabilistic bias analysis resulted in only weak to moderate bias-adjusted HRs for both (HR = 1.17 for PFS; HR = 1.29 for OS). Finally, while most previous studies have used CD68 for detection of CLS-B [16, 19‐21], it does not distinguish between types of macrophages (M1 vs M2) that could be associated with risk factors and prognosis differently.

This study further demonstrates the consistency and strength of association between BMI and detection and density of CLS-B in both African-American and White women diagnosed with breast cancer. Similar to previous studies, we did not find evidence that CLS-B was associated with tumor characteristics at diagnosis. However, in contrast to two previous investigations, we did not observe worse overall or progression-free survival among those with CLS-B present, raising questions related to differences in methodology in CLS-B assessment and whether CLS-B is in fact related to worse clinical outcomes.