New evidence for neutrophil extracellular traps, neutrophil-related mediators and proinflammatory cytokines in human thyroid cancer: from the least aggressive to the most aggressive type

Thyroid cancer (TC) is one of the most common endocrine malignancies, representing the eighth most commonly diagnosed cancer worldwide; its occurrence has risen in the last decade. Chronic low-grade inflammation is related to tumorigenesis. This correlation has begun to be studied for over a decade. Until now, T lymphocytes were considered the prevalent and most important immune cells in TC patients. Emerging roles for neutrophils and their mediators in cancer-related inflammation have been described. Activated human neutrophils release several de novo synthesized and preformed mediators. Recently, it has been shown that activated human neutrophils release extracellular fibrillary networks, named neutrophil extracellular traps (NETs), constituted by nuclear elements (DNA and histones) and proteins from primary, secondary and tertiary granules [such as myeloperoxidase (MPO), pentraxin 3 (PTX3) and matrix metalloproteinase 9 (MMP-9)]. NETs seem to be central elements in cancer-related inflammation, and to take part into a more aggressive behavior by cancer cells. Here, we studied the possible roles of neutrophils and NETs as indicators of disease progression in TC patients, from healthy controls to the most aggressive TC, through intermediate severity forms. In particular, we assessed the expression of NETs biomarkers, dsDNA, nucleosomes, citrullinated histones H3 (CitH3) and MPO-DNA complexes, in our patients’ sera. Furthermore, we conducted a characterization of neutrophil modulating factors, by measuring in our patients’ sera a panel of neutrophil-related biomarkers (MPO, PTX3, MMP-9, GM-CSF and CXCL8) as well as of inflammatory cytokines (IL-6, CXCL8, CXCL10, CCL2, CXCL9, TNF-a, IFN-g, IL-17A, IL-1b, IL-2 and MIP-1b), in order to define their roles in predicting the progression of TC. Our results showed that NETs appear to be related to the malignancy and severity of TC. These finding, in addition to the levels of neutrophil-related mediators found to be elevated in TC compared to multinodular goiter (MNG) and healthy controls, confirm that neutrophilic inflammation may be involved in TC. We also demonstrated a positive correlation between NETs biomarkers and clinical-pathological features of TC patients, like male sex, advanced age and presence of metastatic disease at diagnosis. Moreover, we confirmed the known involvement of chronic inflammation in TC by finding increased serum levels of IL-6 and TNF-a in anaplastic TC (ATC) patients compared to dedifferentiated TC (DeDTC) patients, differentiated TC (DTC) patients and MNG patients, suggesting that they could be considered as the most specific biomarkers of malignancy among those evaluated. Furthermore, we found increased serum levels of CXCL9 in ATC patients and DeDTC patients compared to DTC patients, suggesting that it could characterize the most aggressive forms of TC. Finally, we tested the relationship between patient age and TC type, finding a higher mean age among ATC patients, thus confirming the higher prevalence of ATC in older patients. To conclude, increasing circulating levels of NETs biomarkers, neutrophil-related mediators and proinflammatory cytokines/chemokines could reflect the complex involvement of neutrophilic inflammation in TC. The results of this project aim to lay the foundations for further studies with the ultimate objective of the identification of new neutrophil-related biomarkers, in order to orientate the therapeutic choice and to optimize the patient follow-up.