Christof Hofele

Recurrent copy number gain of transcription factor SOX2 and corresponding high protein expression in oral squamous cell carcinoma

Gene copy number aberrations are involved in oral squamous cell carcinoma (OSCC) development. To delineate candidate genes inside critical chromosomal regions, array‐CGH was applied to 40 OSCC specimens using a microarray covering the whole human genome with an average resolution of 1 Mb. Gene copy number gains were predominantly found at 1q23 (9 cases), 3q26 (11), 5p15 (13), 7p11 (7), 8q24 (17), 11q13 (15), 14q32 (8), 19p13 (8), 19q12 (7), 19q13 (8), and 20q13 (9), whereas gene copy number losses were detected at 3p21‐3p12 (15), 8p32 (11), 10p12 (8), and 18q21‐q23 (10). Subsequent mRNA expression analyses by quantitative real time polymerase chain reaction found high mRNA expression of candidate genes SOX2 in 3q26.33, FSLT3 in 19p13.3, and CCNE1 in 19q12. Tissue microarray (TMA) analyses in a representative OSCC collection found gene copy number gain for SOX2 in 52% (115/223) and for CCNE1 in 31% (72/233) of the tumors. Immunohistochemical analyses on TMA sections of the corresponding proteins detected high expression of SOX2 in 18.1% (49/271) and of CyclinE1 in 23.3% (64/275) of tumors analyzed. These findings indicate that SOX2 and CCNE1 might be activated via gene copy number gain and participate in oral carcinogenesis. The combination of array‐CGH with TMA analyses allows rapid pinpointing of novel promising candidate genes, which might be used as therapeutic stratification markers or target molecules for therapeutic interference.

Recurrent coamplification of cytoskeleton-associated genes EMS1 and SHANK2 with CCND1 in oral squamous cell carcinoma.

Chromosomal band 11q13 is frequently amplified in oral squamous cell carcinoma (OSCC) and assumed to be critically involved in tumor initiation and progression by proto‐oncogene activation. Though cyclin D1 (CCND1) is supposed to be the most relevant oncogene, several additional putative candidate genes are inside this chromosomal region, for which their actual role in tumorigenesis still needs to be elucidated. To characterize the 11q13 amplicon in detail, 40 OSCCs were analyzed by comparative genomic hybridization to DNA microarrays (matrix‐CGH) containing BAC clones derived from chromosomal band 11q13. This high‐resolution approach revealed a consistent amplicon about 1.7 Mb in size including the CCND1 oncogene. Seven BAC clones covering FGF3, EMS1, and SHANK2 were shown to be frequently coamplified inside the CCND1 amplicon. Subsequent analysis of tissue microarrays by FISH revealed amplification frequencies of 36.8% (88/239) for CCND1, 34.3% (60/175) for FGF3, 37.4% (68/182) for EMS1, and 36.3% (61/168) for SHANK2. Finally, quantitative mRNA expression analysis demonstrated consistent overexpression of CCND1 in all tumors and of EMS1 and SHANK2 in a subset of specimens with 11q13 amplification, but no expression of FGF3 in any of the cases. Our study underlines the critical role of CCND1 in OSCC development and additionally points to the functionally related genes EMS1 and SHANK2, both encoding for cytoskeleton‐associated proteins, which are frequently coamplified with CCND1 and therefore could cooperatively contribute to OSCC pathogenesis.

Ala228 variant of trail receptor 1 affecting the ligand binding site is associated with chronic lymphocytic leukemia, mantle cell lymphoma, prostate cancer, head and neck squamous cell carcinoma and bladder cancer.

Allelic loss of chromosome 8p21–22 is a frequent event in various human cancers including mantle cell lymphoma (MCL), prostate cancer, head and neck squamous cell carcinoma (HNSCC) and bladder cancer. The tumor necrosis factor‐related apoptosis inducing ligand (TRAIL) receptors, including TNFRSF10A and TNFRSF10B, are located within this chromosomal region. Since recent studies demonstrate that chronic lymphocytic leukemia (CLL) and prostate cells are TRAIL induced apoptosis, TRAIL‐receptors are strong tumor suppressor candidate genes in human cancers exhibiting loss of chromosomal material in 8p21.3. However, no mutation of the TRAIL receptor genes has been reported in CLL, MCL, prostate cancer, HNSCC so far. In this study we analyzed the complete coding region of TNFRSF10A and TNFRSF10B in a series of 32 MCL and 101 CLL samples and detected a single nucleotide polymorphism (SNP) in TNFRSF10A (A683C) with tumor specific allele distribution. We examined allele distribution in 395 samples of different tumor entities (prostate cancer, n = 43; HNSCC, n = 40; bladder cancer, n = 179) and compared them to 137 samples from healthy probands. We found the rare allele of TNFRSF10A is more frequent in CLL, MCL, prostate cancer, bladder cancer and HNSCC. The A683C polymorphism did not cosegregate with other TNFRSF10A polymorphisms previously described. Thus screening for 683A→C nucleotide exchanges may become important in diagnosis and/or treatment of these malignancies.

Amplification of Cyclin L1 is associated with lymph node metastases in head and neck squamous cell carcinoma (HNSCC).

Overrepresentation of chromosomal bands 3q25–q29 has been associated with shortened disease-specific survival in head and neck squamous cell carcinoma (HNSCC). To assess the prevalence of copy number gains (>4 signals per cell) and high-level amplifications (>8 signals per cell) from putative oncogenes in this chromosomal region (CCNL1, SNO, PIK3CA, TP73L), tissue microarray analysis was applied on 280 HNSCCs by fluorescence in situ hybridization. Overall frequency of additional copy numbers was 34.3% for CCNL1, 31.8% for SNO, 39.0% for PIK3CA and 38.3% for TP73L, respectively. In general, gains were more frequently detected in stage IV compared to stage I–III tumours. Performing multivariate logistic regression analysis, a significant association of CCNL1 gains and the presence of lymph node metastases was found, which was independent of anatomical site and T-stage of the primary tumour (P=0.049). Site-specific subgroup analysis further showed that copy number gains of CCNL1 and SNO occurred more frequently in oral carcinomas in advanced clinical stages as compared to N0 oral lesions (CCNL1: P=0.03; SNO: P=0.03). Finally, Kaplan–Meier analysis revealed that high-level amplifications of CCNL1 correlated with shorter overall survival of the patients. Our results indicate that CCNL1 plays a critical role in the loco-regional progression of HNSCC and may serve as an indicator for occult advanced tumour stages.

Clinical use of navigation based on cone-beam computer tomography in maxillofacial surgery

Image-guidance in maxillofacial surgery is based predominantly on computed tomographic (CT) images. Its main disadvantage is the considerable amount of radiation to which the patient is exposed, and dental metal artefacts. Recently, a new class of devices based on the concept of cone-beam computed tomography (CBCT) has been introduced for maxillofacial imaging, which we have investigated. In a clinical study, the first seven patients to be operated using a navigation system based on CBCT images, were evaluated. In all cases patient to image recording was uneventful and the surgical objective was reached. The guidance given by the navigation system was helpful. CBCT is an alternative to conventional CT, gives a lower dose of radiation, and costs less. Limitations in the quality of the images and the size of the field of view may restrict its use. It is suitable for image-guided surgery using a navigation system as long as the images show enough of the relevant anatomy and pathology.

Cytogenetic characterization of head and neck squamous cell carcinoma cell lines as model systems for the functional analyses of tumor-associated genes

Head and neck squamous cell carcinoma (HNSCC) is a solid malignant neoplasm exhibiting aggressive phenotypes and high recurrence rates. To improve its clinical management, understanding the molecular basis of HNSCC development is of critical importance. For the investigation of tumor-associated genes, functional analyses in well-characterized tumor cell systems are required. To establish an experimental platform, a set of 20 HNSCC cell lines was screened for genetic imbalances by chromosomal comparative genomic hybridization (cCGH). Frequent DNA copy number gains were detected on 3q26.3-qter, 5p, 7p11-p13, 8q23-qter, 9p11-p13, 9q31-qter, 11q13 and 20q13.1, whereas copy number losses were found on 3p, 4p, 4q32.1-qter, 8p11-p12 and 18q22 in agreement with previous observations on genetic aberrations detected in primary HNSCC specimens. Subsequent mRNA expression analysis of 11q13 candidate genes CCND1 and CTTN revealed that HNSCC cell lines exhibiting a DNA copy number gain on 11q13 had a higher transcript level of CCND1 and CTTN compared with HNSCC cell lines without 11q13 copy number gain (P = 0.014 and P = 0.009, respectively). Furthermore, CCND1 and CTTN amplification as detected by fluorescence in situ hybridization correlated with protein expression as assessed by immunocytochemistry. In summary, the cytogenetic characterization illustrates that this set of HNSCC cell lines is representative for the HNSCC genome and provides tumor model systems for detailed analysis of genes with a possible role in the pathomechanism of head and neck tumors.

Prospektive Phase-II-Studie zur neoadjuvanten Radiochemotherapie fortgeschrittener, operabler Mundhöhlenkarzinome

Hintergrund. Die simultane Radiochemotherapie hat das Ziel, die lokoregionäre Tumorkontrolle zu erhöhen und die Rate an Fernmetastasen zu senken. Therapieregimes, die Cisplatin/5-FU enthalten, sind allgemein als Standardtherapie bei fortgeschrittenen Kopf-Hals-Karzinomen anerkannt. Die meisten Studien berichten über interessante Ansprechraten und Überlebensraten, gleichzeitig aber auch über eine beträchtliche Mukosatoxizität. Taxol zeigte in den bisherigen Studien sowohl in der Mono- als auch in der Kombinationstherapie eine bemerkenswerte Aktivität bei Kopf-Hals-Karzinomen. In der vorliegenden ambulanten Phase-II-Studie wurde die Kombination von Taxol, Carboplatin und Strahlentherapie bis 40 Gy als neoadjuvantes Konzept bei fortgeschrittenen, operablen Mundhöhlen- und Oropharynxkarzinomen (Stadium III/IV) geprüft. Patienten und Methode. Im Zeitraum von Mai 1998–Oktober 2000 wurden insgesamt 53 Patienten gemäß Studienprotokoll rekrutiert und erhielten wöchentlich Taxol (40 mg/m2) und Carboplatin (AUC 1,5) für 5 Zyklen mit simultaner Strahlentherapie (40 Gy) in konventioneller Fraktionierung. Innerhalb von 3–4 Wochen nach Abschluss der Radiochemotherapie erfolgte die Resektion des Primärtumors und der regionären Halslymphknoten. Ergebnisse. 52 Patienten waren bezüglich Toxizität und Ansprechrate auswertbar. Komplettremissionen wurden bei 31/52 Patienten (CR 60%), partielle Remissionen bei 21/52 Patienten (40%) beobachtet. Histopathologische Komplettremissionen im Resektat wurden bei 30/52 Patienten (pCR 58%) gefunden. Die 1-, 2- und 3-Jahres-Überlebensrate beträgt 84%. Schlussfolgerung. Die vorliegenden Ergebnisse zeigen beeindruckende klinische und pathologische Remissionsraten einer simultanen Radiochemotherapie mit Taxol/Carboplatin als präoperatives Behandlungskonzept bei fortgeschrittenen Mundhöhlen- und Oropharynxkarzinomen. Purpose. The purpose of simultaneous chemoradiotherapy is to increase local-regional control and to decrease the incidence of distant metastases. Regimens containing cisplatin/5-FU chemotherapy are widely accepted as standard treatment in advanced head and neck cancer. Most studies reported promising response and survival data, but also severe mucosal toxicity. In recent years the newly developed drug Taxol demonstrated interesting activity in head and neck cancer as a single agent as well as in combination drug regimens. In the present outpatient phase II trial, we investigated the combination of Taxol/carboplatin with 40 Gy radiotherapy in a neoadjuvant setting of operable stage III/IV squamous cell carcinoma of the oral cavity and oropharynx. Patients and methods. Fifty-three patients were enrolled in this trial during the period from May 1998 to October 2000 and received five cycles weekly of Taxol (40 mg/m2) and carboplatin (AUC 1,5) with conventional radiotherapy (40 Gy). Within 3–4 weeks after chemoradiotherapy resection of the primary tumor and the regional neck nodes was performed. Results. Fifty-two patients were evaluable for toxicity and response. Complete response was observed in 31 of 52 patients (CR 60%), and partial remission was seen in 21 of 52 patients (PR 40%). In 30 of 52 patients complete pathologic response (pCR 58%) was documented in the resection specimens. The 1-, 2-, and 3-year overall survival rate was calculated as 84%. Conclusion. Our present results demonstrated impressive clinical and pathological response rates of concurrent Taxol/carboplatin and radiotherapy as a preoperative treatment modality in advanced oral and oropharyngeal cancer.

Oral health-related quality of life and depression/anxiety in long-term recurrence-free patients after treatment for advanced oral squamous cell cancer

This report focuses on the association between oral health-related quality of life (OHRQoL) and depression/anxiety of a homogeneous group of cancer patients who were recurrence-free for 8 years after treatment for advanced oral squamous cell. Participants were 24 patients (mean age 55 years, 75% men) treated with neoadjuvant concurrent radiochemotherapy followed by surgery with a mean recurrence-free period of 95 months (from 39 to 164 months). The OHRQoL (OHIP) and the anxiety/depression (HADS) were assessed twice (1 year between t1 and t2). OHRQoL was impaired in this group (mean OHIP score 65 units). In cross-lagged correlation analysis, the correlation between OHRQoL to t1 and depression to t2 was significant and greater than the non-significant correlation for depression to t1 and OHRQoL to t2 indicating that OHRQoL predicts depression better than vice versa. However, the difference in the correlation coefficients was not significant (ZPF-test). The same was true for OHRQoL and anxiety. The OHRQoL measured with the OHIP was impaired in comparison to the normal population. In the limitations of the study design and bearing the small sample size in mind, the results give evidence that OHRQoL predicts psychological outcomes, namely depression and anxiety, better than vice versa.

Möglichkeiten und chancen der Gewebechiptechnologie bei Kopf-Hals-Tumoren. Eine neue Technik zur schnellen Analyse von potenziellen Tumormarkern.

Plattenepithelkarzinome der Mundhöhle haben trotz moderner adjuvanter und neoadjuvanter Therapieformen nach wie vor eine schlechte Prognose. Sie entwickeln sich aus normaler Mundschleimhaut über einen mehrstufigen Prozess, zu dem u. a. Deletionen von Tumorsuppressorgenen und die Amplifikation von Onkogenen gehören. Die molekularen und zellulären Ereignisse während der klonalen Entwicklung eines invasiven Karzinoms sind komplex, und es existieren bis jetzt keine zytogenetischen Marker für spezifische Stadien der Tumorentwicklung, die für eine genauere Typisierung der Läsionen notwendig wären. In der vorliegenden Studie wird erstmals der Einsatz der neuartigen Gewebechiptechnologie bei 293 Plattenepithelkarzinomen der Mundhöhle dargestellt. Untersucht wurden das Onkogen Cyclin D1 mittels Fluoreszenz-in-situ-Hybridisierung (FISH) sowie die Proteine Cyclin D1, p53, p16, CDK4, Bcl2, Mdm2, und Rb mittels Immunhistochemie (IHC). Die Gewebechiptechnologie erwies sich als ein verlässliches und schnelles Screeningverfahren zur Charakterisierung von molekularen und zellulären Veränderungen bei Mundhöhlenkarzinomen. Oral squamous cell carcinomas (OSCC) are malignant tumors with a poor prognosis and low long-term survival rates, even when using modern adjuvant and neoadjuvant therapy forms in addition to surgery. For the clinical estimation of each tumor, it is necessary to define stage-dependent molecular and/or cellular parameters as it is known that OSCC develop along a multistep pathway including the loss of tumor suppressor genes and the amplification of oncogenes which result in changes in protein expression. In order to establish a reliable pattern of molecular and cellular biomarkers, a large number of tumor specimens from different stages of the disease need to be analysed. In this study, biopsies of a collective of 293 OSCC in different stages were screened with the novel technique of tissue chip microarrays by fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC). FISH-analysis was performed on the oncogene cyclin D1 and IHC-analysis on the proteins cyclin D1, p53, p16, cdk4, bcl2, mdm2 and rb. Tissue chip technology was shown to facilitate rapid screening for molecular and cellular alterations in different stages of OSCC and revealed reliable and reproducible results that may allow the definition of a multistep pathway model for tumor progression in OSCC.

Recurrent craniofacial dermatofibrosarcoma protuberans: long-term prognosis after close surgical removal.

Dermatofibrosarcoma protuberans (DFSP) is a low-grade malignant neoplasm of the dermis that rarely manifests in the craniofacial area. In this retrospective analysis, we investigated the long-term survival of 7 patients with recurrent craniofacial DFSP. This study includes all patients in our department with recurrences of DFSP between 1989 and 2006. All patients were treated by radical surgery with 1-cm free safety margin in every direction and remained in routine long-term follow-up for tumor patients. Two of the 7 patients showed a local recurrence, which was again successfully treated surgically with the same technique. Advanced reconstruction with free full-thickness skin transfers, regional flaps, and forearm flaps, respectively, was required in 5 of the 7 patients. The other 2 patients were reconstructed locally. The long-term prognosis of craniofacial DFSP can be assessed optimistically even if the tumor already reoccurred. All 7 patients included in this study are still alive and so far not suffering from local recurrence. Advanced reconstructive techniques are often required in the management of reoccurring craniofacial DFSP. Late recurrences have been reported; therefore, a long-term follow-up for these patients should be considered.