Microarchitecture of medication-related osteonecrosis of the jaw (MRONJ); a retrospective micro-CT and morphometric analysis.

Medication-related osteonecrosis of the jaw (MRONJ) is a severe side effect of antiresorptive (AR) drugs such as bisphosphonates (BP) and denosumab (Dmab). Although several risk factors are described, the etiology of MRONJ is still not fully elucidated. Bone-strengthening is the primary aim of antiresorptive therapy; however, overly increased bone mass and microcrack accumulation are also discussed in MRONJ etiologies. The aim of this study is to evaluate the microarchitecture of jaw bones with micro-computed tomography (micro-CT) in AR-treated patients with or without MRONJ. Human jaw bone samples of AR-treated patients were separated into 11 groups by AR treatment bisphosphonate (BP), denosumab (Dmab), both (M) and control groups. Subgroups were divided according to the clinical localization as AR-exposed vital jaw bone (BPexp, Dmabexp, Mexp), osteonecrosis-margin of a sequestrum (BPOmar, DmabOmar, MOmar) and osteonecrosis-sequestrum (BPOseq, DmabOseq, MOseq). Healthy jaw bone (CHB) and osteoporotic jaw bone (COP) represent control groups. Samples underwent retrospective micro-CT and morphometric analysis in representative units by bone volume fraction (BV/TV), bone surface density (BS/BV), trabecular thickness (Tr.Th.), trabecular number (Tr.N.), trabecular space (Tr.Sp.), Euler characteristic for bone connectivity, bone mineral density (BMD) and tissue mineral density (TMD). A total of 141 samples from 78 patients were analyzed. BV/TV of Mexp group (mean: 0.46 ± 0.27) was significantly higher than in the COP group (mean: 0.14 ± 0.05; p = 0.0053). Tr.Th. differed significantly between the BPexp group (mean: 0.32 ± 0.15) and the Mexp group (mean: 0.57 ± 0.20; p = 0.0452) as well as between the BPOseq group (mean: 0.25 ± 0.10) and the MOseq group (mean: 0.39 ± 0.18; p = 0.0417). Signs of trabecular thickening and unorganized trabecular microarchitecture from AR-exposed- to sequestrum groups, were analyzed in 3D reconstructions. However, BS/BV, Tr.N., and Tr.Sp. showed no significant differences. Euler characteristic of the BPOseq group (median: 7.46) doubled compared to that of the BPexp group (median: 14.97; p = 0.0064). Mineralization parameters BMD and TMD were similar in all groups. Findings show evidence of enhanced bone mass and suspect microarchitecture in some AR-treated jaw bone compared to osteoporotic jaw bone. Despite increased bone mass, some MRONJ samples showed decreased trabecular connectivity by Euler characteristic compared to AR-treated jaw bone. These samples may indicate extensive ossification and ineffective bone mass with superficially higher bone mass without existing or even reduced mechanical stability, indicated by connectivity loss. This result might also suggest a high risk to microcrack accumulation. At some point, possibly some kind of over-ossification could lead to under-nourishment and microarchitectural weakness, creating instability, subsequently increasing vulnerability to MRONJ.

Histologic analysis of medication-related osteonecrosis of the jaw compared with antiresorptive-exposed bone and other infectious, inflammatory, and necrotic jaw diseases.

OBJECTIVE: This study characterized histologic features of medication-related osteonecrosis of the jaw (MRONJ) through analysis of tissues from patients and healthy individuals. STUDY DESIGN: Bone biopsies were collected from various infectious, inflammatory, and necrotic jaw diseases. Samples were divided into bone exposed to bisphosphonates or denosumab, as well as bisphosphonate-related osteonecrosis of the jaw (BRONJ), denosumab-related osteonecrosis of the jaw (DRONJ), and mixed necrosis, enabling us to identify features of single agent necrosis without influence from previous therapies. Hematoxylin and eosin (H&E), receptor activator of nuclear factor κ-Β ligand (RANKL), tartrate-resistant acid phosphatase (TRAP), osteoprotegerin, toluidine blue, CD14, and CD68 staining and micro-computed tomography (micro-CT) analysis were performed. Groups were compared by using analysis of variance (ANOVA). RESULTS: In total, 156 bone samples were collected from 105 patients. MRONJ variants exhibited more infectious infiltration. Bisphosphonate (P < .001) and mixed necrosis (P = .002) demonstrated more RANKL- and TRAP-positive osteoclasts. Denosumab necrosis (P = .007), and bone exposed to bisphosphonates (P = .028) in combination with denosumab (P = .022) demonstrated significantly lower numbers of osteocytes per area. CD14 and CD68 positivity was increased for BRONJ (P = .008; P < .001, respectively). MRONJ variants exhibited the widest trabecular width and decreased medullary space to bone. No diminished vascular network in MRONJ samples was observed. CONCLUSIONS: Histologic features differ among MRONJ variants, with oversuppressed bone turnover, dysfunctional bone resorption, and a disturbed osteocyte network as potential mechanisms of pathogenesis.

Gingival fibroblasts and medication-related osteonecrosis of the jaw: Results by real-time and wound healing in vitro assays.

OBJECTIVE: This study investigated the effects of bisphosphonates and denosumab on human gingival fibroblasts (HGFs) that could influence inflammation, wound healing, and angiogenesis in medication-related osteonecrosis of the jaw (MRONJ). METHODS: A real-time in vitro assay was performed on HGFs with and without the addition of bacterial lipopolysaccharide and a mononuclear cell co-culture to observe the effects of zoledronate, ibandronate, alendronate, clodronate, denosumab, and combinations of zoledronate and denosumab at varied concentrations. A wound healing assay was performed, and gene and protein expression was analyzed for inflammatory, angiogenic, and osteoclastogenic cytokines and mediators including interleukin (IL)-1ß, IL-6, tumor necrosis factor alpha (TNFα), IL-8, vascular endothelial growth factor (VEGF), RANKL, and osteoprotegerin. RESULTS: Higher concentrations of antiresorptives resulted in impaired wound healing and HGF death, which also occurred without mechanical damage. These effects were increased with bacterial lipopolysaccharide and mononuclear cells. Increased levels of IL-1ß, TNFα, IL-8, VEGF, osteoprotegerin, and decreased levels of IL-6 were observed. CONCLUSIONS: Antiresorptive exposure was associated with HGF death and delayed wound healing, which could be attributed to an elevated inflammatory response and immune dysfunction contributing to MRONJ development. There was no evidence of anti-angiogenic effects. Our experiments present the first results of denosumab with HGFs.

Clinical course and therapeutic outcomes of operatively and non-operatively managed patients with denosumab-related osteonecrosis of the jaw (DRONJ).

PURPOSE: Details regarding risk factors, onset, and outcomes for denosumab-related osteonecrosis (DRONJ) are sparse. This study examines the clinical characteristics and operative and non-operative therapeutic outcomes in patients with DRONJ not previously exposed to other antiresorptives. METHODS: A retrospective medical record review was conducted, and data were collected, including clinical findings, management, healing outcomes, and radiologic, histologic, and micro-computed tomography (CT) analyses. RESULTS: Seventeen patients were treated with denosumab, with 14.1 ± 8.3 doses before DRONJ onset. The majority of lesions were observed at sites of dental prostheses (41%) and dental extractions (35%). Sixteen patients were managed non-operatively (10/16) or operatively (6/16) with either major (5/6) or minor surgery (1/6) and included in the follow-up analysis. Complete healing was significant in patients treated with major surgery (80%) compared to the non-operative group (20%; p < 0.035). Denosumab was discontinued in 60% of non-operative patients and major surgery patients with no effect on healing. Histologic findings of 4 patients analyzed exhibited a decreased number of osteocyte lacunae, and micro-CT of 3 patients scanned revealed trabecular thickening. CONCLUSION: DRONJ lesions occurred mostly at sites of prostheses sores after a mean of 14 doses of denosumab. Major surgery demonstrated more complete healing than non-operative management, and denosumab cessation did not improve healing outcomes.

Estrogen and Progesterone hormone receptor expression in oral cavity cancer

BACKGROUND: Recent studies have shown an increase in the incidence of oral squamous cell carcinoma (OSCC) in younger patients. The hypothesis that tumors could be hormonally induced during pregnancy or in young female patients without the well-known risk factors alcohol or tobacco abuse seems to be plausible. MATERIAL AND METHODS: Estrogen Receptor alpha (ERá) and Progesterone Receptor (PR) expression were analyzed in normal oral mucosa (n =5), oral precursor lesions (simple hyperplasia, n = 11; squamous intraepithelial neoplasia, SIN I-III, n = 35), and OSCC specimen. OSCCs were stratified in a young female (n = 7) study cohort and older patients (n = 46). In the young female study cohort three patients (n = 3/7) developed OSCC during or shortly after pregnancy. Breast cancer tissues were used as positive control for ERá and PR expression. RESULTS: ERá expression was found in four oral precursor lesions (squamous intraepithelial neoplasia, SIN I-III, n = 4/35, 11%) and in five OSCC specimen (n = 5/46, 11%). The five ERá positive OSCC samples were older male patients. All patients within the young female study cohort were negatively stained for both ERá and PR. CONCLUSIONS: ER expression could be regarded as a seldom risk factor for OSCC. PR expression seems to be not relevant for the development of OSCC

Standardized pretreatment inflammatory laboratory markers and calculated ratios in patients with oral squamous cell carcinoma.

Analyzing the inflammatory microenvironment has become an important issue in the management of oral squamous cell carcinoma (OSCC). Pretreatment C-reactive protein (CRP) levels, leucocytes, monocytes, lymphocytes, neutrophils, basophils, eosinophils, platelets, neutrophil-to-lymphocyte ratio (NLR), derived NLR (dNLR), lymphocyte-to-monocyte ratio (LMR), and platelet-to-lymphocyte ratio (PLR) derived from the peripheral blood were analyzed. Receiver operating characteristic (ROC) curves determined a cut-off value for each parameter in 146 patients with OSCC compared with 93 controls and the results were associated with clinicopathological characteristics. CRP expression of tumors was measured by immunohistochemistry. ROC analysis determined cut-off values for CRP levels, leucocytes, monocytes, lymphocytes, neutrophils, NLR, dNLR, LMR, PLR and showed significant differences between the OSCC and control group. Compared with single laboratory tests calculated ratios were superior in measuring sensitivity and specificity of OSCC disease. NLR was significant directly associated and correlated with PLR. LMR was significant inversely associated and correlated with NLR and PLR. Immunohistochemical analysis did not show CRP expression of OSCCs. This study highlights the first analysis for cut-off values of pretreatment single laboratory tests and calculated ratios, which are strongly needed for a follow-up of cancer patients. Additionally, the calculated baselines can be used as a goal for successful immunotherapies in the future. The links between NLR, LMR, and PLR might be helpful for the clinical course (monitoring) of cancer patients and have been first described for OSCC in this study. Taken together, analyzing these data provides an additional practical guideline of further postoperative OSCC management.

Effect of bisphosphonates on macrophagic THP-1 cell survival in bisphosphonate-related osteonecrosis of the jaw (BRONJ).

OBJECTIVES: Immune deficiency and bacterial infection have been suggested to play a role in the pathophysiology of bisphosphonate-related osteonecrosis of the jaw (BRONJ). Zoledronate was previously found to promote THP-1 cell death. To examine this hypothesis with all commonly prescribed bisphosphonates, we tested the effect of (nitrogen-containing) ibandronate, risedronate, alendronate, pamidronate, and (non-nitrogen-containing) clodronate on macrophagic THP-1 cells. STUDY DESIGN: Activated THP-1 cells were exposed to .5 to 50 µM of nitrogen-containing bisphosphonates and .5 to 500 µM of clodronate. Cell adherence and survival were assessed in vitro using the xCELLigence real-time monitoring system. Results were confirmed histologically and verified with Live/Dead staining. RESULTS: All bisphosphonates inhibited THP-1 cell adherence and survival dose and time dependently, significant for zoledronate, alendronate, pamidronate, and clodronate in high concentrations (50 µM and 500 µM; P < .05). Low concentrations (0.5 µM) of risedronate, alendronate, and pamidronate prolonged the inflexion points of THP-1 cell survival compared with controls (P < .05). THP-1 cells exhibited no cytomorphologic changes at all concentrations. CONCLUSIONS: Commonly prescribed bisphosphonates inhibit the survival of macrophagic THP-1 cells dose-dependently without altering morphology. This may suggest a local immune dysfunction reflective of individual bisphosphonate potency leading to the pathogenesis of BRONJ.

Immunophenotyping of patients with oral squamous cell carcinoma in peripheral blood and associated tumor tissue.

The immune system is important for elimination of cancer cells. Tumors including oral squamous cell carcinoma (OSCC) are capable of escaping detection by host immune cells through apoptotic depletion of tumor-infiltrating lymphocytes (TILs). Circulating peripheral blood lymphocytes (PBLs) and corresponding TILs of tumor specimen were evaluated before and after curative tumor resection (n = 30) compared with PBLs of controls (n = 87). PBLs were characterized for the total number of T cells (CD3(+)), T helper cells (Th, CD3(+)/CD4(+)), regulatory T cells (Treg, CD4(+)/CD25(+)/CD127(low)), cytotoxic T cells (Tc, CD3(+)/CD8(+)), activated T cells (CD3(+)/HLA-DR(+)), and natural killer (NK) cells (CD3(-)/CD16(+)/CD56(+)). In tumor tissue, the prevalence of CD3(+), CD4(+), and CD8(+) TILs was assessed using immunohistochemistry, whereas the incidence of apoptosis was assessed using terminal deoxynucleotidyl transferase deoxyuridinetriphosphate nick-end labeling (TUNEL) assay. In PBLs of pretreated OSCC patients, a highly significant decrease in total number of T cells (p = 0.0001), Th cells (p < 0.0001), Treg cells (p < 0.0001), Tc cells (p < 0.0001), and NK cells (p = 0.0037) were found compared with controls. Decreased PBLs of OSCC patients were correlated with decreased numbers of corresponding TILs, which were associated with increased detection of apoptosis in the tumor tissue. Compared with the controls, the total number of T cells remained unchanged after surgery but the total number of NK cells significantly increased. Standardized immunophenotyping of OSCC may help to identify patients likely to benefit from cancer immunotherapy strategies and/or chemoradiation. Finally, future attempts to enhance an effective tumor-reactive immune response by immunotherapy or vaccination should be made by promoting tumor-specific Th and/or Tc cell/NK cell responses.

Evaluation of a biomarker based blood test for monitoring surgical resection of oral squamous cell carcinomas.

INTRODUCTION: The potential use of determination of biomarkers in blood for the monitoring of surgical removal of oral squamous cell carcinomas (OSCC) was evaluated using the epitope detection in monocytes (EDIM) technology. MATERIALS AND METHODS: In tumor specimen, elevated Apo10 and transketolase-like 1 (TKTL1) expression was analyzed by immunohistochemistry. Apo10 and TKTL1 biomarkers have been used prospectively for EDIM blood test in patients with primary and/or recurrent OSCC (n = 92) before surgery and after curative tumor resection (n = 45). RESULTS: There were highly significant (p < 0.0001) correlations found between EDIM blood scores and the tissue expression of both biomarkers measured by immunohistochemistry (Apo10: n = 89/92, 97%; TKTL1: n = 90/92, 98%). EDIMApo10 and EDIM-TKTL1 scores were positive in 92% (EDIM-Apo10: n = 85/92) and 93% (EDIM-TKTL1: n = 86/92), respectively, in patients with OSCC before surgery. The combined score EDIM-Apo10/EDIM-TKTL1 increased significantly the detection rate of tumors to 97% (n = 89/92). After surgery, the EDIM-TKTL1 and EDIMApo10 scores significantly decreased in 75.6 and 86.7% of the patients (p < 0.0001), respectively, in the aftercare. CONCLUSIONS: The correlation of TKTL1 and Apo10 immunohistochemistry with the blood test results indicates that the EDIM blood test could serve as a non-invasive diagnostic tool (liquid biopsy) to assess surgical removal of OSCC by determination of two biomarkers. CLINICAL RELEVANCE: This is the first study that has been demonstrated a reliable and successful monitoring of OSCC cancer patients by a blood test. The specific and significant decrease of EDIM-TKTL1 and EDIM-Apo10 scores after surgery could serve as a new tool for monitoring surgical removal of OSCC.

Altered macrophagic THP-1 cell phagocytosis and migration in bisphosphonate-related osteonecrosis of the jaw (BRONJ).

OBJECTIVES: Local immune dysfunction via macrophages is a proposed aetiology of bisphosphonate-related osteonecrosis of the jaw (BRONJ). This study aimed to clarify the effects of various bisphosphonates on macrophage function using a THP-1 monocytic model to examine migration, phagocytosis, and fibrin structure. MATERIALS AND METHODS: THP-1 cell migration was measured in the presence and absence of zoledronate, ibandronate, risedronate, alendronate, pamidronate (0.5, 5 and 50 µM) and clodronate (125, 250 and 500 µM) using the real-time xCELLigence system. Phagocytosis and actin fibre assays were performed after 72 h with zoledronate, ibandronate, alendronate and clodronate. RESULTS: Time to maximum migration for THP-1 cells was significantly reduced (p < 0.05) for high dosages of zoledronate, ibandronate and alendronate compared to controls. All dosages of clodronate and a low dose of zoledronate exhibited prolonged migrations. Phagocytic capacity was significantly reduced in high dosages of all bisphosphonates and for 5 µM zoledronate and ibandronate (p < 0.05). Low bisphosphonate exposure was accompanied by overcharged phagosoms. Altered appearance in F-actin fibrin structure was observed in bisphosphonate-exposed cells. CONCLUSIONS: All bisphosphonates altered the migration of THP-1 cells dose-dependently. Low doses also prolonged migration and altered cell morphology. These findings support the idea of a disturbed local immune function of macrophages even in jaw bone exposed to low concentrations of bisphosphonate. CLINICAL RELEVANCE: These are the first real-time results for disrupted migration and function of macrophagic THP-1 cells in high doses. Low dosages also demonstrated altered macrophage phagocytosis and cell morphology, suggesting a disturbed local immune function in BRONJ pathogenesis.

Glutaminolysis and carcinogenesis of oral squamous cell carcinoma.

Glutaminolysis is a crucial factor for tumor metabolism in the carcinogenesis of several tumors but has not been clarified for oral squamous cell carcinoma (OSCC) yet. Expression of glutaminolysis-related solute carrier family 1, member 5 (SLC1A5)/neutral amino acid transporter (ASCT2), glutaminase (GLS), and glutamate dehydrogenase (GLDH) was analyzed in normal oral mucosa (n = 5), oral precursor lesions (simple hyperplasia, n = 11; squamous intraepithelial neoplasia, SIN I-III, n = 35), and OSCC specimen (n = 42) by immunohistochemistry. SLC1A5/ASCT2 and GLS were significantly overexpressed in the carcinogenesis of OSCC compared with normal tissue, while GLDH was weakly detected. Compared with SIN I-III SLC1A5/ASCT2 and GLS expression were significantly increased in OSCC. GLDH expression did not significantly differ from SIN I-III compared with OSCC. This study shows the first evidence of glutaminolysis-related SLC1A5/ASCT2, GLS, and GLDH expression in OSCC. The very weak GLDH expression indicates that glutamine metabolism is rather related to nucleotide or protein/hexosamine biosynthesis or to the function as an antioxidant (glutathione) than to energy production or generation of lactate through entering the tricarboxylic acid cycle. Overcoming glutaminolysis by targeting c-Myc oncogene (e.g. by natural compounds) and thereby cross-activation of mammalian target of rapamycin complex 1 or SLC1A5/ASCT2, GLS inhibitors may be a useful strategy to sensitize cancer cells to common OSCC cancer therapies.

Serum vitamin D levels of patients with oral squamous cell carcinoma (OSCC) and expression of vitamin D receptor in oral precancerous lesions and OSCC

BACKGROUND: Resistance to programmed cell death (apoptosis) is a crucial factor for the carcinogenesis of oral squamous cell carcinoma (OSCC). Vitamin D (calcitriol) may overcome apoptosis resistance in tumor cells of OSCC. Vitamin D receptor (VDR) expression in oral precancerous lesions of OSCC has not been analyzed and serum vitamin D level seems to be a predictor of cancer development. MATERIAL AND METHODS: Expression of VDR was analyzed in normal oral mucosa (n=5), oral precursor lesions(simple hyperplasia, n=11; squamous intraepithelial neoplasia, SIN I-III, n=35), and OSCC specimen (n=42) by immunohistochemistry (IHC). Moreover, serum vitamin D levels were measured by 25(OH)D3 (calcidiol) in patients with OSCC (n=42) and correlated with IHC results. RESULTS: Expression of VDR was significantly increased in precancerous and OSCC compared with normal tissue. Compared with SIN I-III lesions VDR expression significantly decreased in OSCC. Severe vitamin D deficiency was detected in our OSCC patient cohort but there was no significant correlation analyzed between serum vitamin D levels and corresponding immunohistochemically detected VDR expression in OSCC. CONCLUSIONS: Our survey provides the first evidence of VDR expression in precancerous lesions of OSCC. Apoptosis induction of VDR+ cells in oral precancerous lesions and OSCC by natural vitamin D or synthetic vitamin D compounds could be useful for chemoprevention. Moreover, systemically and/or locally applied, these compounds may act as sensitizers for apoptosis mediated by radio-, and chemotherapy treatment in OSCC

Serum vitamin D levels of patients with oral squamous cell carcinoma (OSCC) and expression of vitamin D receptor in oral precancerous lesions and OSCC.

BACKGROUND: Resistance to programmed cell death (apoptosis) is a crucial factor for the carcinogenesis of oral squamous cell carcinoma (OSCC). Vitamin D (calcitriol) may overcome apoptosis resistance in tumor cells of OSCC. Vitamin D receptor (VDR) expression in oral precancerous lesions of OSCC has not been analyzed and serum vitamin D level seems to be a predictor of cancer development. MATERIAL AND METHODS: Expression of VDR was analyzed in normal oral mucosa (n=5), oral precursor lesions (simple hyperplasia, n=11; squamous intraepithelial neoplasia, SIN I-III, n=35), and OSCC specimen (n=42) by immunohistochemistry (IHC). Moreover, serum vitamin D levels were measured by 25(OH)D3 (calcidiol) in patients with OSCC (n=42) and correlated with IHC results. RESULTS: Expression of VDR was significantly increased in precancerous and OSCC compared with normal tissue. Compared with SIN I-III lesions VDR expression significantly decreased in OSCC. Severe vitamin D deficiency was detected in our OSCC patient cohort but there was no significant correlation analyzed between serum vitamin D levels and corresponding immunohistochemically detected VDR expression in OSCC. CONCLUSIONS: Our survey provides the first evidence of VDR expression in precancerous lesions of OSCC. Apoptosis induction of VDR+ cells in oral precancerous lesions and OSCC by natural vitamin D or synthetic vitamin D compounds could be useful for chemoprevention. Moreover, systemically and/or locally applied, these compounds may act as sensitizers for apoptosis mediated by radio-, and chemotherapy treatment in OSCC.

Co-expression of CD44+/RANKL+ tumor cells in the carcinogenesis of oral squamous cell carcinoma.

Receptor activator of nuclear factor-kappa (RANK)/receptor activator of nuclear factor-kappa B ligand (RANKL) signaling helps putative cancer stem cells (CSC) to maintain their stemness. Expression of CD44 and RANKL was analyzed in oral squamous cell carcinoma specimen (n = 191). Moreover, RANKL expression was measured in cancer cell lines (BICR3, BICR56) by immunohistochemistry and western blot analysis. Scanned images were digitally analyzed using ImageJ and the immunomembrane plug-in. CD44 and RANKL expression on protein level was correlated with clinical characteristics and impact on survival. RANKL was co-labeled with CD44 in immunohistochemical and immunofluorescence double labeling experiments. Although high CD44+/RANKL+ co-expression was significantly associated with clinicopathological factors and worse survival, multivariate analysis did not demonstrate high CD44+/RANKL+ co-expression as independent prognostic factor. Immunohistochemical and immunofluorescence double labeling experiments revealed RANKL expression by CD44+ cancer cells. RANKL specificity was confirmed by western blot analysis. For the first time, this study provides evidence that RANKL expression in OSCC might be associated with disease recurrence and a cell compartment measured by CD44+/RANKL+ co-expression within the mucosal epithelial basal layer cells.

Apoptosis resistance-related ABCB5 and DNaseX (Apo10) expression in oral carcinogenesis.

BACKGROUND: Apoptosis resistance is a crucial factor for the carcinogenesis of oral squamous cell carcinoma (OSCC). METHODS: Expression of apoptosis resistance-related ATP-binding cassette (ABC) transporter ABCB5 [subfamily B (MDR/TAP) member 5] and DNaseX (Apo10) were analyzed in normal oral mucosa (n = 5), oral precursor lesions (simple hyperplasia, n = 11; squamous intraepithelial neoplasia, SIN I-III, n = 35), and OSCC specimen (n = 42) by immunohistochemistry. RESULTS: Expression of ABCB5 and Apo10 were significantly increased in the carcinogenesis of OSCC compared with normal tissue. Compared with SIN I-III, ABCB5 expression was significantly decreased in OSCC. Apo10 expression did not significantly differ from OSCC compared with SIN I-III. CONCLUSIONS: This study provides the first evidence of the expression of ABCB5 and Apo10 in the multi-step carcinogenesis of OSCC. Overcoming drug resistance of ABCB5+ and Apo10+ cells in precursor lesions and tumors by natural compounds may act as sensitizers for apoptosis or could be useful for chemoprevention.

Zoledronate but not denosumab suppresses macrophagic differentiation of THP-1 cells. An aetiologic model of bisphosphonate-related osteonecrosis of the jaw (BRONJ).

OBJECTIVES: Bisphosphonates and denosumab are antiresorptive drugs used for the treatment of osteoporosis and oncological tumors. A severe side effect is osteonecrosis of the jaw. Monocyte/macrophage dysfunction is considered to play a distinct role in osteonecrosis. THP-1 monocytic cells were used in this study to elucidate the influence of zoledronate and denosumab on phorbol-12-myrisate-13-acetate (PMA)-induced macrophage differentiation and function in real-time. MATERIALS AND METHODS: Macrophagic differentiation of the THP-1 suspension cells was measured by cell adherence in the presence or absence of different concentrations of zoledronate (0.5, 5, 50 µM) and denosumab (1, 10, 20, 40 µg/mL) using the real-time xCELLigence system. Additionally, a live/dead staining was performed by fluorescence microscopy. RESULTS: THP-1 cells demonstrated a regular initial PMA-induced differentiation to macrophages by live measurements of cell adherence and by an increase in CD68 surface expression as detected by flow cytometry. The addition of zoledronate led to cell detachment of the THP-1-derived macrophages in a dose-dependent manner in contrast to denosumab. Cell detachment was based on cell death as confirmed by live/dead staining, revealing elevated numbers of dead cells following addition of high zoledronate concentrations. However, denosumab did not deteriorate THP-1 cell viability. CONCLUSION: Our results demonstrate that zoledronate but not denosumab suppresses monocytic THP-1 cell viability after macrophagic differentiation dose-dependently. CLINICAL RELEVANCE: This is the first real-time study providing evidence for a dose-dependent immunosuppressive effect of zoledronate in contrast to denosumab on local macrophages.

Association of cancer metabolism-related proteins with oral carcinogenesis - indications for chemoprevention and metabolic sensitizing of oral squamous cell carcinoma?

BACKGROUND: Tumor metabolism is a crucial factor for the carcinogenesis of oral squamous cell carcinoma (OSCC). METHODS: Expression of IGF-R1, glycolysis-related proteins (GLUT-1, HK 2, PFK-1, LDHA, TKTL1), mitochondrial enzymes (SDHA, SDHB, ATP synthase) were analyzed in normal oral mucosa (n = 5), oral precursor lesions (simple hyperplasia, n = 11; squamous intraepithelial neoplasia, SIN I-III, n = 35), and OSCC specimen (n = 42) by immunohistochemistry and real-time polymerase chain reaction (qPCR) analysis in OSCC cell lines. Metabolism-related proteins were correlated with proliferation activity (Ki-67) and apoptotic properties (TUNEL assay) in OSCC. Specificity of antibodies was confirmed by western blotting in cancer cell lines. RESULTS: Expression of IGF-R1, glycolysis-related proteins (GLUT-1, HK 2, LDHA, TKTL1), and mitochondrial enzymes (SDHA, SDHB, ATP synthase) were significantly increased in the carcinogenesis of OSCC. Metabolic active regions of OSCC were strongly correlated with proliferating cancer (Ki-67+) cells without detection of apoptosis (TUNEL assay). CONCLUSIONS: This study provides the first evidence of the expression of IGF-R1, glycolysis-related proteins GLUT-1, HK 2, PFK-1, LDHA, and TKTL1, as well as mitochondrial enzymes SDHA, SDHB, and ATP synthase in the multi-step carcinogenesis of OSCC. Both, hypoxia-related glucose metabolism and mitochondrial oxidative phosphorylation characteristics are associated with the carcinogenesis of OSCC. Acidosis and OXPHOS may drive a metabolic shift towards the pentose phosphate pathway (PPP). Therefore, inhibition of the PPP, glycolysis, and targeted anti-mitochondrial therapies (ROS generation) by natural compounds or synthetic vitamin derivatives may act as sensitizer for apoptosis in cancer cells mediated by adjuvant therapies in OSCC.

GLUT-1(+)/TKTL1(+) coexpression predicts poor outcome in oral squamous cell carcinoma.

OBJECTIVE: Tumor hypoxia is a crucial negative prognostic factor associated with outcome of oral squamous cell carcinoma (OSCC). STUDY DESIGN: Expression of glucose transporter 1 (GLUT-1) (solute carrier family 2 [facilitated glucose transporter], member 1 [SLC2A1]) was analyzed in OSCC specimen (n = 161) and cancer cell lines by immunohistochemistry and Western blotting. GLUT-1 expression on protein level was correlated with transketolase-like 1 (TKTL1) expression, clinical characteristics, and effect on survival. Subgroup analysis was performed for GLUT-1/TKTL1 coexpression. RESULTS: GLUT-1 expression was significantly correlated with TKTL1 expression (P < .0001) and recurrence of the tumor (P = .001). Multivariate analysis did not find GLUT-1 expression to be an independent prognostic factor (P = .2478). GLUT-1(+)/TKTL1(+) subgroup showed the worst effect on survival compared with the GLUT-1(-)/TKTL1(-) subgroup (P = .0002). CONCLUSIONS: This study provides evidence that tumors linked with combined enhanced glucose uptake (GLUT-1(+)) and hypoxia-related glucose metabolism (TKTL1(+)) characteristics (GLUT-1(+)/TKTL1(+) coexpression) are associated with shorter survival in OSCC.

Immunohistochemical detection of Helicobacter pylori without association of TLR5 expression in oral squamous cell carcinoma.

BACKGROUND: Approximately 15% of human deaths from cancer are associated with chronic viral or bacterial infections. Helicobacter pylori (HP), a flagellated, Gram-negative, spiral, microaerophilic bacteria is considered to be the most common chronic bacterial infection in humans. Toll-like receptor 5 (TLR5) is involved in recognition of bacterial flagella and is thought to promote tumour growth through inflammation-dependent mechanisms in epithelial cells. METHODS: Expression of HP and TLR5 was analysed in OSCC specimen (n = 191) by immunohistochemistry. TLR5 expression specificity was conducted by Western blotting in cancer cell lines (BICR3, BICR56). TLR5-stained sections were scanned and digitally analysed using ImageJ and the immunomembrane plug-in. HP expression and TLR5 expression were associated with clinicopathological characteristics and impact on survival. RESULTS: Helicobacter pylori detection was significantly associated with recurrence of the tumour, whereas TLR5 expression was not. Multivariate analysis demonstrated HP expression as an independent prognostic factor (P = 0.0260). TLR5 specificity was confirmed by Western blot analysis. CONCLUSIONS: For the first time, this study provides evidence that immunohistochemically detected HP expression in OSCC is associated with reduced disease-free survival in a large patient cohort. Although TLR5 was not associated with any clinicopathological characteristics or impact on survival, investigation of the TLR family seems to be reasonable due to the possible existence of other pathogenic bacterial or viral compounds in oral cavity cancer.