Background The role of different subtypes of tumour infiltrating lymphocytes (TILs) in breast ductal carcinoma in situ (DCIS) is still poorly defined. This study aimed to assess the prognostic significance of B and T lymphocytes and immune checkpoint proteins expression in DCIS. Methods A well characterised DCIS cohort ( n = 700) with long-term follow-up comprising pure DCIS ( n = 508) and DCIS mixed with invasive carcinoma (IBC; n = 192) were stained immunohistochemically for CD20, CD3, CD4, CD8, FOXP3, PD1 and PDL1. Copy number variation and TP53 mutation status were assessed in a subset of cases ( n = 58). Results CD3+ lymphocytes were the predominant cell subtype in the pure DCIS cohort, while FOXP3 showed the lowest levels. PDL1 expression was mainly seen in the stromal TILs. Higher abundance of TILs subtypes was associated with higher tumour grade, hormone receptor negativity and HER2 positivity. Mutant TP53 variants were associated with higher levels of stromal CD3+, CD4+ and FOXP3+ cells. DCIS coexisting with invasive carcinoma harboured denser stromal infiltrates of all immune cells and checkpoint proteins apart from CD4+ cells. Stromal PD1 was the most differentially expressed protein between DCIS and invasive carcinoma ( Z = 5.8, p < 0.0001). Dense TILs, stromal FOXP3 and PDL1 were poor prognostic factors for DCIS recurrence, while dense TILs were independently associated with poor outcome for all recurrences (HR = 7.0; p = 0.024), and invasive recurrence (HR = 2.1; p = 0.029). Conclusions Immunosuppressive proteins are potential markers for high risk DCIS and disease progression. Different stromal and intratumoural lymphocyte composition between pure DCIS, DCIS associated with IBC and invasive carcinoma play a potential role in their prognostic significance and related to the underlying genomic instability. Assessment of overall TILs provides a promising tool for evaluation of the DCIS immune microenvironment.

Legumain is a proteolytic enzyme that plays a role in the regulation of cell proliferation in invasive breast cancer. Studies evaluating its role in ductal carcinoma in situ (DCIS) are lacking. Here, we aimed to characterize legumain protein expression in DCIS and evaluate its prognostic significance. Legumain was assessed immunohistochemically in a tissue microarray of a well-characterized cohort of DCIS (n = 776 pure DCIS and n = 239 DCIS associated with invasive breast cancer (DCIS-mixed)). Legumain immunoreactivity was scored in tumor cells and surrounding stroma and related to clinicopathological parameters and patient outcome. High legumain expression was observed in 23% of pure DCIS and was associated with features of high-risk DCIS including higher nuclear grade, comedo necrosis, hormone receptor negativity, HER2 positivity, and higher proliferation index. Legumain expression was higher in DCIS associated with invasive breast cancer than in pure DCIS (p < 0.0001). In the DCIS-mixed cohort, the invasive component showed higher legumain expression than the DCIS component (p < 0.0001). Legumain was an independent predictor of shorter local recurrencefree interval for all recurrences (p = 0.0003) and for invasive recurrences (p = 0.002). When incorporated with other risk factors, legumain provided better patient risk stratification. High legumain expression is associated with poor prognosis in DCIS and could be a potential marker to predict DCIS progression to invasive disease.

Recent discovery showing the presence of microRNAs (miRNAs) in the circulation sparked interest in their use as potential biomarkers. Our previous studies showed the diagnostic potential of miR-451 as a serological marker for inflammatory breast cancer (IBC), miR-337-5p and miR-30b for non-inflammatory breast cancer (non-IBC). The aim of this study is to investigate the prognostic values of circulating miRNAs by comparing the amounts of 12 circulating miRNAs in the serum of IBC and non-IBC from Tunisian breast cancer patients, and by determinating whether correlated pairs of miRNAs could provide useful information in the diagnosis of IBC and non-IBC patients. TaqMan qPCR was performed to detect circulating expression of miRNAs in serum of 20 IBC, 20 non-IBC and 20 healthy controls. Nonparametric rank Spearman rho correlation coefficient was used to examine the prognostic value of miRNAs and to assess the correlation profile between miRNAs expression. Further, a large number of miRNAs were highly correlated (rho>0.5) in both patients groups and controls. Also, the correlations profiles were different between IBC, non-IBC and healthy controls indicating important changes in molecular pathways in cancer cells. Our results showed that miR-335 was significantly overexpressed in premenopausal non-IBC patients; miR-24 was significantly overexpressed in non-IBC postmenopausal patients. Patients with previous parity had higher serum of miR-342-5p levels than those without. Furthermore, patients with HER2+ IBC present lower serum levels of miR-15a than patients with HER2-disease. Together, these results underline the potential of miRNAs to function as diagnostic and prognostic markers for IBC and non-IBC, with links to the menopausal state, Her2 status and parity.