Joonas H. Kauppila

Depth of invasion, tumor budding, and worst pattern of invasion: Prognostic indicators in early-stage oral tongue cancer

Oral (mobile) tongue squamous cell carcinoma (SCC) is characterized by a highly variable prognosis in early‐stage disease (T1/T2 N0M0). The ability to classify early oral tongue SCCs into low‐risk and high‐risk categories would represent a major advancement in their management.

Toll-like receptor 5 (TLR5) expression is a novel predictive marker for recurrence and survival in squamous cell carcinoma of the tongue

Background:Toll-like receptor 5 (TLR5) is an immune receptor recognising bacterial flagellin. Activation of TLR5 results in cancer invasion and cytokine release. As certain bacteria have been linked to oral cancer, we wanted to study TLR5 expression in oral tongue squamous cell carcinoma (OTSCC).Methods:Samples from 119 patients with OTSCC were obtained, including 101 samples of adjacent normal lingual mucosa. The TLR5 histoscore (0–300) was assessed semiquantitatively by immunohistochemistry in a blinded manner.Results:Toll-like receptor 5 was expressed in 84 normal epithelia and 118 cancer samples. Expression of TLR5 was increased in cancer when compared with normal lingual epithelium (median histoscore 15 vs 135). In cancer, higher TLR5 was associated with age of >70 years at the time of diagnosis, female gender and disease recurrence. No association between TLR5 expression and tumour grade, stage or treatment was found. In multivariate analysis, TLR5 was an independent predictor of cancer mortality (hazard ratio (HR) 3.587, 95% confidence interval (CI) (1.632–7.882)) and disease recurrence (HR 4.455, 95% CI (2.168–9.158)).Conclusion:Toll-like receptor 5 has a previously undescribed role in the pathophysiology of OTSCC and might represent a link between bacteria and cancer. It could be a useful marker for predicting recurrence or survival of OTSCC patients.

Increased Toll‐like receptor 9 expression indicates adverse prognosis in oesophageal adenocarcinoma

Kauppila J H, Takala H, Selander K S, Lehenkari P P, Saarnio J & Karttunen T J
(2011) Histopathology59, 643–649

Toll-Like Receptor 9 Is a Novel Biomarker for Esophageal Squamous Cell Dysplasia and Squamous Cell Carcinoma Progression

Background: Stimulation of Toll-like receptor 9 (TLR9) has been linked to invasion in various cancer cells in vitro. We investigated TLR9 expression in normal, dysplastic and malignant esophageal squamous epithelium. Methods: TLR9 expression was analyzed by immunohistochemistry in 46 cases of esophageal squamous cell carcinoma, including 12 cases with adjacent squamous dysplasia and 24 cases with normal esophageal epithelium. TLR9 expression was compared with tumor grade, stage, proliferation, apoptosis and vascular density. Results: In normal esophageal squamous epithelium, TLR9 staining intensity decreased linearly from the basal layers to the superficial layers (p < 0.001). Strong TLR9 expression was detected across full thickness of high-grade dysplasia, the intensity clearly differing from the normal squamous epithelium and squamous cell carcinoma (p < 0.001). All squamous cell carcinomas exhibited TLR9 expression that was positively associated with a high grade (p < 0.05), the presence of lymph node metastases (p < 0.05) and previously undetected distant metastases (p < 0.05). Conclusions: Expression of TLR9 in the basal parts of normal esophageal epithelium suggests a role related to cell proliferation and differentation. TLR9 upregulation detected in dysplastic epithelium and in disseminated carcinomas indicates that this protein may serve as a novel marker for esophageal squamous dysplasia and carcinoma with metastatic potential.

A simple novel prognostic model for early stage oral tongue cancer.

The prognostication of patient outcome is one of the greatest challenges in the management of early stage oral tongue squamous cell carcinoma (OTSCC). This study introduces a simple histopathological model for the prognostication of survival in patients with early OTSCC. A total of 311 cases (from Finland and Brazil) with clinically evaluated early stage OTSCC (cT1-T2cN0cM0) were included in this multicentre retrospective study. Tumour budding (B) and depth of invasion (D) were scored on haematoxylin-eosin-stained cancer slides. The cut-off point for tumour budding was set at 5 buds (low <5; high ≥5) and for depth of invasion at 4mm (low <4mm; high ≥4mm). The scores of B and D were combined into one model: the BD predictive model. On multivariate analysis, a high risk score (BD score 2) correlated significantly with loco-regional recurrence (P=0.033) and death due to OTSCC (P<0.001) in early stage OTSCC. The new BD model is a promising prognostic tool to identify those patients with aggressive cases of early stage OTSCC who might benefit from multimodality treatment.

Short DNA sequences and bacterial DNA induce esophageal, gastric, and colorectal cancer cell invasion.

Toll‐like receptor 9 (TLR9) recognizes both bacterial and self‐DNA and it is abundantly expressed in the gastrointestinal tract. In this study, we investigated the influences of both bacterial DNA and specific short DNA sequences on TLR9‐mediated gastrointestinal cancer cell invasion. We assessed the effect of various DNA ligands on cellular invasion and on TLR9 and matrix metalloproteinase expression of three gastrointestinal cancer cell lines. DNA‐ligands described in this study include CpG‐ODN M362, 9‐mer (hairpin), human telomeric sequence h‐Tel22 G‐quadruplex, and bacterial DNAs from Escherichia coli and Helicobacter pylori. All of the DNAs studied were demonstrated to induce invasion in the studied cells. The DNA‐induced invasion was inhibited with a broad‐spectrum MMP inhibitor and partly also with chloroquine suggesting that it could be mediated via MMP activation, endosomal signaling, and TLR9. Interestingly, H. pylori DNA was shown to induce a more pronounced invasion in a gastric cancer cell line than in the other cell lines. Our results suggest that bacterial DNA as well as deoxynucleotides having stable secondary structures (i.e. hairpins or G‐quadruplex structures) may serve as endogenous, invasion‐inducing TLR9‐ligands and promote local progression and metastasis of cancers in the alimentary tract.

TLR4 and TLR9 are induced in oral lichen planus

BACKGROUND The role of Toll-like receptors (TLRs) has been elucidated in many human infectious, autoimmune and neoplastic diseases. Previously, TLR2 and TLR4 expression in oral lichen planus (OLP) was described. The aim of our study was to examine expression patterns of TLR4 and TLR9 in normal oral mucosa and OLP and describe the effect of topical tacrolimus treatment on the expression of TLR4 and TLR9 in OLP. METHODS Toll-like receptor 4 and TLR9 expression was analysed by immunohistochemistry in five samples of normal oral mucosa and 50 samples of OLP (31 representing clinically white and 19 clinically erythematous/erosive lesions). We evaluated also the effect of topical tacrolimus on TLR4 and TLR9 expression in a patient with OLP. RESULTS Toll-like receptor 4 and TLR9 expression was increased in OLP epithelium compared with normal epithelium (P < 0.001); no significant difference between the two clinical types of OLP was observed. TLR9 expression was strongest in the superficial layer of the epithelium (P < 0.001), while the expression of TLR4 was strongest in the basal layer (P < 0.001). Treatment of OLP lesions with topical tacrolimus resulted in clinical improvement but had no effect on TLR expression levels. CONCLUSIONS Toll-like receptor 4 and TLR9 are induced in OLP; our finding confirms the results of a previous study. TLR4 and TLR9 may play a part in the pathogenesis of OLP. Further studies are needed to dissect the definitive role of TLRs in OLP pathogenesis and progression and to determine the effect of tacrolimus on the function of TLRs.

Cathepsin K Is Present in Invasive Oral Tongue Squamous Cell Carcinoma In Vivo and In Vitro

Objectives Cathepsin K, a lysosomal cysteine protease, is expressed in the tumor microenvironment (TME) of skin carcinoma, but nothing is known about cathepsin K in oral tongue squamous cell carcinoma (OTSCC). Our aim was to describe the expression of cathepsin K in invasive OTSCC in vitro and in a series of clinical cancer specimens. Materials and Methods OTSCC invasion in vitro was studied using invasive HSC-3 tongue carcinoma cells in 3D organotypic models. In total, 121 mobile tongue OTSCCs and 10 lymph node metastases were analyzed for cathepsin K expression. The association between cathepsin K expression and clinicopathological factors was evaluated. Results Cysteine protease inhibitor E64 and cathepsin K silencing significantly (p<0.0001) reduced HSC-3 cell invasion in the 3D models. Cathepsin K was expressed in a majority of carcinoma and metastatic cells, but the expression pattern in carcinoma cells did not correlate with clinical parameters. Instead, the weak expression of cathepsin K in the invasive TME front correlated with increased overall recurrence (p<0.05), and in early-stage tumors this pattern predicted both cancer recurrence and cancer-specific mortality (p<0.05 and p<0.005, respectively). Conclusions Cathepsin K is expressed in OTSCC tissue in both carcinoma and TME cells. Although the diminished activity and expression in aggressive tongue HSC-3 cells reduced 3D invasion in vitro, the amount of cathepsin K in carcinoma cells was not associated with the outcome of cancer patients. Instead, cathepsin K in the invasive TME front seems to have a protective role in the complex progression of tongue cancer.

Low TLR9 expression defines an aggressive subtype of triple-negative breast cancer

Toll-like receptor-9 (TLR9) is a DNA receptor widely expressed in cancers. Although synthetic TLR9 ligands induce cancer cell invasion in vitro, the role of TLR9 in cancer pathophysiology is unclear. We discovered that low tumor TLR9 expression is associated with significantly shortened disease-specific survival in patients with triple negative but not with ER+ breast cancers. A likely mechanism of this clinical finding involves differential responses to hypoxia. Our pre-clinical studies indicate that while TLR9 expression is hypoxia-regulated, low TLR9 expression has different effects on triple negative and ER+ breast cancer invasion in hypoxia. Hypoxia-induced invasion is augmented by TLR9 siRNA in triple negative, but not in ER+ breast cancer cells. This is possibly due to differential TLR9-regulated TIMP-3 expression, which remains detectable in ER+ cells but disappears from triple-negative TLR9 siRNA cells in hypoxia. Our results demonstrate a novel role for this innate immunity receptor in cancer biology and suggest that TLR9 expression may be a novel marker for triple-negative breast cancer patients who are at a high risk of relapse. Furthermore, these results suggest that interventions or events, which induce hypoxia or down-regulate TLR9 expression in triple-negative breast cancer cells may actually induce their spread.

Toll-Like Receptors in Esophageal Cancer

Esophageal squamous cell carcinoma and esophageal adenocarcinoma are cancers of high mortality. EAC develops through Barrett’s esophagus (BE) and columnar dysplasia, preceded by gastro-esophageal reflux disease. The risk of esophageal squamous cell carcinoma is increased by smoking and alcohol consumption. New treatment options for esophageal cancer are desperately needed. Toll-like receptors (TLRs) play a central role in mammalian immunity and cancer. TLRs are activated by microbial components, such as lipopolysaccharide, flagellin, DNA, and RNA, as well as endogenous ligands, including heat-shock proteins and endogenous DNA. This review summarizes the studies on TLRs in esophageal squamous cell carcinoma and EAC. It has been shown that TLRs 1–10 are expressed in the normal esophagus. In esophageal squamous cell carcinoma, TLRs3, 4, 7, and 9 have been studied, showing associations to aggressive disease properties. In BE and EAC, only TLRs4, 5, and 9 have been studied. In the review, we discuss the implications of TLRs in esophageal cancer.