Family History of Breast Cancer Predicts Breastmilk Protein Expression

Dear Editor:

Pregnancy-associated breast cancer (PABC) is traditionally defined as breast cancer (BCa) diagnosed during pregnancy or within 1 year postpartum, although it now appears clear that the influence of pregnancy on BCa risk lasts for a longer period postpartum. ¹ PABC tends to be aggressive, with worse outcomes than BCa overall. The worse outcome has been attributed to the pro-inflammatory, angiogenic environment of the breast present during involution, which is tumor promotional. ¹ The time of breast weaning, involution, and remodeling is thought to significantly influence future BCa risk. ²

A family history (FH) of BCa is known to increase BCa risk. ³ BCa in young women with PABC has a stronger genetic component ⁴ than in women with postmenopausal disease. How genetic factors such as FH influence risk in PABC is unknown. A recent report involving over 60,000 parous women observed that the association of BCa with breastfeeding and risk was modified by whether the women had an FH of BCa (p=0.03); only the group with FH had a protective effect from breastfeeding (hazard ratio 0.41; 95% confidence interval 0.22–0.75). ⁵ The authors proposed that BCa incidence may be higher among women who did not breastfeed because of disordered involution. ⁵ Whereas a coordinated process of apoptosis and remodeling occurs following physiologic weaning, ⁶ in the absence of breastfeeding or suppressive medications, the engorged breast may become inflamed, which increases risk.

We hypothesized that gene products (proteins) present in the breastmilk might provide insight into why those at increased BCa risk might gain more benefit from lactation. Kallikreins (KLKs), serine proteases implicated in endocrine malignancies, ⁷ are detectable in breastmilk, ⁸ as are transforming growth factor (TGF) β-1 and -2, ⁹ which mediate pro-apoptotic effects and increase in level during breast involution. ¹⁰ KLKs 1, 3, and 5–15 are dysregulated in BCa cells and/or serum from BCa patients, with some KLKs having cancer-promoting and others cancer-suppressive effects. ¹¹ Our strategy was to determine how protein biomarkers in breastmilk linked to inflammation and/or cancer are influenced by FH of BCa.

After subjects provided consent to participation in an Institutional Review Board–approved project, three milk samples—beginning of lactation, defined as within 10 days of the initiation of lactation (aka transitional [T] milk); 2 months after lactation started; and when the woman was weaning (W milk)—were requested from healthy women who had delivered a term infant. All participants were cancer free. None reported chronic obstructive lung disease; one had lupus. This was the first full-term pregnancy for 34 participants, the second for 25, and the third or greater for 12. Women were classified as having an FH if one or more first-degree relatives had developed BCa. Samples were immediately frozen after collection. Collected milk was thawed and spun down, the layers were separated, and the specimens were stored as previously described. ¹² Total protein content was determined, ⁸ and the concentrations of KLKs were measured in duplicate by immunoassay, ⁸ as were those of TGFβ-1 and β-2 (R&D Systems, Minneapolis, MN), following the manufacturer's instructions. After natural log transformation, the data were tested by longitudinal analysis using a linear mixed model with subject as a random effect. Values of p were adjusted for multiple tests with Holm's step-down procedure.

Seventy-two women provided one or more breastmilk samples: 71 T samples (nine with an FH), 70 2-month samples (nine with an FH), and 47 W samples (four with an FH). The mean age of participants was 28.3 years. Comparing W with T milk expression, KLK13 expression increased in women with an FH and decreased in women without an FH, which gave a significant difference in trend (p=0.0047) (Fig. 1). When the trend changes over all three time points were examined, expression of KLK5 (p<0.001), KLK14 (p=0.0025), TGFβ-1 (p=0.002), and TGFβ-2 (p<0.001) were significantly associated with FH.

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FIG. 1.

Trend in kallikrein 13 (KLK13) level in breastmilk from the start to the end of lactation (transitional milk) based on the presence (1) or absence (0) of a family history of breast cancer. From start to end of lactation, the KLK13 content increased in women with and decreased in women without a family history of breast cancer.

The findings of our pilot study provide preliminary insight into how FH might preferentially influence the beneficial effect of breastfeeding on BCa risk. KLK13 has been postulated to have anti-angiogenic properties ¹³ and is a favorable prognostic marker in women with BCa. ¹⁴ Although women with an FH started off with significantly (p<0.05) lower KLK13 levels, during weaning KLK13 values increased in women with an FH but decreased in other women. The difference in trend in KLK13 expression based on FH is consistent with the earlier mentioned cohort study in which the BCa-protective effect of breastfeeding was limited to those with an FH. ⁵

For the four proteins (KLK5, KLK14, TGFβ-1, and TGFβ-2) whose expression appears to be influenced by FH, the differential expression was noted primarily comparing 2-month with W breastmilk samples. For women with an FH, expression levels of the proteins declined or stayed similar throughout lactation, whereas for women without an FH protein expression increased with the onset of weaning and involution. KLK5 is down-regulated and KLK14 is up-regulated in BCa.^15,16 There is evidence that KLK5 and KLK14 activate the inactive precursor forms of other KLKs, ¹⁷ suggesting a possible regulatory mechanism of expression between these two KLKs. TGFβ isoforms are pleiotropic, with both cancer-suppressive and cancer-promotional properties. ¹⁰ The increase in their levels was less dramatic in women with an FH.

Clearly our observations are preliminary. We recognize that our sample size is limited and that problems exist in considering FH a marker of genetic susceptibility or risk because only a fraction of women with an FH will have a known BCa gene, whereas in other women the influence of lower-penetrance genes as well as environmental influences may be more important. ⁴ Moreover, we cannot say with certainty that the changes observed in KLK13 would put these women at a lower risk of future BCa because we do not have sufficient follow-up to address this. Nonetheless, we feel it is interesting that expression of the anti-angiogenic protein KLK13 was highest among women with an FH during the critical time of breastfeeding weaning and involution and lowest for women without, as was the observation of differential expression of KLK5, KLK14, TGFβ-1, and TGFβ-2 based on FH. Whether breastfeeding protects against BCa to a greater extent in women at greater risk and the role of KLKs in risk stratification are areas of investigation worthy of further study, for example, using an animal model to analyze tissue in the lactating mammary gland and milk in lactating women.