To our knowledge, this is the first report that identifies good responders to DCV in combination with NAC related to CD8 expression in the needle core biopsy with the established 4% cut-off point. Moreover, this is the first time that biomarker changes in the tumor and its milieu are reported in RD in naïve BC patients treated with standard NAC ± active cell based-immunotherapy, by showing maintained CD4 levels and a trend to a rise in CD8 infiltration (only in TNBC). We have also appreciate a trend to a better outcome when TILs were higher in RD, with less relapse events in the VG. Clinical impact of these findings could be relevant to improve selection of: 1) patients that could benefit from the addition of DCV to NAC; and 2) the best maintenance therapy in TNBC subtypes with RD based on the expression of CD8 and CD4 markers to choose standard capecitabine [8, 25] versus immunotherapy. Modifications on the tumor niche that reflects immune activation after DCV helps to be open wide to less toxic and more specific immunotherapy.
Regarding TILs, TNBC patients reach higher levels than the luminal subtypes in our study, as described before [5]. This could be in part due to specific mutational signatures, copy number variations, stromal metagenes and clonal heterogeneity of TNBC subtype [26–28].
We have shown a non-significant increase in stromal TILs after combined therapies with NAC plus DCV in up to 66% of TNBC patients, but not in the luminal subtypes. Probably our results are not significant due to the small sample size, being one limitation, and conferring the study an exploratory role that should be validated in other cohorts of patients.
Increased TILS could illustrate a reinforcement of the immune niche produced by DCV in BC patients. It can be suggested that vaccines could produce an increase in CD8 T cells post-treatment in TNBC. Waks et al. found that luminal BC are less enriched in CD8 TILs than other BC subtypes and this cell population decreased after NAC [29]. Nowadays, there is not enough knowledge about how immunotherapy could change BC microenvironment. Results of the studies that have worked with paired samples (before and after conventional NAC and no immunotherapy) remain controversial, suggesting different roles of immune cell populations in carcinogenesis, response to therapies, tumor progression and a crosstalk among the tumor and the microenvironment. While some reports described a reduction of CD8 TILs in cancer milieu after NAC [29–32], others shown an increase on TILs count [6, 33, 34] or an inversion on the CD4/CD8 ratio [4]. Luen et al. described an increase in TILs level in 48% of the patients and a decreased in 47% of the patients in RD after NAC [35].
The input of immunotherapy in BC patients in the neoadjuvant scenario looks for a global improvement within the tumor (higher pCR), the niche (hottest tumors) and the systemic immune surveillance. Dendritic cell-based adjuvant immunotherapy has already shown a gain in CD8 T cells in peripheral blood in non-luminal BC with an encouraging PFS improvement, suggesting benefits in the systemic immunity [16]. Vaccination is more effective in the prevention of tumor growth, and probably the clinical advantage could be more relevant in patients with a small tumor burden and with a preserved immune system (naïve of therapies) than in large tumor burden patients, including metastatic scenario, and with an exhausted immune system. Thus, immune cell profile of primary versus metastases of BC patients is different [36, 37].
Current efforts have been made to standardize the quantification of TILs and to produce reliable results [38–41]. Some authors pointed out that the reproducibility of TILs evaluation improves when the categories are simplified to low versus high TILs, based on the concept of lymphocyte-predominant breast cancer phenotype [42] but the standard cut-off points need to be defined. The incorporation of digital analysis (ACIS III) provided a better quantification of the stromal TILs avoiding interobserver bias. Additionally, other aspect to be considered is the selection of the area to be evaluated and how many fields should be selected. Recent works support the fact that the average lymphocyte score from a single biopsy of a tumor is reasonably representative of the whole cancer [43]. Our results showed that a cut-off point of 4% CD8 in the diagnostic specimen could predict a better pathologic response when DCV are added to conventional NAC, with a lower relapse (not significant). As it can be observed in Figure 1, this cut-off could be considered a relatively high density of CD8 cells in the stromal tumor. This optical microscope immunohistochemical image could be used as a reference of what we consider as the minimal density of cells which correlates with response to vaccines.
Qingzhu et al., found in their meta-analysis that memory T lymphocyte infiltration of a tumor site could serve as a indicator for OS and DFS prediction in patients with malignant tumours [44]. Also, Yajima et al., find a relationship between high CD45RO (i.e memory T lymphocyte and marker of activated T cells) expression and a lower pathological stage in BC patients [45]. The importance of CD4 TILs is not clear in BC scenario. No association between CD4 and pCR was found in our study, although in the CG CD4 TILs significantly decreased in patients after NAC (p=0.04) as compared to those who received the experimental therapy which remained stable (p=0.24) (Table 2). However, most of the studies correlate CD4 or CD4/CD8 ratio with an increased pCR and better survival [34, 5], and this fact strengths the role of DCV.
We have not look for tertiary lymphoid structures in this study although they are seen occasionally in the samples. Its neogenesis is not specific for tumors and immune therapies applied in oncology, so they also appear in autoimmune and infectious diseases, trasplanted solid organs, inflammatory disorders as well as after preventive vaccines (eg; HPV).
Although more studies are needed to establish the benefit of DCV addition to chemotherapy, our results suggest that we are moving in the right direction. Identification of predictive and prognostic biomarkers to select patients that could benefit from the addition of immunotherapy to the standard systemic chemotherapy is key to develop more efficient therapeutic strategies [47]. The relationship between lymphocyte-predominant breast cancer patients in the case of the TNBC [48] and the pCR, PFS and OS is clearly established [49–51]. Nonetheless, an outstanding selection of good responders to immunotherapy is complex [52] and the study of biomarkers in RD after NAC plus immunotherapy as well as in peripheral blood is mandatory to improve this limitation.
Evaluation of tumor responses to immune strategies by imaging techniques has become tricky because changes in tumor burden need new response criteria based on special guidelines (iRECIST) [53]. In this way, biologic markers in the blood, the tumor and its milieu could contribute to a more specific information than imaging markers regarding patient selection for immune strategies in the early BC arena.
In conclusion, a 4% cut-off point of CD8 TILs in a TNBC subtype could help to establish which patients can benefit from DCV added to NAC. In the same way, a maintained expression of CD4 as well as an increased infiltration of stromal CD8 after DCV therapy could improve responses to further therapies, as seen in other solid tumors after relapse to IMT strategy when patients were treated with second line chemotherapy in the advanced scenario [6, 7]. A trend to a lower relapse in diagnostic biopsies enriched with TILs has been also shown. Our findings suggest that patients with TNBC especially benefit from the stimulation of the antitumor immune system by using DCV pulsed with tumor antigens. Deeper studies based on immune profiling genomic panels in the tumor and immune cell populations on peripheral blood are still ongoing within our patients and could help us to elucidate the biological behavior of BC as well as the benefits from adding immunotherapy to conventional NAC. Promising combined immune approaches potentiating immune system with DCV added to the blockade of immune checkpoints together with NAC should be tested in clinical trials in this selected population.