Manuel Weber

Small oral squamous cell carcinomas with nodal lymphogenic metastasis show increased infiltration of M2 polarized macrophages – An immunohistochemical analysis

BACKGROUND In solid malignancies the influence of immunological parameters - especially of macrophages - on invasiveness, metastatic potential and prognosis has been shown. There are no studies quantitatively analysing the macrophage polarization in oral squamous cell carcinoma (oscc). The aim of this study was to correlate macrophage polarization in the epithelial and stromal compartment of oscc with histopathologic parameters. METHODS T1 and T2 oscc samples (n = 34) were used. Automated immunohistochemical staining detected CD68, CD11c, CD163 and MRC1 positive cells. All samples were completely digitalized using whole slide imaging and the number of stained cells per area was assessed quantitatively. RESULTS Primary tumours with lymphogenic metastasis (N+) showed a significantly (p < 0.05) increased count of CD68, CD11c, CD163 and MRC1 positive cells in the epithelial fraction compared to N0 tumours. The ratio of CD163 positive cells (M2 macrophages) to CD68 positive cells (M1 and M2 macrophages) was significantly (p < 0.05) increased in N+ tumours. CONCLUSION An increased macrophage infiltration and an increased M2 polarization in primary oral squamous cell carcinomas with lymphogenic metastasis was shown. Macrophages that migrated into the epithelial tumour fraction seem to be of special biological importance. The results indicate a central role of macrophages in the progression of oscc.

Increased malignancy of oral squamous cell carcinomas (oscc) is associated with macrophage polarization in regional lymph nodes – an immunohistochemical study

BackgroundIt is largely accepted that specific immunological parameters in solid malignancies are associated with patient’s prognosis. Recently a correlation of macrophage polarization with histomorphological parameters could also be shown in oral squamous cell carcinoma (oscc). The observed tumor derived peripheral immune tolerance could be associated with the macrophage polarization in regional tumor draining lymph nodes.So far there are no studies analyzing the macrophage polarization in cervical lymph nodes of oscc patients. In the present study we aimed to correlate macrophage polarization in different anatomical lymph node compartments of patients diagnosed with oscc with histopathologic parameters of the primary tumor (T-, N-, L-, V-, Pn-status, grading).MethodsTumor free (n = 37) and metastatic (n = 17) lymph nodes of T1 and T2 oscc patients were processed for immunohistochemistry to detect CD68, CD11c, CD163 and MRC1 positive cells. Samples were digitized using whole slide imaging and the number of cells expressing the aforementioned markers in the region of interest quantitatively analyzed.ResultsThe malignancy of the primary tumor (defined by T-, L-, Pn-status, grading) correlated with the lymph node macrophage polarization. L1 and Pn1 tumor cases displayed a significantly (p < 0.05) decreased M1 and increased M2 polarization in the sinus of the lymph nodes. G3 cases presented a significantly (p < 0.05) increased M2 polarization in the sinus compared to G2 cases. T2 tumors had significantly (p < 0.05) increased M2 polarization in the interfollicular zone of regional lymph nodes compared to T1 tumors. Metastatic and non-metastatic lymph nodes did not differ regarding their macrophage polarization.ConclusionsThe current study revealed for the first time an influence of oscc on the macrophage polarization in regional lymph nodes. Markers of malignant behavior in the primary tumor were associated with a shift of macrophage polarization in lymph nodes from the anti-tumoral M1 type to the tumor-promoting M2 type. As tumor free and metastatic lymph nodes did not differ in terms of their macrophage polarization pattern, there must be other factors influencing the location for lymph node metastasis formation.

Prognostic significance of macrophage polarization in early stage oral squamous cell carcinomas

BACKGROUND Polarization of tumor infiltrating macrophages is associated with the prognosis of solid malignancies and correlates with the occurrence of lymph node metastases in oral squamous cell carcinomas (oscc). Early stage (T1/T2, N0) oscc are characterized by a good prognosis and can be cured by surgery. The postoperative regime usually contains no adjuvant radio-/chemotherapy. The current pilot study was conducted to elucidate whether macrophage polarization in tumor resection specimens and diagnostic biopsies of early stage oscc is associated with tumor outcome. METHODS Patients with T1/T2, N0, and R0>5mm oscc without adjuvant therapy and 3-year follow-up after tumor resection were retrospectively selected. Tissue microarrays (TMA) containing diagnostic biopsies (n=17) and tumor resection specimens (n=17) were processed for immunohistochemistry in this pilot study to detect CD68-, CD11c-, CD163- and MRC1-positive macrophages. Samples were digitized, and the expression of macrophage markers was quantitatively analyzed. RESULTS High infiltration of M2 polarized macrophages correlated with poor tumor outcome in early stage (T1/T2, N0) oscc. This correlation was observed in tumor resection specimens, but was also observed in diagnostic biopsies. M2 macrophage polarization in biopsies - but not in tumor resection samples - correlated with high scores in tumor grading. CONCLUSION Macrophage polarization in early stage oscc is a potential prognostic marker for tumor outcome. The correlation of M2 polarized macrophages with tumor outcome can already be detected in the initial biopsies. Furthermore, M2 polarization of macrophages in biopsies is associated with an increased dedifferentiation.

Implant-based rehabilitation in oncology patients can be performed with high long-term success.

PURPOSE Radiotherapy and compromised vital bone and/or surrounding soft tissue can be a challenge to the successful osseointegration of dental implants. We evaluated the long-term results of dental implants in patients with oral cancer. MATERIALS AND METHODS To address the research purpose, we designed and implemented a retrospective cohort study that included patients with oral cancer who had received dental implants from 2003 to 2011. The data were collected from a clinical oncology database. The predictor variables included a set of heterogeneous variables grouped into logical sets of demographics, surgical treatment, dental rehabilitation, radiotherapy type, and tumor entity. The primary outcome variable was implant survival; the secondary outcome variable was peri-implantitis. The descriptive statistics, survival time analysis, Kaplan-Meier implant survival curves, and Cox hazard proportional modeling were computed. RESULTS The study sample included 59 patients with oral cancer (20 women [33.9%], 39 men [66.1%]; mean age at tumor diagnosis, 55 years), who had had 272 implants placed during the study period. The mean follow-up period was 30.9 months (range 3 to 82). Of the 272 implants, 269 (98.9%) and 264 (97.1%) had survived for 2 and 5 years, respectively. During the observation period, 10 implants were lost (3.7%). Of the implant failures, 82% occurred in transplanted bone (4 fibula flaps, 4 iliac crests, and 2 native mandibles). We observed peri-implantitis caused by insufficiently attached gingiva and bone loss in 182 of the implants (67%). The factors associated with implant failure were peri-implantitis, insufficient soft and hard tissue, muscle dysfunction, and xerostomia. CONCLUSIONS Implant-based rehabilitation in oncology patients can achieve a high long-term success rate, although risk factors such as impaired muscle function and a high frequency of peri-implantitis can affect healing.

Assessment of free microvascular flap perfusion by intraoperative fluorescence angiography in craniomaxillofacial surgery

Microsurgical tissue transfer represents a standard technique for reconstruction in craniomaxillofacial surgery. The transferred tissue is anastomosed to vessels of varying diameters and different physiological conditions. The aim of this study was to evaluate the blood flow in free flaps at their origin and compare this with the flow after reperfusion. In 24 patients undergoing microsurgical procedures (13 radial forearm free flaps (RFFF) and 11 parascapular/scapular free flaps (PSFF)), blood flow was evaluated by intraoperative fluorescence angiography after flap raising and again after reperfusion in the neck area (Flow800, Carl Zeiss AG, Oberkochen, Germany). Flow is expressed by the blood flow index (BFI), maximum intensity (MaxInt) and half-time to MaxInt (t1/2) and was measured in the flap pedicle itself, as well as in the supplying vessels. Following anastomosis of the free flaps in the head and neck area, both the arterial and the venous BFI and MaxInt significantly increased, whereas t1/2 decreased significantly. There was no significant difference in the perfusion parameters between RFFF and PSFF. Intraoperative fluorescence angiography is a reliable method for assessing the perfusion of free microvascular flaps. In the head and neck area, free flaps undergo a significant increase in perfusion but show no differences between varying flap types.

BRONJ-related jaw bone is associated with increased Dlx-5 and suppressed osteopontin—implication in the site-specific alteration of angiogenesis and bone turnover by bisphosphonates

ObjectivesSite-specific suppression of bone remodelling has been implicated in bisphosphonate-(BP)-related osteonecrosis of the jaws (BRONJ). Due to the origin of jaw bone from cranial neural crest, osseous differentiation is regulated specifically by the antagonizing BMP-2-downstream-transcription factors Msx-1 and Dlx-5. Osteopontin has been implicated in bone remodelling and angiogenesis. The osteoblast and osteoclast progenitor proliferation mediating Msx-1 has been demonstrated to be suppressed in BRONJ. In vitro BPs were shown to increase Dlx-5 and to suppress osteopontin expression. This study targeted Dlx-5 and osteopontin in BRONJ-related and BP-exposed jaw bone compared with healthy jaw bone samples at protein- and messenger RNA (mRNA) level, since increased Dlx-5 and suppressed osteopontin might account for impaired bone turnover in BRONJ.Materials and methodsFifteen BRONJ-exposed, 15 BP-exposed and 20 healthy jaw bone samples were processed for real-time reverse transcription polymerase chain reaction (RT-PCR) and for immunohistochemistry. Targeting Dlx-5, osteopontin and glyceraldehyde 3-phosphate dehydrogenase mRNA was extracted, quantified by the LabChip-method, followed by quantitative RT-PCR. For immunohistochemistry, an autostaining-based alkaline phosphatase antialkaline phosphatase (APAPP) staining kit was used. Semiquantitative assessment was performed measuring the ratio of stained cells/total number of cells (labelling index, Bonferroni adjustment).ResultsThe labelling index was significant decreased for osteopontin (p < 0.017) and significantly increased for Dlx-5 (p < 0.021) in BRONJ samples. In BRONJ specimens, a significant fivefold decrease in gene expression for osteopontin (p < 0.015) and a significant eightfold increase in Dlx-5 expression (p < 0.012) were found.ConclusionsBRONJ-related suppression of bone turnover is consistent with increased Dlx-5 expression and with suppression of osteopontin. The BP-related impaired BMP-2–Msx-1–Dlx-5 axis might explain the jaw bone specific alteration by BP.Clinical relevanceThe findings of this study help to explain the restriction of RONJ to craniofacial bones. BRONJ might serve as a model of disease elucidating the specific signal transduction of neural crest cell-derived bone structures in health and disease.

Expression of transforming growth factor beta 1-related signaling proteins in irradiated vessels

AimMicrovascular free tissue transfer is a standard method in head and neck reconstructive surgery. However, previous radiotherapy of the operative region is associated with an increased incidence in postoperative flap-related complications and complete flap loss. As transforming growth factor beta (TGF-β) 1 and galectin-3 are well known markers in the context of fibrosis and lectin-like oxidized low-density lipoprotein 1 (LOX-1) supports vascular atherosclerosis, the aim of this study was to evaluate the expression of TGF-β1 and related markers as well as LOX-1 in irradiated vessels.Materials and methodsTo evaluate the expression of galectin-3, Smad 2/3, TGF-β1, and LOX-1, 20 irradiated and 20 nonirradiated arterial vessels were used for immunohistochemical staining. We semiquantitatively assessed the ratio of stained cells/total number of cells (labeling index).ResultsExpression of galectin-3, Smad 2/3, and TGF-β1 was significantly increased in previously irradiated vessels compared with nonirradiated controls. Furthermore, LOX-1 was expressed significantly higher in irradiated compared with nonirradiated vessels.ConclusionFibrosis-related proteins like galectin-3, Smad 2/3, and TGF-β1 are upregulated after radiotherapy and support histopathological changes leading to vasculopathy of the irradiated vessels. Furthermore, postoperative complications in irradiated patients can be explained by increased endothelial dysfunction caused by LOX-1 in previously irradiated patients. Consequently, not only TGF-β1 but also galectin-3inhibitors may decrease complications after microsurgical tissue transfer.ZusammenfassungEinführungDer freie mikrovaskuläre Gewebetransfer gilt heute als fester Standard in der rekonstruktiven Kopf-Hals-Chirurgie. Es zeigte sich jedoch, dass im Falle einer stattgehabten Bestrahlung im Operationsgebiet mit einer erhöhten Rate an transplantatbezogenen Komplikationen gerechnet werden muss. Sowohl TGF-β1 als auch Galektin-3 sind bekannte Mediatoren in Bezug auf die Fibroseentstehung, wohingegen LOX-1 eine unterstützende Rolle bei der Entwicklung von Atherosklerose einnimmt. Das Ziel dieser Studie war es, zu untersuchen, wie sich die Expression von TGF-β1 und verwandten Fibrosemediatoren sowie LOX-1 im bestrahlten Gefäß verhält.Material und Methoden20 bestrahlte und 20 nichtbestrahlte Arterien wurden mittels immunhistochemischer Färbungen auf die Expression von Galektin-3, Smad2/3, TGF-β1 sowie LOX-1 hin untersucht. Semiquantitativ wurde das Verhältnis gefärbte Zellen pro Gesamtzellzahl (Färbungsindex) bestimmt.ErgebnisseDie Expression von Galektin-3, Smad2/3 sowie TGF-β1 war in bestrahlten Arterien signifikant höher als in nichtbestrahlten Gefäßen. LOX-1 wurde in vorbestrahlten Gefäßen ebenso signifikant höher exprimiert.KonklusionMediatoren der Fibroseentstehung wie Galektin-3, Smad2/3 und TGF-β1 sind in bestrahlten Gefäßen erhöht exprimiert und tragen mutmaßlich zu der Entstehung der radiogen induzierten Vaskulopathie bei. Des Weiteren können postoperative Komplikationen bei bestrahlen Patienten durch die verstärkte endotheliale Dysfunktion, unterstützt durch LOX-1, erklärt werden. Folglich könnten nicht nur TGF-β1- sondern auch Galektin-3-Inhibitoren einen minimierenden Effekt auf die Komplikationen nach mikrovaskulärem Gewebetransfer haben.

Macrophage polarization differs between apical granulomas, radicular cysts, and dentigerous cysts

ObjectivesApical periodontitis can appear clinically as apical granulomas or radicular cysts. There is evidence that immunologic factors are involved in the pathogenesis of both pathologies. In contrast to radicular cysts, the dentigerous cysts have a developmental origin. Macrophage polarization (M1 vs M2) is a main regulator of tissue homeostasis and differentiation. There are no studies comparing macrophage polarization in apical granulomas, radicular cysts, and dentigerous cysts.Materials and methodsForty-one apical granulomas, 23 radicular cysts, and 23 dentigerous cysts were analyzed in this study. A tissue microarray (TMA) of the 87 consecutive specimens was created, and CD68-, CD11c-, CD163-, and MRC1-positive macrophages were detected by immunohistochemical methods. TMAs were digitized, and the expression of macrophage markers was quantitatively assessed.ResultsRadicular cysts are characterized by M1 polarization of macrophages while apical granulomas show a significantly higher degree of M2 polarization. Dentigerous cysts have a significantly lower M1 polarization than both analyzed periapical lesions (apical granulomas and radicular cysts) and accordingly, a significantly higher M2 polarization than radicular cysts. Macrophage cell density in dentigerous cysts is significantly lower than in the periapical lesions.ConclusionsThe development of apical periodontitis towards apical granulomas or radicular cysts might be directed by macrophage polarization. Radicular cyst formation is associated with an increased M1 polarization of infiltrating macrophages. In contrast to radicular cysts, dentigerous cysts are characterized by a low macrophage infiltration and a high degree of M2 polarization, possibly reflecting their developmental rather than inflammatory origin.Clinical relevanceAs M1 polarization of macrophages is triggered by bacterial antigens, these results underline the need for sufficient bacterial clearance during endodontic treatment to prevent a possible M1 macrophage-derived stimulus for radicular cyst formation.

Macrophage and osteoclast polarization in bisphosphonate associated necrosis and osteoradionecrosis

PURPOSE Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is a complication of antiresorptive therapy with nitrogen-containing bisphosphonates (BP). With various suggestions as to pathogenesis, the etiology of BRONJ is not sufficiently understood. Osteoclasts and their precursors, that is, macrophages, are the main target cells of BP. BP can repolarize regeneration- and healing-associated M2 macrophages towards the tissue destructive M1-type. The current study aims to elucidate differences in macrophage and osteoclast polarization in BRONJ, osteoradionecrosis (ORN) and healthy control specimens. MATERIALS AND METHODS A total of 39 jaw bone samples (18 BRONJ, 8 ORN and 13 healthy controls) were processed for immunohistochemistry to detect CD68-, CD11c- and CD163-positive cells. Macrophages and osteoclasts were distinguished on the basis of morphological differences. Samples were digitized, and the macrophage and osteoclast cell counts were quantitatively analyzed. RESULTS In jaw bone affected by BRONJ, a significantly increased macrophage infiltration and M1 polarization of macrophages can be seen. The density of CD68-expressing osteoclasts is significantly increased in BRONJ specimens compared to ORN and to healthy controls. CONCLUSIONS A bisphosphonate-derived shift of macrophage polarization towards M1-polarized macrophages might impair bone tissue homeostasis and thus contribute to the pathogenesis of BRONJ. The observed increase in osteoclast density might be caused by BP-induced prolonged osteoclast survival.

Galectin 3 expression in primary oral squamous cell carcinomas

BackgroundImmunologic factors can promote the progression of oral squamous cell carcinomas (oscc). The phylogenetic highly conserved protein Galectin 3 (Gal3) contributes to cell differentiation and immune homeostasis. There is evidence that Gal3 is involved in the progression of oscc and influences the regulation of macrophage polarization. Macrophage polarization (M1 vs. M2) in solid malignancies like oscc contributes to tumor immune-escape. However, the relationship between macrophage polarization and Gal3 expression in oscc is not yet understood. The current study analyzes the association between histomorphologic parameters (T-, N-, L- Pn-status, grading) and Gal3 expression resp. the ratio between Gal3 expressing cells and CD68 positive macrophages in oscc specimens.MethodsPreoperative diagnostic biopsies (n = 26) and tumor resection specimens (n = 34) of T1/T2 oscc patients were immunohistochemically analyzed for Gal3 and CD68 expression. The number of Gal3 expressing cells and the ratio between CD68 and Gal3 expressing cells was quantitatively assessed.ResultsIn biopsy and tumor resection specimens, the number of Gal3 positive cells as well as the Gal3/CD68 ratio were significantly (p < 0.05) higher in T2 oscc compared to T1 cases. In biopsy specimens, a significantly (p < 0.05) increased Gal3 expression and Gal3/CD68 ratio was associated with the progression marker lymph vessel infiltration (L1). Tumor resection specimens of cases with lymph node metastases (N+) had a significantly (p < 0.05) increased Gal3 expression. Additionally, a high Gal3/CD68 ratio correlated significantly (p < 0.05) with higher grading (G3) in tumor resection specimens.ConclusionHigh Gal3 expression in oscc is associated with tumor size (T-status) and parameters of malignancy (N-, L-status, grading). Gal3 might contribute to M2 macrophage mediated local immune tolerance. Gal3 expression shows association with prognosis in oscc and represent a potential therapeutic target.