Contamination by murine fibroblasts was reduced by short applications of trypsin-EDTA taking advantage of stronger adherence of malignant epithelial cells to the culture support. Microsatellite analysis For fingerprint experiments, DNA has been isolated from cell pellets using the DNeasy tissue kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. adapted to em in vitro /em culture and is currently available to the scientific community with reliable identification based on microsatellite genotyping. Both Capi1 and Capi3 have histological characteristics of adenocarcinomas and display intense expression of EMA, CEA and cytokeratin 7. Multifish chromosome analysis demonstrated a translocation involving chromosomes 4 and 21 in both specimens. Distinct rare missense mutations of the em TP53 /em gene were detected in Capi1 (codon 312) and Capi3 (codon 181); the codon 181 mutation is consistent with a previously reported similar finding in a small series of CUP specimens. Finally, intense membrane expression of c-kit was recorded in Capi3. Conclusion Our data suggest that xenografted tumors can be obtained from a substantial fraction of CUP clinical specimens. The hypothesis of a preferential association of CUPs with em TP 53 /em mutations of codon 181 deserves further investigations. The Capi3 cell line will be a useful tool for assessment of novel c-kit inhibitors. Background The pathogenesis of the carcinomas of unknown primary site (CUPs) remains one of the most enigmatic topics in the field of metastasis research [1,2]. They are defined as biopsy proven metastases of a malignancy in the absence of an identifiable primary site after clinical examination, radiological imaging and biological workup. Although there is no consensus about their incidence, it is reasonable to estimate that CUPs account for 2C3 % of all newly diagnosed patients with cancer [2,3]. In all described series, this disease appears to be extremely aggressive with a median survival below 9 months [2]. Biological mechanisms underlying the CUP phenomenon remain almost entirely unknown. With regard to histological characteristics, they are predominantly classified as adenocarcinomas (50C70%) or poorly differentiated carcinomas (20C30%). Only 5C8% are squamous cell carcinomas [3]. Though CUPs comprise a heterogeneous group of tumours with widely varying natural histories, the clinical picture of CUP demonstrates common characteristics. Patients predominantly present with a short history of nonspecific complaints (anorexia, weight loss, etc…). The primary tumor remains unidentified in most cases throughout the patient’s life [4]. The pattern of metastatic spread tends to be different in CUPs compared to metastasized known primary tumors. Approximately 30% of patients with CUP present with three or more organs involved in contrast with less than 15% in classical metastatic syndromes [4-6]. With the exception of some treatable subgroups C for example young men with extra-gonadal germ cell tumors C patients with CUP have a very poor prognosis [7,8]. In some recent, mostly phase II studies with patients selected from poor prognostic groups, a median survival of 8C13 weeks was reached [9,10]. Despite these intriguing characteristics and the severity of the prognosis, biology of CUPs has been poorly investigated. Surprisingly, there are only few em in vitro /em cell lines representative of this category of tumors. To our knowledge, only one such cell collection is available in the American Type Tradition Collection but it is definitely uncharacterized (CRL-7431). It is well worth noting that none of the NCI-60 cell collection panel utilized for systematic em in vitro /em testing of anti-cancer compounds in the National Tumor Institute (Developmental Therapeutics System) is derived from a CUP [11]. In order to obtain biological material required for biological and pharmacological investigations of CUPs, we attempted to create xenografted tumor lines derived from new medical specimens (biopsies or medical specimens). Successful xenografts were accomplished in 2 instances out of 4. One of them (Capi1) was lost after only 2 passages whereas the additional one (Capi3) has been adapted to em in vitro /em tradition and is available to the medical community. We here report biological characteristics of Capi3 along with some data on Capi1. Rare mutations of the em TP53 /em gene were recorded for both Capi1 (exon 5) and Capi3 (exon.A distinct em TP 53 /em mutation was found at codon 312 (Thr to Ser) in the Capi1 xenograft. whereas the additional one (Capi3) has been adapted to em in vitro /em tradition and is currently available to the medical community with reliable identification based on microsatellite genotyping. Both Capi1 and Capi3 have histological characteristics of adenocarcinomas and display Eletriptan hydrobromide intense manifestation of EMA, CEA and cytokeratin 7. Multifish chromosome analysis shown a translocation including chromosomes 4 and 21 in both specimens. Distinct rare missense mutations of the em TP53 /em gene were recognized in Capi1 (codon 312) and Capi3 (codon 181); the codon 181 mutation is definitely consistent with a previously reported related finding in a small series of CUP specimens. Finally, intense membrane manifestation of c-kit was recorded in Capi3. Summary Our data suggest that xenografted tumors can be obtained from a substantial fraction of CUP medical specimens. The hypothesis of a preferential association of CUPs with em TP 53 /em mutations of codon 181 deserves further investigations. The Capi3 cell collection will be a useful tool for assessment of novel c-kit inhibitors. Background The pathogenesis of the Eletriptan hydrobromide carcinomas of unfamiliar main site (CUPs) remains probably one of the most enigmatic topics in the field of metastasis study [1,2]. They may be defined as biopsy verified metastases of a malignancy in the absence of an identifiable main site after medical exam, radiological imaging and biological workup. Although there is no consensus about their incidence, it is sensible to estimate that CUPs account for 2C3 % of all newly diagnosed individuals with malignancy [2,3]. In Eletriptan hydrobromide all explained series, this disease appears to be extremely aggressive having a median survival below 9 weeks [2]. Biological mechanisms underlying the CUP phenomenon remain almost Eletriptan hydrobromide entirely unfamiliar. With regard to histological characteristics, they are mainly classified as adenocarcinomas (50C70%) or poorly differentiated carcinomas (20C30%). Only 5C8% are squamous cell carcinomas [3]. Though CUPs comprise a heterogeneous group of tumours with widely varying natural histories, the medical picture of CUP demonstrates common characteristics. Patients mainly present with a short history of nonspecific issues (anorexia, weight loss, etc…). The primary tumor remains unidentified in most cases throughout the patient’s existence [4]. The pattern of metastatic spread tends to be different in CUPs compared to metastasized known main tumors. Approximately 30% of individuals with CUP present with three or more organs involved in contrast with less than 15% in classical metastatic syndromes [4-6]. With the exception of some treatable subgroups C for example young men with extra-gonadal germ cell tumors C individuals with CUP have a very poor prognosis [7,8]. In some recent, mostly phase II studies with patients selected from poor prognostic organizations, a median survival of 8C13 weeks was reached [9,10]. Despite these intriguing characteristics and the severity of the prognosis, biology of CUPs has been poorly investigated. Remarkably, there are only few em in vitro /em cell lines representative of this category of tumors. To our knowledge, only one such cell collection is available in the American Type Tradition Collection but it is definitely uncharacterized (CRL-7431). It is well worth noting that none of the NCI-60 cell collection panel utilized for systematic em in vitro /em testing of anti-cancer compounds in the National Tumor Institute (Developmental Therapeutics System) is derived from a CUP [11]. In order to obtain biological material required for biological and pharmacological investigations of CUPs, we attempted to create xenografted tumor lines derived from new medical specimens (biopsies or medical ENSA specimens). Successful xenografts were accomplished in 2 instances out of 4. One of them (Capi1) was lost after only 2 passages whereas the additional one (Capi3) has been adapted to em in vitro /em tradition and is available to the medical community. We here report biological characteristics of Capi3 along with some data on Capi1. Rare mutations of the em TP53 /em gene were recorded for both Capi1 (exon 5) and Capi3 (exon 9). Methods Tumor transplantation in immunodeficient mice Fragments of tumor biopsies or medical specimens were obtained with authorized educated consent from 4 individuals and grafted on irradiated Swiss Eletriptan hydrobromide nude (5 Gy) and/or NOD-SCID mice. Two to six tumor fragments of about 4 mm3 were implanted subcutaneously in the recipient animals. em In vitro /em tradition A xenografted Capi3 tumor was.