Η TLR-4 και TLR-9 - εξαρτώμενη ανοσιακή απάντηση στο ακανθοκυτταρικό καρκίνωμα γλώσσας

Abstract

Objectives: Squamous cell carcinoma (SCC) is the most common malignant neoplasm of the oral cavity, most frequently affecting the tongue. SCC is considered to be an aggressive tumor with high rate of regional metastasis and a guarded prognosis. Elucidating the molecular mechanisms implicated in oral carcinogenesis including a better understanding of the interaction of tumor cells with their microenvironment and its implications for cancer development and growth, is a major research objective. Toll-like receptors (TLRs) are arguably the best studied members of the family known as Pattern Recognition Receptors (PRRs). TLRs recognize exogenous components of a wide variety of pathogens (Pathogens Associated Molecular Patterns; PAMPs) or endogenous molecules, released from damaged tissues (Damage Associated Molecular Patterns; DAMPs) regulating the innate and adaptive immunity. Due to these functions TLRs play a pivotal role in many types of cancer, either supporting tumor progression by upregulating of the NF-κB pathway or cancer inhibition by inducing type I interferons. Recent studies have implicated TLRs as important players in OSCC. The aim of our study was to investigate TLR4 and TLR9 functional role in Τongue Squamous Cell Carcinoma (TSCC) samples of variable tumor grade and HPV status. Materials and Methods: Fifty-eight formalin-fixed and paraffin-embedded biopsy specimens of TSCCs samples and five in situ carcinomas, corresponding to 29 males and 34 females (mean age 59.3 years, and age range of 26-83 years), were selected from the archives of the Biopsy Service of the Department of Oral Medicine and Pathology, University of Athens, Greece. Immunohistochemical expression of TLR4, TLR9, NF-κΒ(p65) and IFN-β was graded in a semi-quantitative manner, according to the intensity and percentage of positive tumor and inflammatory cells and correlated with the degree of differentiation (based on the WHO 2017 criteria), the IHC expression in the adjacent morphological normal mucosa and the HPV status (with the criteria of PCR positivity and p16 INK4aexpression in >70% of tumor cells). In addition, IHC expression of the molecules studied was correlated (only in invasive SCCs cases), with the five individual histopathologic parameters of modified Anneroth system (i.e.keratinization, nuclear pleomorphism, number of mitoses, pattern of invasion, and inflammation). IHC scores for TLR4, TLR9, NF-κB and INF-β were compared among different histopathologic categories (well, moderately and poorly differentiated TSSCs), separately for tumor cells and inflammatory cells with the application of Kruskal-Wallis and Dunn’s tests. Comparative analysis of the IHC scores between tumor cells and adjacent morphologically normal mucosa was performed with the application of Wilcoxon Signed Ranks test. The Spearman's correlation coefficient (rho) was calculated to measure the association of IHC expression levels between the different studied molecules, the HPV positivity status and the histopathologic parameters evaluated by the modified Anneroth system. Results: Histopathologically, 5 cases (7.9%) were classified as CIS, 9 cases (14.3%) as superficially invasive SCC, and 49 cases as fully invasive SCC, including 20 (31.7%) well differentiated SCC, 23 (35.6%) moderately differentiated SCC and 6 (9.5%) poorly differentiated SCC. For further analysis, samples were also re-grouped into 3 broader histopathologic categories, as follows: Group A: CIS and superficially invasive SCC (14 cases), Group B: well differentiated SCC (20 cases), and group C: moderately and poorly differentiated SCC (29 cases). Combining HPV DNA PCR and p16INK4a IHC results, HPV positivity was determined in 10 out of 63 samples (15.9%) and the most prevalent subtype was HPV 31 (in 6 cases, 60.0%), followed by HPV 70, 16, 35, 45, 40 and 68. A positive correlation between HPV positivity and increased pleomorphism (RHO=0.453, p=0.014) in group C (moderately and poorly differentiated carcinomas) was noticed. All molecules were expressed at different levels in both tumor and inflammatory cells in the vast majority of cases. The mean values of IHC scores for TLR4, NF-κΒ and IFN-β (but not TLR9) showed a statistical significant increase with progression towards higher histological grades (p<0.05) and all the four of them from the adjacent to cancerous mucosa (p<0.001). In the whole sample, as well as in groups A and C, the total score for TRL4 expression was positively correlated with the total scores of NF-κΒ score (RHO=0.422, p=0.001, RHO=0.711, p=0.004 and RHO=0.620, p=0.000, respectively). TLR4 total score was also positively correlated with IFN-β in the whole sample and group C (RHO=0.239, p=0.061 and RHO=0.577, p=0.001, respectively). TLR4 expression in tumor inflammatory cells showed a negative correlation (RHO = -0.419, p=0.003 for percentage score; RHO=-0.375, p=0.008 for intensity score; and RHO=-0.393, p=0.005 for total score) with the number of tumor cell mitoses. The percentage score of TLR9 expression in tumor inflammatory cells showed a negative correlation with the parameters of keratinization (RHO=-0.298, p=0.037), type of invasion (RHO=-0.309, p=0.031) and number of mitoses (RHO=-0.344, p=0.015). HPV presence was positively associated with TLR9 percentage score in the inflammatory cells (RHO=0.274, p=0.030) as well as the percentage, intensity and total score of NF-κB cytoplasmic expression in tumor cells of group A (RHO=0.847, p=0.00, for all three scores). Discussion-Conclusions: The study attempted to shed some light on the role of TLR4 and TLR9 in oral, especially mobile tongue, cancer. Elevated levels of TLR4 and TLR9 in tumor cells, compared to the adjacent, morphologically normal epithelium (as well as in fully invasive cases compared to CIS and SI SCC), suggest that these molecules may participate in oral carcinogenesis. Our results are in accordance with previous studies suggesting that upregulation of TLRs expression possibly triggers NF-κB activation in tumor cells, participating in oral mucosa neoplastic transformation and loss of differentiation. On the other hand, although TLR4 and TLR9 expression in tumor inflammatory cells is also upregulated, a negative correlation with tumor grade and more aggressive histopathologic features is detected thus supporting an anti-tumor activity, possibly through IFN-β production. Not with standing that HPV infection may play an important role only in a minority oforal cancer cases, the positive correlations of HPV positivity with TLR9 and NF-κΒ levels suggest that, in HPV-related OTSCCs, the virus may be recognized by TLR9 participating, possibly manipulating the activation of the immune response. Obviously, the intricacies of TLR signaling in oral cancer and its microenvironment, its correlation with microbial and viral influences and its effects on tumor promotionand/or inhibition need to be further explored. Especially, the clinical significance ofthese molecular events awaits further confirmation through large scale studies thatwill specifically assess their prognostic and predictive value and will test the usefulness of their manipulation for therapeutic purposes.

Εισαγωγή: Οι υποδοχείς τύπου Toll (TLRs) διαδραματίζουν σημαντικό ρόλο σε πολλούς τύπους καρκίνου, είτε προάγοντας την ανάπτυξη του όγκου μέσω της ενεργοποίησης της οδού NF-κΒ είτε αναπτύσσοντας αντινεοπλασματική δράση μέσω της παραγωγής ιντερφερονών τύπου Ι. Στην μελέτη, διερευνήθηκε ο λειτουργικός ρόλος των υποδοχέων TLR4 και TLR9 σε ακανθοκυτταρικά καρκινώματα γλώσσας διαφορετικού βαθμού διαφοροποίησης και HPV θετικότητας. Υλικά και Μέθοδοι: 59 μερικές βιοψίες ΑΚΓ και πέντε in situ καρκινώματα γλώσσας, που αντιστοιχούσαν σε 29 άρρενα και 34 θήλυ άτομα (μέση ηλικία 59,3 έτη και ηλικιακό εύρος 26-83 έτη) συμπεριελήφθησαν στη μελέτη. Η ανοσοïστοχημική έκφραση των μορίων TLR4, TLR9, NF-κΒ(ρ65) και IFN-β αξιολογήθηκε ως προς την ένταση και το ποσοστό θετικότητας της χρώσης στα κύτταρα των όγκων και στα φλεγμονώδη κύτταρα και συσχετίστηκε με ιστολογικές παραμέτρους των όγκων, την έκφραση των μορίων στον παρακείμενο των βλαβών μορφολογικά υγιή βλεννογόνο και με την παρουσία του ιού HPV (ανίχνευση HPV DNA με PCR και ανοσοϊστοχημική έκφραση του μορίου p16 INK4a σε ποσοστό > 70% των καρκινικών κυττάρων). Αποτελέσματα: Όλα τα μόρια εκφράστηκαν σε διαφορετικό βαθμό στη συντριπτική πλειονότητα των περιπτώσεων, τόσο στα κύτταρα των όγκων όσο και στα φλεγμονώδη κύτταρα. Οι μέσες τιμές του IHC score για τα μόρια TLR4, NF-κΒ και IFN-β (όχι για το TLR9) έδειξαν αύξηση με τη μετάβαση σε χαμηλότερου βαθμού ιστολογική διαφοροποίηση (p<0,05) και από το παρακείμενο στον καρκινικό βλεννογόνο (p<0,001). Η παρουσία HPV ανιχνεύθηκε στο 15,9% των δειγμάτων και συσχετίστηκε θετικά με την έκφραση του TLR9 στα φλεγμονώδη κύτταρα (RHO = 0,274, p = 0,030). Συμπέρασμα: Στατιστικά σημαντική έκφραση των μορίων TLR4 και TLR9 παρατηρήθηκε στο ΑΚΓ η οποία επηρεάζοντας την έκφραση σημαντικών κατιόντων μορίων όπως ο NF-κB και της IFN-β, πιθανότατα διαδραματίζει σημαντικό ρόλο στην καρκινογένεση του στόματος.