Joachim E. Zöller

Recurrent chromosomal imbalances detected in biopsy material from oral premalignant and malignant lesions by combined tissue microdissection, universal DNA amplification, and comparative genomic hybridization

Biopsies routinely performed for the histopathological diagnosis of oral epithelial lesions before treatment were screened for chromosomal imbalances by comparative genomic hybridization. Comparative genomic hybridization was performed on 12 oral premalignant lesions (OPLs; dysplasias and carcinomas in situ) and 14 oral squamous cell carcinomas (OSCCs). Eight biopsies displayed areas of different histopathological appearance, so that OPLs and OSCCs from the same patient were analyzed. To avoid contamination with nonneoplastic cells, defined cell populations were isolated by micromanipulation with a glass needle. Before comparative genomic hybridization analysis, universal DNA amplification was performed using the DOP-polymerase chain reaction protocol. In the 14 OSCCs examined, the average number of chromosomal imbalances was significantly higher than in the 12 OPLs (mean +/- SEM: 11.9 +/- 1.9 versus 3.2 +/- 1.2; P = 0.003). The DNA copy number changes identified in more than one OPL were gains on 8q (3 of 12) and 16p (2 of 12), as well as losses on 3p (5 of 12); 5q (4 of 12); 13q (3 of 12); and 4q, 8p, and 9p (2 of 12 each). In more than 30% of OSCCs, gains of chromosomal material were identified on 20q (8 of 14); 8q, 11q, 22q (7 of 14 each); 3q, 15q, and 17p (6 of 14 each); and 14q, 17q, and 20p (5 of 14 each), and losses were identified on 3p and 4q (9 of 14 each), 5q (7 of 14), 13q (6 of 14), and 2q and 9p (5 of 14 each). These results were validated by positive and negative control comparative genomic hybridization experiments and microsatellite analysis for the detection of allelic loss. The vast majority of genomic alterations found in OPLs were again identified in OSCCs from the same biopsy, supporting the hypothesis that multiple lesions in the same patient are clonally related. In summary, we show that comprehensive information on the genomic alterations in oral epithelial lesions can be obtained from small biopsies. Such data may identify prognostic indicators that could eventually assist in designing therapeutic strategies.

Impact of podoplanin expression in oral squamous cell carcinoma: clinical and histopathologic correlations

Cervical lymph node metastases reduce the overall survival of patients with oral squamous cell carcinoma (OSCC) and require a neck dissection. However, elective management of a clinical N0 neck remains a controversial topic, as there are no reliable factors available predicting cervical lymph node metastases. Recent studies suggest an impact of podoplanin expression on metastatic spread to the cervical lymph nodes. Our aim was to investigate the influence of podoplanin expression on prognosis and metastatic lymphatic spread. In our retrospective study, podoplanin expression was examined in a set of 80 patients with OSCC by immunhistochemistry. We analysed associations between the level of podoplanin expression and various clinicopathologic parameters. In 67 patients (84%), podoplanin was expressed on the tumour cells. Nineteen patients (24%) showed high levels of expression. The 5-year overall survival (31%) for patients with high levels of podoplanin expression was significantly lower (p < 0.001) than for patients with low and moderate expression of podoplanin (93% and 65%, respectively). There was an association between podoplanin expression and the frequency of cervical lymph node metastases. Cervical lymph node metastases were found in 79% of the patients with high podoplanin expression, while patients with weak podoplanin expression had metastases in only 22% (p < 0.001). None of the 13 patients without podoplanin expression had cervical lymph node metastases. We concluded that podoplanin is expressed frequently in OSCC and that podoplanin expression correlates with cervical lymph node metastases and clinical outcome.

EXPRESSION OF CD44 SPLICE VARIANTS IN SQUAMOUS EPITHELIA AND SQUAMOUS CELL CARCINOMAS OF THE HEAD AND NECK

Splice variants of the adhesion molecule CD44 have been described as essential for the lymphatic spread of rat tumour cells and are claimed to be involved in the metastatic spread of several human tumours. Immunohistochemistry has been used to analyse the expression pattern of CD44 standard (CD44s) and variant (CD44v) isoforms in normal and dysplastic squamous epithelia, as well as in primary and metastatic squamous cell carcinomas (SCCs), which spread predominantly by way of the lymphatic system. Frozen sections of squamous epithelia and of squamous cell carcinomas were stained with a panel of monoclonal antibodies recognizing epitopes of CD44s as well as of the variant exons v5, v6, v7, v7–v8, and v10. The stratum basale and stratum suprabasale of squamous epithelia stained with all antibodies; the stratum spinosum stained with anti‐CD44v5, anti‐CD44v6, anti‐CD44v7–8 and anti‐CD44v10; the lower layers of the stratum corneum stained with anti‐CD44v5. This expression profile was seen in epithelia of the lip, the tongue, the gingiva, the hard palate, the floor of the mouth, the buccal mucosa, and the pharynx. The same pattern of expression was also noted in dysplastic epithelia, but expression of the variant exons v7, v8, and v10 was significantly downregulated in primary squamous cell carcinomas and was not detected at all in the majority of metastasis‐derived specimens. Expression of CD44v5 and CD44v6, on the other hand, was mainly unaltered. Thus, epithelial cell layers representing different stages of differentiation express distinct sets of CD44 variant isoforms, where especially exons v8–v10 might be required for the maintenance of the structural integrity of squamous epithelium. Downregulation of these exons on tumour cells could indicate that they are irrelevant for tumour progression or may even hamper infiltration of surrounding tissue or of lymphatics.

Growth and Proliferation of Human Osteoblasts on Different Bone Graft Substitutes An In Vitro Study

The purpose of this study was to investigate the effect of different bone graft substitutes onto the growth and proliferation pattern of bone cells derived from human iliac cancellous bone. Five different bone graft materials were used to investigate the effect on the proliferation of osteoblasts in vitro: phytogene hydroxyapatite (Algipore®), &agr;-Tricalcium phosphate (Bio-Base®), bovine hydroxyapatite (low temperature) (Bio-Oss®), bovine hydroxyapatite (high temperature) (Osteograf®), and bovine hydroxyapatite (high temperature) enhanced with p-15, synthetic peptide (PepGen p-15®). The osteoblasts were derived from human iliac cancellous bone and seeded with the different bone substitutes. The cell proliferation and viability (WST-1), alkaline phosphatase as an early marker of osteoblast proliferation, was evaluated after 6 and 9 days. The cultures were examined for cell growth pattern and morphology by normal light and scanning electron microscopy. The human osteoblasts showed a different proliferation pattern according to the type of applied bone graft substitute. PepGen P-15 showed the highest proliferation and differentiation rate followed by Osteograf, Algipore, and Bio-base. Bio-Oss showed the lowest. These results were confirmed by electron microscopy and light microscopy evaluation in which similar growth pattern were observed. Distinct bone graft materials have different impact onto the proliferation pattern of human osteoblasts in vitro.

Computer-assisted orthognathic surgery: feasibility study using multiple CAD/CAM surgical splints

OBJECTIVE We present a virtual planning protocol incorporating a patented 3-surgical splint technique for orthognathic surgery. The purpose of this investigation was to demonstrate the feasibility and validity of the method in vivo. MATERIALS AND METHODS The protocol consisted of (1) computed tomography (CT) or cone-beam computed tomography (CBCT) maxillofacial imaging, optical scan of articulated dental study models, segmentation, and fusion; (2) diagnosis and virtual treatment planning; (3) computed-assisted design and manufacture (CAD/CAM) of the surgical splints; and (4) intraoperative surgical transfer. Validation of the accuracy of the technique was investigated by applying the protocol to 8 adult class III patients treated with bimaxillary osteotomies. The virtual plan was compared with the postoperative surgical result using image fusion of CT/CBCT dataset by analysis of measurements between hard and soft tissue landmarks relative to reference planes. RESULTS The virtual planning approach showed clinically acceptable precision for the position of the maxilla (<0.23 mm) and condyle (<0.19 mm), marginal precision for the mandible (<0.33 mm), and low precision for the soft tissue (<2.52 mm). CONCLUSIONS Virtual diagnosis, planning, and use of a patented CAD/CAM surgical splint technique provides a reliable method that may offer an alternate approach to the use of arbitrary splints and 2-dimensional planning.

Lymph node density in oral cavity cancer: results of the International Consortium for Outcomes Research

Background:Lymph node density (LND) has previously been reported to reliably predict recurrence risk and survival in oral cavity squamous cell carcinoma (OSCC). This multicenter international study was designed to validate the concept of LND in OSCC.Methods:The study included 4254 patients diagnosed as having OSCC. The median follow-up was 41 months. Five-year overall survival (OS), disease-specific survival (DSS), disease-free survival (DFS), locoregional control and distant metastasis rates were calculated using the Kaplan–Meier method. Lymph node density (number of positive lymph nodes/total number of excised lymph nodes) was subjected to multivariate analysis.Results:The OS was 49% for patients with LND⩽0.07 compared with 35% for patients with LND>0.07 (P<0.001). Similarly, the DSS was 60% for patients with LND⩽0.07 compared with 41% for those with LND>0.07 (P<0.001). Lymph node density reliably stratified patients according to their risk of failure within the individual N subgroups (P=0.03). A modified TNM staging system based on LND ratio was consistently superior to the traditional system in estimating survival measures.Conclusion:This multi-institutional study validates the reliability and applicability of LND as a predictor of outcomes in OSCC. Lymph node density can potentially assist in identifying patients with poor outcomes and therefore for whom more aggressive adjuvant treatment is needed.

Erste Ergebnisse bei der vertikalen Distraktionsosteogenese des atrophischen Alveolarkamms

Zusammenfassung Neben herkömmlichen Augmentationsverfahren zur Therapie defizienter oder atrophischer Kieferabschnitte steht seit neuem die Distraktionsosteogenese als Alternative zur Verfügung. Zur Anwendung kam bei der Distraktion im zahnlosen Alveolarfortsatz eine neu entwickelte vertikale Distraktionsapparatur basierend auf den beschriebenen Vorteilen der Distraktionsosteogenese in den letzten Jahren. Die neue Technik der „vertikalen Distraktionsosteogenese (VDO)“ wurde in unserer Arbeitsgruppe entwickelt und zur vertikalen Distraktion von bezahnten und unbezahnten Alveolarfortsatzsegmenten mit monokortikaler Befestigung der Apparatur über Mikroplatten angewendet. Bei 9 Patienten wurde nach benignen oder malignen Tumorerkrankungen nach Kastenresektion, nach traumatischem Knochenverlust oder bei Alveolarkammatrophie eine vertikale Distraktion des Alveolarfortsatzes vorgenommen. Die Segmentbreite betrug im Durchschnitt 23,7 mm, die durchschnittliche vertikale Distraktionsstrecke 9,9 mm. Bei 7 Patienten wurden Unterkiefersegmente verlagert; bei 2 Patienten erfolgte eine Distraktion von atrophen Oberkiefersegmenten. Bei allen Patienten konnten wir eine ausreichende Stabilität und die gewünschte Bewegung des Alveolarfortsatzsegments erreichen. Im Gegensatz zu Knochentransplantationen ist bei der Kallusdistraktion eine Mineralisation des zugewonnenen Knochens bereits frühzeitig erreicht. Dieses konnten wir radiologisch und histologisch nachweisen und daraufhin die Implantatversorgung von 5 Patienten bereits 12 Wochen nach der Distraktion durchführen. Wesentliche Vorteile der vertikalen Distraktionsosteogenese sind: 1. keine Knochenentnahme, 2. geringere Resorption, 3. geringe Morbidität im Vergleich zu konventionellen Techniken, 4. geringe Infektionsrate, 5. Implantatversorgung nach 3 Monaten, 6. Weichgewebsgewinn durch Distraktion. Summary Besides several conventional therapeutic regimens to treat severe forms of alveolar ridge atrophy the distraction osteogenesis is an alternative method. We introduced a new technique for the treatment of alveolar ridge atrophy encouraged by the advantages of distraction osteogenesis in the last decade. A new technique called „vertical distraction osteogenesis (VDO)“ has been developed by our study group to move dentolous and edentolous segments of the alveolar process vertically with a device in microplate-design. Vertical distraction osteogenesis was completed successfully in nine patients with segments length ranging from 6.5 mm to 43 mm with an average of 23.7 mm. The vertical distraction rate was 9.9 mm on average in seven mandibular and two maxillar segments. In all cases we found good stability and the predicted movement of the segments. In contrast to bone transplantation an earlier mineralization in the vertically distracted area could be seen by radiological examination and biopsy. Five patients were treated with dental implants 12 weeks after distraction procedure. Main advantages of vertical distraction osteogenesis are: 1. No bone harvesting, 2. decreased resorption tendency, 3. lower morbidity compared with conventional techniques, 4. lower infection rate and 5. feasibility to insert dental implants 12 weeks after distraction procedure, 6. gain of soft tissue.

Comparison of cone-beam volumetric imaging and combined plain radiographs for localization of the mandibular canal before removal of impacted lower third molars

OBJECTIVE The removal of third molars requires information about the relative position of the root tips and the mandibular nerve. The diagnostic value of conventional radiologic procedures using a panoramic radiograph and symmetrical PA cephalometric radiograph (PAN&PA) was compared with that of a cone-beam volumetric imaging (CBVI) device (Galileos; Sirona, Bensheim, Germany). STUDY DESIGN Six observers evaluated 30 PAN&PA and 30 CBVI for the position of root tips. Diagnostic information was rated from 1 to 5 (excellent to poor). RESULTS With PAN&PA, 3 times more scans showed nondetectable information for horizontal position compared with CBVI. The diagnostic information in the vertical dimension received a median rating of 2 (good) for CBVI and for PAN & PA; for the horizontal dimension, CBVI received a median rating of 2 (good), compared with a significantly worse median rating of 3 (sufficient) for PAN & PA (P = .000). The variance was highest for the horizontal dimension with PAN & PA (1.27). CONCLUSION These findings indicate that cone-beam technology improves the localization of third molar for presurgical planning.

Accuracy of a newly developed integrated system for dental implant planning

OBJECTIVES To evaluate the accuracy of the first integrated system for cone-beam CT (CBCT) imaging, dental implant planning and surgical template-aided implant placement. MATERIALS AND METHODS On the basis of CBCT scans, a total of 54 implant positions were planned for 10 partially edentulous anatomical patient-equivalent models. Surgical guides were ordered from the manufacturer (SICAT). Two different types of guidance were assessed: for assessment of the SICAT system inherent accuracy vendors titanium sleeves of 2 mm internal diameter and 5 mm length were utilized for pilot drills. The guide sleeves of the NobelGuide system were implemented for fully guided surgery and implant insertion. Deviations perpendicular to the implant axes at the crestal and apical end, as well as the angle deviations between the virtual planning data and the surgical results, were measured utilizing a follow-up CBCT investigation and referential marker-based registration. RESULTS The SICAT system inherent mean deviation rates for the drilled pilot osteotomies were determined to be smaller than 500 mum even at the apical end. Mean angle deviations of 1.18 degrees were determined. Utilizing the NobelGuide sleeve-in-sleeve system for fully guided implant insertion in combination with the investigated template technology enabled to insert dental implants with the same accuracy. Crestal deviations, in general, were significantly lower than the apical deviations. CONCLUSION Although hardly comparable due to different study designs and measurement strategies, the investigated SICAT systems inherent accuracy corresponds to the most favourable results for computer-aided surgery systems published so far. In combination with the NobelGuide surgical set for fully guided insertion, the same accuracy level could be maintained for implant positioning.

Repair of human skull defects using osteoinductive bone alloimplants

To estimate the efficacy of cranioplasty in clinical practice, autolyzed, antigen-extracted, allogenic (AAA) bone was prepared from cortical bones of human organ donors. AAA bone implants consisted of completely demineralized bone powder, completely demineralized pliable bone chips, surface-demineralized bone chips with pliable crevices, surface-demineralized rigid bone chips, or combinations thereof. 21 patients received AAA bone cranioplasties and were followed-up for between 12 and 58 months (average: 29 months). No infection or rejection of any of the AAA bone implants occurred. X-ray assessments as well as bone scintigraphies revealed osseous integration and remodelling of the AAA bone implants with minimal resorption, with the exception of completely demineralized AAA bone chips which showed partial resorption (2 cases). However, the partial resorption of completely demineralized AAA bone chips ceased after the implants had been remodelled. In 4 cases, the osteosynthesis material was removed between 10 and 18 months after the cranioplasty. In another case, a re-entry was necessary because of recurrence of an intracranial tumor. All of these five AAA bone reconstructions showed bleeding surfaces and osseous consolidations at the time of re-entry. A bone biopsy taken from one of these cranioplasties showed osteoinduction on the surface of the AAA bone implants. This first clinical review of cranial reconstructions using osteoinductive AAA bone implants emphasizes the therapeutical application of AAA bone for cranioplasty. Large AAA bone chips from human skull bones facilitate the reproduction of the skulls convexity especially when combined with preoperative stereolithography-based planning.