Seventeen patients (5.9%) had metastases on initial presentation. up data available in 163 cases. Results EGFR expression frequency ranged between 0.3% and 52.9%, depending on the antibody and scoring method used. In all, 3.5% of the tumours showed gene amplification by FISH, which correlated with EGFR expression for three antibodies. Only one antibody had independent prognostic value in multivariate analysis and correlated with an unfavourable outcome; gene amplification status showed no correlation with clinical Fosphenytoin disodium features. Conclusions Frequency of EGFR immunopositivity in STS strongly depends on the antibody used, and only one of five antibodies tested predicted an unfavourable clinical outcome. This indicates that choice of primary antibody and scoring system have a substantial impact on the determination of EGFR immunoreactivity. amplificationDuda DNA significantly impaired their proliferative ability.26 The application of imatinib mesylate (Gleevec) in gastrointestinal stromal tumours (GIST) and dermatofibrosarcoma protuberans has already shown a selective blockade of the tyrosine kinases c\abl, PDGFR, and c\kit, with significant improvement in the clinical outcome.27,28,29 However, the use of these new substances generally demands a reliable prediction of patients who might experience clinical benefit.30,31,32 As for c\gene amplification status measured by FISH analysis. Methods Samples In all, 302 specimens of malignant soft tissue tumours from 287 patients were retrieved from the files of the Institute of Pathology, University of Mnster. All consecutive cases of malignant soft tissue tumour of the trunk and limbs received between 1988 and 2000 were included. Biopsy specimens and cases of Ewing sarcoma were disregarded. The collection consisted mainly of primary tumour tissue samples (97%), but also included six recurrent tumour specimens and three metastases. All patients were treated according to the same surgical protocol. Tumour specimens were used for investigation after informed consent had been obtained. The use of tumour tissue was also approved by the local ethics committee. The samples were formaldehyde fixed and embedded in paraffin. Specimens were classified according to standard protocols.4,33 The most common entities encountered were malignant fibrous histiocytoma, liposarcoma, and leiomyosarcoma. The frequencies of other tumour entities are listed in table 2?2.. Fourteen per cent of tumours were classified as grade 1, 30% as grade 2, and 55% as grade 3; 73% had a diameter of more than 5?cm, 27% a diameter of 5?cm or less. Clinical follow up data were available in 163 cases. The mean follow up time was 46?months (range 3 to 235). Mean age at diagnosis was 47 years (range 2 to 87), with a standard deviation of 19.2 years. Thirty four per dent of patients developed local recurrence, 40.9% developed metastases, and 34.6% died of the disease. Seventeen patients (5.9%) had metastases on initial presentation. Of the patients with available clinical data, all had surgical resection of the tumour, 46.9% were treated with additional radiotherapy, 35.2% with additional chemotherapy (different protocols), and 8.6% and 14.8% received neoadjuvant radiotherapy or chemotherapy, respectively. Table Fosphenytoin disodium 2?Frequencies of included soft tissue tumour entities Angiosarcoma72.3%Fibrosarcoma165.3%Haemangioendothelioma41.3%Malignant fibrous histiocytoma7023.2%Leiomyosarcoma3611.9%Liposarcoma4414.6%Neurogenic sarcoma93%Rhabdomyosarcoma3210.6%Malignant peripheral nerve sheath tumour247.9%Synovial sarcoma3411.3%Sarcoma NOS72.3%Other196.3%(n?=?302) Open in a separate window NOS, not otherwise specified. Tissue microarray construction A tissue microarray (TMA) was composed,34,35 consisting of more than 600 cores with a diameter of 0.6?mm each and a distance of 0.2?mm. To locate representative tumour areas, haematoxylin and eosin stained sections were prepared from each original tumour block. Two cores per specimen were Fosphenytoin disodium punched out using a dedicated TMA instrument (Beecher Instruments, Silver Spring, Maryland, USA). Immunohistochemistry Five primary antibodies (table 3?3)) were used for immunohistochemical evaluation of EGFR expression. Extensive testing was conducted using consecutive sections of a squamous cell carcinoma as Fosphenytoin disodium positive control to determine where possible the optimal pretreatment, dilution, and antibody detection system of primary antibodies (table 3?3).). Procedures were modified until high concordance was achieved among the Fosphenytoin disodium different antibodies. Benign tumours and non\neoplastic tissues such as skin, included in the TMA, were used as additional positive and negative controls. Table 3?Overview of characteristics and tissue processing issues of 5 different antibodies to EGFR applied to soft tissue sarcomas detection was derived from homo sapiens PAC clone containing the whole gene (GenBank accession No “type”:”entrez-nucleotide”,”attrs”:”text”:”AC006977″,”term_id”:”5931479″,”term_text”:”AC006977″AC006977). The procedure was carried out as previously published.36 For each core, 20 non\overlapping intact tumour cell nuclei were selected for scoring, as previously reported.37 The cut Mouse monoclonal to SMN1 off frequency for amplification was defined as four signals per nucleus. Statistical analysis Statistical analysis and tests were undertaken using SPSS Version 11.5.1. Correlations between EGFR expression, amplification, and clinical variables were tested with cross tables applying the 2 2 test,2 and correlation analysis was done according to Kendall (Tau b). For survival analysis, KaplanCMeier analysis, log rank tests, and multivariate survival analysis.