Triple-negative breast cancer (TNBC) is defined as a breast carcinoma without estrogen receptor (ER) and progesterone receptor (PR) immunoexpression, and with a lack of amplification of human epidermal growth factor receptor-2 (HER2). The disease accounts for ~15% of all breast carcinomas (1–3). TNBC shows biological aggressiveness and a higher recurrence rate, with no benefit from endocrine or HER2-targeted therapies (1–3). A number of studies have previously been performed to identify additional prognostic markers to better classify TNBC and stratify it into subgroups with different clinical courses (3–8).

Lactoferrin (LF), an iron binding 78- to 80-kDa glycoprotein usually present in mammalian milk, has been immunohistochemically revealed in numerous human neoplastic conditions of different sites (9–22). An immunopositive LF rate with a large variability, ranging from 7.5 to 42% of cases, has previously been identified in breast carcinoma (23–27); however, LF was more often observed in low-grade ER/PR-positive ductal carcinomas, confirming a decrease in LF immunostaining in less differentiated and more aggressive breast carcinomas (25–27). Moreover, it has been hypothesized that increased LF levels LF may be associated with reduced ERα and PR expression, and possibly reduced HER2 expression, and could therefore contribute to TNBC phenotype development (24). Consequently, taking into consideration the downregulation of ER, PR and HER2 at post-transcriptional levels in TNBC cell lines (24), the present study analyzed the immunohistochemical distribution of LF in a cohort of human surgical TNBC patients.

The routinely stained H&E sections exhibited good morphology, confirming the histopathological diagnosis of ductal invasive breast carcinoma (BC) in all cases. The immunoistochemical confirmed the diagnosis of TNBC.

Clinicopathological and immunohistochemical data for LF for the 65 available TNBC cases analyzed are shown in Table I. The follow-up time ranged from 3 to 112 months (mean, 54.5 months).

LF immunostaining was mainly localized in the cytoplasm of neoplastic elements and occasionally in the nucleus of the same cells; 17 cases (26.15%) exhibited an ID score of >2 and were therefore considered as positive for LF (Fig. 1). Statistical analyses showed that LF positivity was significantly associated with a low Ki-67 LI (<20%; P=0.040), the absence of recurrence (P=0.010) and alive status (P=0.020).

Univariate analysis showed that a high pT stage (P=0.040) and the absence of LF immunoexpression (P=0.030) were significantly associated with shorter DFS times (Table II). Multivariate analysis for DFS demonstrated that patient age, pT stage and Ki-67 LI were independent variables (Table I).

Univariate analysis for CSS showed that an age >70 years (P=0.007) and a high pT stage (P=0.030) were significant negative prognostic factors (Table III). The absence of LF immunoexpression was not associated with shorter CSS times, since statistical significance was not reached (P=0.060; Table III). At multivariate analysis, patient age (P=0.004), pT stage (P=0.008) and Ki-67 LI (P=0.040) emerged as independent variables (Table III).

It is well known that lactating breast tissue, as well as ductules and intralobular duct epithelial cells in normal and dysplastic tissue, strongly stain for LF (25); consequently, LF has been considered a potential marker for glandular or acinar differentiation, similar to its use in other malignancies (9,18,22). However, in breast cancer, the association between LF expression and clinical parameters is not well defined (24), even if it has been hypothesized that tumors with high LF expression retain a certain degree of physiological control over cell growth, which may explain a good prognosis (25).

In the present TNBC series, an LF immunostaining rate (ID score) of >2 was found in ~26.15% of cases, and was significantly associated with a low Ki-67 LI (<20%), the absence of recurrence and an alive status. Therefore, given the aggressive course of TNBC, the presence of LF-immunopositive cases may identify a more indolent subtype of the disease with peculiar clinical characteristics, including less aggressive biological behavior and a more favorable prognosis; by contrast, shorter DFS and CSS times were significantly associated with the absence of LF immunoexpression.

However, a similar capability to select a TNBC subgroup with low biological aggressiveness has been attributed to androgen (AR) expression (6,7); in particular, AR has been shown to be a favorable prognostic factor of DFS and overall survival, with significantly decreased recurrence and mortality risks (6,7,32). Furthermore, in a series of 105 TNBC patients with stage II or III disease treated with neoadjuvant chemotherapy based on docetaxel and doxorubicin, the prognostic and the predictive role of Ki-67 has been analyzed, identifying two distinct subgroups of TNBC with different Ki-67 expression, responses and prognoses (33). In the present study, Ki-67 emerged as an independent variable for DFS and CSS in TNBC, further highlighting its prognostic value. In addition, TNBC LF-positive cases always expressed lower levels of Ki-67, displaying a significant inverse association between Ki-67 LI and LF immunoreactivity, thus representing a further element to assess a favorable group of TNBC characterized by a low risk of recurrence and a better prognosis.

In conclusion, the results of the present study appear to be notable with regard to the field of TNBC, although further validation in large prospective studies is required to enable LF to be a promising biomarker. In fact, together with previous studies, the results of the present study show that the first immunohistochemical application of LF in TNBC appears to favor the selection of patients with a less aggressive behavior, particularly in combination with Ki-67 status and AR expression. However, additional investigations are also required with regard to the potential of LF in cancer treatment, due to its nutraceutical function and its ability to potentiate chemotherapy.