Risk modifiers for development of breast cancer are both genetic and environmental. The known germline genetic risk modifiers identified thus far are few (e.g., BRCA1 and BRCA2) and overall account for only 2–10% of all cases [1]–[3]. Environmental risk modifiers including but not limited to reproductive factors, endogenous and exogenous hormones, anthropometric characteristics, and certain lifestyle factors are known to impact a woman's risk for breast cancer development. Mechanisms underlying these effects are still largely unclear and under active investigation. A prior history of pregnancy has been noted as a consistent protective factor in breast cancer but the magnitude of this reduced risk is relatively modest [4], [5]. Thus, it is conceivable that there are undefined characteristics of prior pregnancies that could underlie why some parous women experience a reduced risk of breast cancer while others do not.

We and others have shown that a natural consequence of normal pregnancy is the acquisition and stable persistence of small numbers of fetal cells in the mother's body, a phenomena known as fetal microchimerism (FMc) [6]–[9]. FMc has been identified in the peripheral blood within subsets of immune cells [7], [8]. Although the specific effects or consequences of FMc remain to be demonstrated in healthy women, FMc has been implicated in “alloimmunity” in some classically autoimmune diseases, for example systemic sclerosis and thyroiditis [8], [10]–[12]. One possible role of FMc in healthy women might be the provision of allogeneic immune surveillance for (pre-)malignant cells. Allogeneic cells, particularly mismatched or haploidentical cells, in the context of hematopoietic transplantation have been found to provide potent immune surveillance for both hematologic and solid tumor malignancies in a process referred to as graft-versus-tumor [13]–[15]. By integrating the aforementioned observations of FMc in autoimmunity and transplantation immunobiology, we reasoned that allogeneic FMc might provide immune surveillance for breast cancer in parous women. In a prospective, pilot study we previously identified a decreased prevalence of FMc in the peripheral blood mononuclear cells of parous women with breast cancer compared to controls [16]. We demonstrate here in a larger case-control study that women with breast cancer are deficient both by presence and quantity of FMc in the peripheral blood compared to carefully matched controls. This finding supports the hypothesis that parous women with breast cancer fail to harbor a potential source of naturally acquired allogeneic immunity.

Baseline characteristics of breast cancer patients and control participants are shown in Table 1. The two groups did not differ with respect to most characteristics considered. Women diagnosed with breast cancer, however, were significantly older at the time they first gave birth (p = 0.03), an observation consistent with the original analysis of this case-control study [7]. Women with breast cancer were also more likely to have used oral contraception (p = 0.04) than control women.

Overall, FMc was found in 39 women, including 25 of 45 controls (56%) and 14 of 54 breast cancer patients (26%). Table 2 shows the prevalence of FMc and the association of FMc and risk of breast cancer. Compared to women who were FMc-negative, women harboring FMc were significantly less likely to have had breast cancer (OR = 0.20, 95% CI 0.08–0.53; p = 0.001, adjusting for age, number of children, birth of a son and history of miscarriage). The total number of cell equivalents tested for detection of FMc was higher in the controls than in the breast cancer cases (mean 8.2×104 (95% CI 7.2–9.2×10⁴) in controls versus 5.6×104 (95% CI 4.9–6.3×10⁴) in cases). This factor acted as a moderate confounder, slightly diminishing the effect of FMc in the final model (OR = 0.29, 95% CI 0.11–0.83; p = 0.02).

Concentrations of FMc in peripheral blood in women with and without breast cancer are depicted in Figure 1. Median concentrations were 2.0 versus 0 FMc cells per million maternal cells in healthy women and breast cancer patients, respectively. FMc concentrations were significantly higher in the controls than in the women with history of breast cancer (p = 0.01) in a model of the ranked values adjusted for age, number of children, birth of a son, history of miscarriage, oral contraceptive use, and total number of genomes tested.

Among cases, analyses were conducted to examine whether breast cancer specific features were associated with FMc prevalence (Table 3). The results suggest that women without FMc were more likely to have advanced disease features than women with FMc although none of these associations achieved statistical significance.

In this case-control study of breast cancer among parous women, women with breast cancer were more frequently deficient in FMc when compared to control women. Both overall prevalence and quantity of FMc were significantly different between parous women with and without breast cancer. Other known risk modifiers for breast cancer such as age at first pregnancy, history of oral contraceptive use, breast feeding, and smoking did not meaningfully confound the overall association of FMc absence with breast cancer. Furthermore, in case-only analyses, women with adverse breast cancer features (for example, higher stage or hormone receptor negativity) consistently had lower prevalence of FMc although no single characteristic reached statistical significance because power was limited for such analyses.

In prior pilot studies we similarly found a decreased prevalence of FMc in women with breast cancer [16]. When comparing the current data to our prior pilot study, the current data strengthens the overall inverse association of FMc and breast cancer. First, the study here considers more cases for analysis and permitted both examination of proportions and quantities of FMc when compared to the prior pilot study. The current findings replicate the principal finding of the pilot and add to it by demonstrating a difference in FMc concentrations between cases and controls. Second, because we utilized specimens from a matched case-control study, reproductive and other risk factors were more similar than in the prior pilot and allowed for multivariable modeling to adjust for any potential confounders.

When comparing the current data to prior studies of FMc, some additional differences merit discussion. FMc results could vary depending on the compartment of blood tested. Most prior studies of FMc examined Ficoll-purified peripheral blood mononuclear cells. To our knowledge, there are no studies of FMc in buffy coat. The overall prevalence and concentrations of FMc in controls described here were somewhat higher when compared to findings from Ficoll-purified cells. The key constituents more greatly represented in buffy coat compared to Ficoll-purified cells are large granular cells such as neutrophils or dendritic cells. Our data then indirectly suggest that FMc in healthy women might be represented in cells that are part of the innate immune response and/or in cells involved in antigen presentation. Second, in prior work, we have been able to employ an extensive panel of allelic quantitative PCR assays to catalog all potential sources of FMc regardless of sex of the child [19]. We did not have access to samples from children of probands in the current study for the purpose of polymorphism typing to identify informative assays for all potential FMc. On the other hand, control women and women with breast cancer were similar for having given birth to children of either sex and for total number of children.

The protection from breast cancer afforded by parity is incomplete. We provide data that suggest there is a failure to acquire or maintain allogeneic immune cells (FMc) transmitted from the fetus to the mother during pregnancy in women who develop breast cancer. These allogeneic cells have the potential to participate in immune surveillance for breast or other cancers as observed in clinical hematopoietic stem cell transplantation. Additional studies are required to identify the specific mechanisms by which FMc could participate in the protection from breast or other cancers. Knowledge gained from mechanistic studies may contribute to the design of allogeneic vaccine-based or adoptive immunotherapy for breast cancer. We acknowledge that replication of our findings in larger study populations is required because the sample size is small. Nonetheless, if our results are replicated, testing for FMc could be used to identify healthy women who might be at risk for developing breast cancer. Finally, the work presented here is of potential interest to evolutionary biologists as it provides additional support in human females for the concept of long-term fitness benefit through reproduction.