## 4 Discussion
In this regard, we found that only the patients (6/47) who underwent disease progression after adjuvant chemotherapy plus trastuzumab had TILs <25%. Moreover, neoadjuvant treatment failed to achieve a pathological complete response in all BC cases from our series (6/47); interestingly these latter cases exhibited low TILs. Previous studies have documented high TILs levels in aggressive breast cancer subtypes, and showed a possible relation between high TILs, Ki-67, ER and PR immunohistochemical supplemental expression [9; 24; 25]. Similarly, in the present study, we observed a correlation between TILs distribution, proliferative index (Ki-67) and hormone receptors. In detail, intermediate and high-levels of TILs were significantly related to a Ki-67 index <20%. We selected a 20% Ki-67 cutoff according to previous studies by other authors on TILs in HER-2 positive BC [19; 23]; moreover, a Ki-67 index greater than 20% is generally considered the watershed between luminal A and luminal B tumors [3; 4]. Regarding hormone receptors' expression, intermediate-high TILs were significantly related to BC cases showing nuclear staining for both estrogen and progesterone receptors. Finally, there are limited data regarding the concordance or the variability of immune profiles between CNBs and whole tumor sections from surgical procedures [26]. Some authors have reported immune-activation after CNB, with TILs being higher in SRS than in CNB [26]; other studies performed on mouse models suggested a biopsy-mediated development of an increasingly immunosuppressive tumor microenvironment [27]. Different hypotheses have been postulated in order to explain the possible reasons of TILs changes: (1) the CNB process as primary cause of immune-activation; (2) the concept of intratumor heterogeneity; (3) younger age and longer surgery time interval as indicators of the higher chance of TILs' increase after CNB [26; 27; 28]. In our study, we observed a high level of concordance between preoperative biopsies and surgical samples in TILs' distribution. In detail, almost perfect agreement was observed in the intermediate and high TILs groups. We can postulate that the reason for this high concordance rate could rely on the adequate bioptical sampling of the neoplastic tissue (at least four bioptic fragments from different points of the tumoral tissue, in almost the totality of our study cases). On the other hand, two cases were preoperatively classified as low TILs in the CNB evaluation but changed from a low immune status to an intermediate TILs status on surgical samples and experienced a worse breast cancer-free interval. In particular, in these non-concordant cases only 1-2 bioptic fragments were obtained in the preoperative setting. This TILs' change may be explained by: (1) the spatial heterogeneity of lymphocytic infiltrate or the regional segregation of immune cells in some tumors, which can be missed in bioptic cores; (2) the not completely adequate bioptical sampling; (3) a dysfunctional immune infiltrate. The bad prognosis observed in these two cases was quite different from the common view that TILs' increase is related to better survival (especially in TNBCs) but is similar to a recent study on HER2+ tumors in which TILs' heterogeneity was associated with poor prognosis [28].