The role of miRNA as non-invasive biomarkers has been proposed in different type of diseases including cancer. miRNA are considered ideal markers because of their high stability in extreme conditions such as low pH or high temperatures [
44]. Their presence in body fluids other than plasma and serum, such as urine [
45], saliva [
46] and pancreatic juice [
47] has been shown to have a solid diagnostic value [
48]. Independent studies have also established the value of whole blood miRNA profiling in early phases of cancer development [
26,
27,
49,
50]. In this study the relative level of a panel of miRNAs with oncogenic or tumor suppressor properties, in part also acting as immune system modulators, was measured in post-operative EBC patients and compared to healthy donors or within patient sub-groups. We showed that the levels of miR-19a, miR-21, miR-22 and miR-127 could significantly discriminate post-operative non-metastatic EBC patients from healthy donors before and/or after adjuvant chemotherapy and at 2 years follow up, indicating their potential diagnostic value. For both miR-19a and miR-21, we did not detect any difference between their relative levels measured before and after therapy, suggesting that changes in the expression profile were independent of treatment. On one hand, miR-21 was significantly downregulated at T0. This result was somehow surprising since miR-21 is usually showing higher levels in serum or plasma of BC patients. However, since we monitored the miRNA level in whole blood, a drastic post-operative decrease in miR-21 levels could indicate a deregulation of the immune and/or inflammatory process, possibly enhancing the neoplastic disease and promoting proliferation and migration [
35,
51]. In other words the relative upregulation of miR-21 in cancer cells could have been “diluted” by the higher number of PBMC characterized by an evident miR-21 downregulation. Further studies will be nevertheless necessary to confirm this hypothesis. miR-19a, on the other hand, showed significantly higher mean values before and after therapy. Since miR-19a expression is higher in activated lymphocytes [
17,
20,
52], we reasoned that a better prognosis as suggested by survival curve analysis in post-operative patients could have been influenced by a stronger early immune response. With regard to the tumor suppressor miR-127, we found no significant difference between EBC patients and healthy donors at the earlier time points, while a significant lower level was detected at 2-year follow-up. In addition, miR-127 levels measured repeatedly for the same patients decreased significantly from T0 to T2. miR-127 has been shown to be downregulated in BC tissue compared with corresponding healthy tissue and to correlate with an advanced clinical stage and metastasis development [
32]. The principal target of miR-127 is the proto-oncogene BCL6 [
31], which plays a direct role in survival, proliferation and differentiation of B lymphocytes [
53]. Consequently, lower levels of miR-127 in whole blood might indicate a parallel activation of B-cells at a later time point. Surprisingly, miR-127 gave a different result when the patients were stratified according to the presence or absence of CTCs at T0. In this case, miR-127 displayed to be upregulated in CTC positive patients compared to CTC negative patients. Other studies have already shown a correlation between CTCs and upregulation of miRNAs with tumor suppressor activity as is the case with the miR-200 family [
54]. Over-expression of miR-200 s supports the metastatic potential of CTCs inducing the mesenchymal-epithelial-transition (MET), an essential step for starting and developing new metastases. The fourth miRNA which showed significant differences between EBC patients and healthy controls was miR-22, a potent activator of EMT and cell proliferation. miR-22 was upregulated in patients after chemotherapy and at 2-year follow up, a finding which might suggest a selective therapeutic pressure favoring the development of aggressive chemotherapy-resistant tumor cells and possibly micrometastasis with mesenchymal characteristics. Finally, miR-20a and miR-200b, although always detectable, failed to significantly differentiate EBC patients from healthy controls or between patients at different time points, and therefore showed no prognostic value. However, both miRNAs showed some grade of correlation with the primary tumor’s characteristics. miR-20a could differentiate post-operative patients with less aggressive luminal A from those with more aggressive luminal B primary cancer, while miR-200b showed lower mean values in pN1-3 patients compared to pN0 patients. Further studies will be necessary to confirm these preliminary findings.
Although our results are promising, some limitations in this work must be mentioned and addressed in future experimental work. Sampling has been performed retrospectively and the size of the patient’s cohort should be expanded to unravel patients’ subgroups or treatment regimens for which miRNAs could prove to be potential predictive markers. In addition, SUCCESS A clinical trial protocol missed an early pre-operative time point for blood collection; therefore we cannot completely exclude that variations in miRNAs levels are a consequence of surgery after systemic immune response. Most of the patients (62.5%) received nevertheless breast conserving surgery, a less invasive and less stressing procedure with respect to mastectomy and blood samples were collected several days (between 23 and 173) post-surgery, a time frame long enough to assume that post-operative immune functions had reverted to physiological conditions [
27,
43] and that inflammation associated miRNAs were disappeared [
27,
55]. Furthermore, miRNAs have been isolated not only from PBMCs but from whole cellular blood fractions, including platelets, granulocytes and red blood cells. Further studies will be necessary to establish if contamination from cells other than PBMCs can negatively affect the analysis and should be eliminated in some way [
56]. In addition, due to the relative small cohort size, the data must be further validated with a larger number of cases allowing a more robust statistical testing. Finally the detection of CTCs was based on an immunostaining method and not on the FDA-cleared CellSearch® system, till now considered the gold standard for CTC isolation and enumeration [
57].