Quantification of plasma epstein-barr virus dna in patients with nasopharyngeal carcinoma: Preliminary results of a prospective study

Posters

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carcinoma. 92% of the adenoid cystic carcinomas expressed KIT. Materials/Methods: Mutation analysis was performed of 12 adenoid cystic carcinomas to identify mutation in codon 816 of c-kit leading to resistance against Imatinib. Results: Disease specific survivel of patients with and without expression of KIT was evaluated. There was no correlation between KIT-expression and malignancy grade, tumor size, tumor stage, or survivel. We found no mutation in the c-kit gene. Conclusions:We conclude that KIT-expression is not qualified as a prognostic marker in patients with primary parotid carcinoma. The KIT-positive adenoid cystic carcinomas did not have mutations in codon 816 of the c-kit gene. 80 poster PREDICTIVE MARKERS FOR RADIOTHERAPY RESPONSE IN ORAL SQUAMOUS CARCINOMA CELL LINES L. Farnebo. K. Roberg, L. Norberg-Spaak UNIVERSITYHOSPITAL,Departement of Otorhinolaryngology, Link6ping,

Sweden Purpose/Objective: A combination of radiotherapy and surgery or definitive chemoirradiation is the primary mode of treatment of squamous cell carcinoma (SCC) of the head and neck. Radioresistance and local recurrence are significant problems following radiotherapy and therefore there is a paramount need for predictive markers. Loss of growth control and a marked resistance to apoptosis are probably major mechanisms driving tumor progression. Proteins involved in these mechanisms are also known to affect the treatment sensitivity of tumor cells. The present study was undertaken to evaluate the possibility of using the expression of a panel of proteins involved in apoptosis and in growth control as predictive markers for radiotherapy response. Materials/Methods: Ten recently established oral cavity SCC cell lines were selected for this study to represent different parts of the spectrum of in vitro radiosensitivity. The average intrinsic radiosensitivity, measured with the 96 well plate clonogenic assay in a large panel of head and neck SCC cell lines was 1.8 for oral cavity cancers (N=14) and 1.9 for head and neck cancer of all sites (N=37). The selected cell lines have an intrinsic radiosensitivity between 1.4 to 2.6. The expression of proteins was assessed using western blot analyses, ELISA analyses and a mutation analyses was performed on the p53 gene. Results: A panel of proteins involved in apoptosis was investigated e.g. Bcl-2, BcI-XL, Bax, Bad, Bak, Puma, survivin, COX-2, Hsp70, MDM2 and p53 as well as proteins involved in growth control e.g. EGFR, cyklin D1 and SMAD4. Our results suggest that BcI-XL, survivin, COX2, p53 and EGFR are thought to more importantly influence the correlation to radiosensitivity in oral tumor cells. Furthermore, also the balance between pro- and anti-apoptotic members of the Bcl-2 family (e.g. Bcl-2, BcI-XL, Bax and PUMA) seems to have impact on the radiosensitivity in these cells. The expression of the p53 protein is probably of minor importance to radiosensitivity, whereas our results indicate that mutations in the p53 genome is a prognostic marker for radioresistance. Conclusions: Our results show that both the number and the amount of above mentioned proteins as well as p53 mutations have an impact on the radiosensitivity in oral squamous carcinoma cell lines. 81 poster PROTEOMICS PROFILING OF TUMOUR CELLS IN HEAD AND NECK SQUAMOUS CELL CARCINOMA FOR PREDICTING THE RESPONSE TO RADIOTHERAPY L. Albarzangi~,C. Scrase2,A. Poynter~,M. Salam4,J. Norton ~ IUNIVERSITYOFESSEX,Department of Biological Sciences,Colchester, Unit-

ed Kingdom, Zl-HEIPSWICHHOSPITALNHS TRUST,Department of Clinical Oncology, Ips-

wich, United Kingdom, ~FHEIPSWICHHOSPITALNHS TRUST,Department of Medical Physics,Ipswich,

United Kingdom, 4THEIPSWICHHOSPITALNHS TRUST,Department of SpecialSurgery,Ipswich,

United Kingdom Purpose/Objective: Protein profiling of tumour cells by mass spectrometry is becoming established as an approach for identifying candidate prognostic markers of disease that can be evaluated in prospective studies. With the aim of developing predictive markers of radiosensitivity / radiotherapy response in head and neck squamous cell carcinoma (HNSCC), we are investigating the use of mass spectrometry to generate molecular profiles of the tumour cell phosphoprotein fraction, as a component of the cellular proteome that is intimately involved in radiation signalling and that is commonly disrupted in malignant cells. Initially, we sought to establish conditions for optimal spectral acquisition and to determine the extent and magnitude of changes in the cellular phosphoproteome induced by in vitro irradiation that can be detected by mass spectrometry. Materials/Methods: Using the tongue squamous cell carcinoma cell line, (PE/CA-PJ 15), together with primary tumour cells from a patient with HNSCC (StageT4 base of tongue SCC),total cellular protein was isolated following detergent lysis and the phosphoprotein fraction further purified by using a commercial metal affinity matrix column (Qiagen). Spectra from total protein and phosphoprotein fractions were acquired on a Bruker-Daltonics Reflex IV Matrix Assisted Laser Desorption / Ionisation Time of Flight (MALDI-TOF) mass spectrometer. In some experiments, PE/CA-PJ 15 cells were exposed to a 2Gy dose of ionising radiation, then cultured for a further 1-2 hours prior to lysis. Results: In several experiments, complex spectra comprised of several hundred peaks in the m/z range 1-30Kda were clearly resolved. There were significantly greater numbers of the more abundant peaks in total protein (69) than in the phosphoprotein fraction (55). However, in response to irradiation, more changes (~3 fold increase/ decrease) in abundant peaks were observed in the phosphoprotein fraction (34) than in the total cellular protein fraction (25). Conclusions: Our preliminary experiments have optimised experimental conditions for generating mass spectrometry profiles of total protein and phosphoprotein fractions as tomour-specific 'fingerprints' of HNSCC. Changes in the spectral profile of the tumour cell phosphoproteome provide an exquisitely sensitive molecular correlate of radiation exposure, at least in vitro. Future work will evaluate the relation between phosphoprotein spectral profile, in vitro radiosensitivity and patient survival as part of a prospective study for developing molecular markers for monitoring different radiotherapy treatment modalities (particularly intensity modulated radiotherapy) and for predicting the response to radiotherapy. 82 poster QUANTIFICATION OF PLASMA EPSTEIN-BARR VIRUS DNA IN PATIENTS WITH NASOPHARYNGEAL CARCINOMA: PRELIMINARY RESULTS OF A PROSPECTIVE STUDY E. (~zyarl, M. Gultekin1,A. AIp2,G. Hascelik2, S. HosaP, l. Atahan ~ IHACETTEPEUNIVERSITY,Radiation Oncology,Ankara, Turkey, 2HACETrEPEUNIVERSITY,Microbiology and Clinical Microbiology, Ankara,

Turkey, 3HAcETrEPEUNIVERSITY,Ear, Noseand Throat Diseases,Ankara, Turkey

Purpose/Objective: Recently, quantification of plasma EBV DNA was shown to be useful for monitoring patients with nasopharyngeal carcinoma (NPC) and predicting the outcome of treatment. We designed a prospective study, to investigate the correlation between plasma EBV DNA levels and clinical status of the patients with NPC. Materials/Methods: A total of 144 patients with NPC and healthy controls were enrolled between February 2004 and July 2006. The levels of circulating EBV DNA were measured in 3 NPC patient

Posters groups and healthy controls. Group A (34 patients): non-metastatic patients treated with curative intent and measurements were made at diagnosis and every 3 months after treatment. Group B (78 patients): patients without initial EBV DNA measurement but in clinical remission with conventional follow-up examinations and measurements were made at follow-up. Group C (13 patients): patients with evident clinical/radiological local and/or distant relapse. Group D: 19 healthy volunteers were selected as control group. Results: Group A: EBV DNA was detected quantitatively in plasma samples of 24 (70%) out of 34 patients at diagnosis.The median concentration of plasma EBV DNA at the time of initial diagnosis was 582 copies per milliliter (interquartile range, 41 to 15,599). The median EBV DNA concentration decreased to 0 copies per milliliter after the completion of treatment in all but four patients. Group B: During follow-up period, a quantitative increase in EBV DNA concentrations was detected (range 0-13,731 copies/ml) in 7 (9%) out of 78 patients. The imaging of these patients revealed distant metastasis in 2, regional recurrence in 2, local relapse in 2 and false positive in 1 patient. Group C: EBV DNA concentrations were measured quantitatively in seven (54%) of 13 patients with Iocoregional relapse or distant metastases. Group D: All healthy individuals were negative for plasma EBV DNA. Conclusions: This study showed that quantitative plasma EBV DNA can be detected in 70% of the NPC patients at diagnosis.The plasma EBV DNA levels were persistently undetectable or low in patients with clinical remission. These results suggest that quantitative analysis of plasma EBV DNA may be a useful clinical tool in the screening and monitoring of NPC patients. However, longer follow-up is needed for long term results. This study is supported by Hacettepe University Scientific Research Project Unit with project number HUTF BAB 0401101007. 83 poster THE LEVELS OF METALLOPROTEINESESAND THEIR ENDOGENIC INHIBITORS IN PATIENTSWITH LARYNGEAL CANCER E. Klisho ~, O. Savenkova 2, I. Kondakova~, V. Perelmuter2, E. Choinzonov3,D. Shishkin 3 /INSTITUTEOF ONCOLOGY,Laboratory of Tumor Biochemistry, Tomsk,Rus-

sia, 2INSTITUTEOFONCOLOGY,Morfology, Tomsk,Russia, 3INSTITUTEOFONTOLOGY,Headand NeckCancer,Tomsk,Russia Purpose/Objective: Currently, metalloproteinases pretend to the role of prognostic factors of the tumor progression. The analysis of the levels of metalloproteinases (MMP-2,-9) and their endogenic inhibitors (TIMP-1,-2) in tumor tissue and blood serum of patients with squamous-cell laryngeal carcinoma was carried out. The relationship of these levels with clinical-morphological parameters and response to radiation therapy was studied. Materials/Methods: The study analyzed the production of MMP-9 and TIMP-1 in serum of 26 patients with laryngeal cancer in the age range 43-70 years. Serum levels of MMP-9 and TIMP-1 were assessed using the kits for immuno-enzymatic assay (R&D Systems, USA). Expression ofTIMP-1, MMP-9, MMP-2 and TIMP-2 in tumors was evaluated by immunohistochemical staining. Results: Immunohistochemical investigation revealed that MMP-9 andTIMP-1 expressions were higher than MMP-2 andTIMP-2 expressions in tumor tissue (76.9% and 100% cases versus 23.8% and 33.3% of cases, respectively), therefore, only the MMP-9 and TIMP-1 levels in blood serum were analyzed. The TIMP-1 level in serum of patients was significantly higher than that of healthy persons (p=0.023). High level of TIMP-lwas observed in blood serum of patients with stage Ill laryngeal cancer and it was significantly differed from the inhibitor level of the control group (p=0.009), however, no correlation between this level and metalloprtoteinases in blood serum and tumor size was found. High level of TIMP-1 was observed in blood serum of patients having no metastases in regional lymph nodes (124.5+39.5 ng/ml) and it was decreased in patients with regional metastases

$27 (102.7+10.9 ng/ml). A significant difference in TIMP-I level between cancer patients and healthy persons was found (p=0.023).The MMP9 expression in tumor tissue was also related to the presence of metastatses in regional lymph nodes (p=0.0S4). In blood serum of patients with 75-I 00% tumor regression after radiation therapy the TIMP-I level was higher than in patients with low response rate to radiation therapy (p=0.072). Conclusions: Thus, the determination of the TIMP-I level in blood serum and MMP-9 in tumor tissue may have an informative value together with the standard parameters for assessment of the tumor involvement. Besides, the determination of TIMP-I in blood serum may be useful for predicting the response to radiation therapy in laryngeal cancer patients. 84 poster THE ROLE OF ADDITIONAL HISTOLOGICAL STUDIES FORTHE PROGNOSlS OF HEAD AND NECK SQUAMOUS CELL CARCINOMAS W. Golusinski, E. Wasniewska, D. Jarmolowska-Jurczyszyn, P. Majewski, M. Biczysko, A. Marszalek POZNANUNIVERSITYOF MEDICALSCIENCES,Department of Otolaryngology,

Poznan,Poland Purpose/Objective: There are several head and neck squamous cell carcinomas (SCC) classifications, many do not satisfy clinician who need not only histological description of the tumor but also looks for features of tumor growth and prediction of metastases. Materials/Methods: In this work we study SCC cases from tongue and oral cavity floor (80) and larynx (84). Samples were evaluated using Jokobsson classification, TNM and follow up. In 80 cases we did transmission electron microscopy (TEM). Ultrastructural features of squamous cell epithelium differentiation were used: cytokeratins - free/in bundles, acanthotic cells intercellular bridges, desmosomes or other junctions, size and shape of nuclei with hetero-/euchromatine, nucleoli and segregation of its material. Results: This studies revealed in highly differentiated cases groups of cells forming keratin pearls. At tumor periphery small cells organized in rows with focal basement membrane-like (BM-like) structures or focally directly invading stroma were present. Cells producing BM-like material (with cytokeratins adhering irregularly to the cell membrane) were not forming regular hemidesmosomes. Structures which should fix cell in the stroma were not found. Often there were bridges-like structures without cellular junctions. At tumor periphery foci of cells without junction with each other and with the stroma were seen. Mainly euchromatic nuclei with single nucleoli had irregular nuclear membrane with pores. We observed, and aggregates of RNA material around pores. In some laryngeal cases in cells with bundles of cytokeratins neurosecretory granules were seen. Conclusions: TEM studies make possible more detailed evaluation of cellular changes, to find cells with migratory properties and with high activity. It has an impact on more appropriate diagnosis and prognosis. Nowadays, a simple routine histological diagnosis is not sufficient for predicting the outcome of tumors. Extended evaluation of additional criteria and classifications should be implemented at least in complicated cases. 85 poster THE TUMOR MICROENVlRONMENT IN HEAD AND NECK SQUAMOUS CELL CARCINOMAS: PREDICTIVEVALUE AND CLINICAL RELEVANCE OF MOLECULAR MARKERS l.J. Hoogsteen I, K.I. Wijffels 2, H.A.M. Marres 2, F.J.A. van den Hoogen2, A.J. van der KogeI~,J.H.A.M. Kaanders I IRADBOUDUNIVERSITYNIJMEGENMEDICALCENTRE,Radiotherapy,nijmegen, the

netherlands, 2RADBOUDUNIVERSITYNIJMEGENMEDICALCENTRE,Otorhinolaryngology/head

and necksurgery, nijmegen, the netherlands