In this report, we demonstrated that, after adjusting for known breast cancer risk factors, high mitotic index in cultured blood lymphocytes was significantly associated with an increased risk of breast cancer. This result seems support the concept that cellular hyper- proliferation in response to mitogens is associated with an increased risk of breast cancer. Although the endogenous factors that contribute to PHA-induced mitotic index phenotype are likely complex and remain to be elucidated by future studies, our data provided some clues that reproductive or other hormonal exposures may partly contribute to the mitotic index phenotype.
Our data is not in agreement with a previous report by Wiltschke et al showing that PHA-induced lymphocyte proliferation was lower in breast cancer patients compared with that in healthy women controls.20
There are several limitations in this previous small study (90 cases and 60 controls). First, recruitment of the study subjects was not described and it is unknown what type of control subjects were included in the analysis. Second, the characteristics of the study population were not described, thus it is hard to evaluate the comparability of the case-control population. Third, other known reproductive and host factors were not considered in the analysis. It is also worth to note that the study by Wiltschke et al used purified mononuclear cells by Ficoll gradient centrifugation for lymphocyte culture, while the present study used whole blood for lymphocyte cultures. It is possible that lymphocyte growth as measured by mitotic index in our culture system is affected by circulating hormonal and other factors in the blood serum.
We found that mitotic index was positively associated with number of full-term pregnancies and age at first full-term pregnancy, suggesting exposure to very high level of reproductive hormones during pregnancy may increase PHA-induced mitotic index in blood lymphocytes. The ability of the ovarian hormones estrogen and progesterone to promote cell proliferation in the normal breast epithelium can explain key epidemiologic observations regarding reproductive history and breast cancer risk.25
However, whether the reproductive hormones influence the proliferative potential of blood lymphocytes is unknown, although estrogen receptors are present on blood lymphocytes.26
Pregnancy is associated with very high levels of estrogen and progesterone that induce both cell proliferation and differentiation and therefore pregnancy is related to a dual effect on breast cancer; a short term increase in risk for up to 10 years after full term pregnancy and long term decrease.27
The high levels of circulating reproductive hormones during pregnancy result in the differentiation of the terminal duct-lobular unit and confers a protective effect.28
Early age at pregnancy,29
and prolonged lactation are found to be protective against breast cancer.31
Additionally, it is well known that during pregnancy the maternal immune system and cytokine profile are modified in order to achieve immune tolerance towards paternal antigen expressed on fetal cells.32
It is therefore possible that the observed associations between reproductive factors and the PHA-induced mitotic index in blood lymphocytes are the result of pregnancy-related endocrine and immune-function changes.
We are surprised to find a positive correlation between PHA-induced mitotic index and age at menarche and an inverse correlation between PHAinduced mitotic index and BMI. Both early age at menarche and obesity at post-menopausal result in prolonged exposure to physiological level estrogens and progesterone. Prolonged exposure to estrogens and progesterone are well documented risk factors for breast cancer. Early age at menarche (less than 12 years of age) has been associated with a 10%–20% increase in breast cancer risk compared to late age (>14 years of age) at menarche.33
Obesity has a complex relationship with breast cancer that was modulated by menopausal status. In general, BMI has been found to be positively associated with breast cancer risk among postmenopausal women, whereas it is inversely associated with breast cancer risk among premenopausal women.34
Since adipose tissue is an important source of estrogens35
in postmenopausal women, it is expected that obese postmenopausal women have higher levels of endogenous estrogen than lean women. Our data seem to indicate that prolonged exposure to physiological levels of estrogens decreases PHA-induced mitotic index in blood lymphocytes, suggesting that the mitotic index phenotype might be a breast cancer risk factor independent of low level reproductive hormonal exposures.
We also found that the mitotic index was significantly lower in African American women than in white women, which is consistent with African American women having lower overall breast cancer incidence compared with white women.3
Lower breast cancer incidence in African American women can be partially explained by the differences in distributions of the two key reproductive risk factors, younger age at first full term pregnancy and parity.25
Approximately 50% of black women have their first child before age 20, whereas only 20% of white women have their first child at age <20.36,37
Also, a higher proportion of white women have one or two children and black women more often have three or more children.37
African American women are also less likely to breastfeed their children.38
In agreement with current literature we observed racial differences in reproductive factors and these could explain the differential effect of mitotic index as a marker of breast cancer risk. Given the fact that African American women tend to have their first and last child at an earlier age than white women and are less likely to breastfeed, the cumulative effect of reproductive hormones on lymphocyte proliferation during pregnancy would be shifted to an earlier age, thus may not be captured in our study population in which greater than 90% of the women were older than 40 years of age. The small number of African American cases in our study precluded a detailed analysis to understand racial differences in the association between mitotic index and breast cancer risk. Thus future larger studies are needed to characterize the relationship between PHA-induced mitotic index and breast cancer risk in African American women.
Given that this is a case-control study, a theoretical concern is that mitotic index in blood lymphocytes is affected by case status (reverse causality). To evaluate whether having breast cancer increases blood lymphocyte mitotic index, we examined if surgical removal of breast tumor affect the mitotic index among cases. We found that the mean mitotic index in blood samples collected before surgery (3.6%, n = 23) was similar to blood samples collected after surgery (3.6%, n = 89). We further examined the effect of time since surgery on the mitotic index in post-surgical cases. We observed that there were no significant changes in mitotic index between cases who’s blood was drawn within 30 days of post-surgery (3.5%, n = 32) and cases who’s blood was drawn between 30–60 days of post-surgery (3.8%, n = 34) and cases who’s blood was drawn between 60–180 days of post-surgery (3.2%, n = 23). There were no significant differences in mitotic index by stage of the disease (DCIS 3.9%, stage I–II 3.7% and stage III–IV 3.4%, P = 0.22) among cases. Our study is limited by its moderate sample size and do not have sufficient power for subgroup analyses. Thus, our results need to be replicated by large independent studies for more precise risk estimation. Other more relevant mitogens such as estrogens and interleukins should also be tested in future studies.
In summary, our study revealed that high PHA- induced mitotic index in blood lymphocytes was significantly associated with an increased risk of breast cancer. If confirmed by future studies, mitotic index of cultured blood lymphocytes may serve as an independent biomarker for breast cancer risk. Our data also suggested that this association may be modulated by menopausal status and BMI and its interactive effects with lifetime exposures to reproductive and other hormones on breast cancer risk warrants further investigation.