Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Improvement of treatment efficacy and decreased side effects through tumor-targeted drug delivery would be desirable. By ...panning with a phage-displayed cyclic random peptide library we selected a peptide with strong affinity for RMS in vitro and in vivo. The peptide minimal binding motif Arg-X-(Arg/Lys)(Arg/Lys) identified by alanine-scan, suggested the target receptor to be a proprotein convertase (PC). Expression profiling of all PCs in RMS biopsies and cell lines revealed consistent high expression levels for the membrane-bound furin and PC7. Direct binding of RMS-P3 peptide to furin was demonstrated by affinity chromatography and supported by activity and colocalization studies. Treatment of RMS in mice with doxorubicin coupled to the targeting peptide resulted in a two-fold increase in therapeutic efficacy compared to doxorubicin treatment alone. Our findings indicate surface-furin binding as novel mechanism for therapeutic cell penetration which needs to be further investigated. Furthermore, this work demonstrates that specific targeting of membrane-bound furin in tumors is possible for and suggests that RMS and other tumors might benefit from proprotein convertases targeted drug delivery.
Clonal evolution of the leukemogenic compartment may contribute to alter the therapeutic response in acute lymphoblastic leukemia (ALL). Using xenotransplantation of primary leukemia cells, we ...evaluated the phenotypic and genetic composition of de novo resistant very high risk precursor B-cell ALL, a subgroup defined by the persistence of minimal residual disease despite intensive chemotherapy. Analysis of copy number alterations (CNAs) showed that the xenografted leukemia, even when reconstituted from 100 cells, remained highly related to the diagnostic sample, with minor changes in CNAs, mostly deletions, emerging in most cases in the first passage into mice. At the single-cell level, the pattern of monoallelic and biallelic deletions of the CDKN2A locus revealed distinct leukemia subpopulations, which were reproducibly tracked in xenografts. In most very high risk ALL cases, the predominant diagnostic clones were reconstituted in xenografts, as shown by multiplex polymerase chain reaction analysis of immunoglobulin and T-cell receptor loci. In other cases, the pattern in CNAs and immunoglobulin and T-cell receptor rearrangement was less concordant in xenografts, suggesting the outgrowth of subclones. These results unequivocally demonstrate the existence of clonally closely related but distinct subsets of leukemia initiating cells in ALL, which has important implications for drug development and preclinical disease modeling.
Rhabdomyosarcoma (RMS) is classified into two main subgroups: the embryonal (ERMS) and the alveolar (ARMS) form. The majority of the ARMSs are associated with specific chromosomal translocations ...(pARMS). Because ARMS is much more aggressive than ERMS, RMS subclassification has clinical relevance. However, diagnosis of RMS subgroups on the basis of histology or molecular biology can be difficult, and supplementing diagnostic methods would be desirable. The aim of this study was to establish a panel of markers for RMS subgroup classification by immunohistochemistry.
Gene expression data were used for selection of subgroup-specific markers. Single sections of RMS with available expression data were used for establishment of the immunohistochemistry. Evaluation of the sensitivity and specificity of the markers was carried out using a tissue array representing 252 RMSs. Kaplan-Meier survival curves were calculated for determination of differences in overall survival of the different staining subgroups.
AP2beta and P-cadherin were selected as markers for pARMS, and epidermal growth factor receptor (EGFR) and fibrillin-2 as markers for ERMS. EGFR + fibrillin-2 detected ERMS with a specificity of 90% and with a sensitivity of 60%. AP2beta + P-cadherin detected pARMS with a specificity of 98% and a sensitivity of 64%, and allowed the detection of several misclassified tumors. The EGFR + fibrillin-2-positive group is associated with a favorable outcome, and the AP2beta + P-cadherin-positive group is associated with an unfavorable outcome.
The presented set of marker proteins detects RMS subgroups with high specificity and may be useful in routine subtype classification of RMS.
To analyze the clinical course, treatment, complications, outcome, and quality of life (QOL) in patients with perineal/perianal rhabdomyosarcoma (PRMS) treated within the CWS-86, -91, -96, and -2002P ...trials.
Although multiple international study trials exist for the treatment of rhabdomyosarcoma, only very limited information is given on treatment, outcome, and QOL in PRMS.
A total of 35 patients suffering from PRMS were treated with neoadjuvant chemotherapy. Local therapy with radiation and/or surgery was performed, followed by adjuvant chemotherapy. Functional long-term follow-up was evaluated by a gastrointestinal/QOL survey.
Thirty-two patients were evaluated (exclusion n = 3). Eight patients had embryonal histology, and 24 patients had alveolar histology. The median age was 108 months (median follow-up: 5.8 years). The 5-year overall survival was 47% (95% confidence interval: 29-64). Sixteen IRS (Intergroup Rhabdomyosarcoma Study) III and IV patients had locoregional lymph node involvement at diagnosis. Seven patients were treated with chemotherapy/surgery alone 5-year event-free survival (EFS): 85.7%. Eleven patients received only radiochemotherapy (5-year EFS: 27.3%). Combined radiochemotherapy/surgery was used in 12 patients (5-year EFS: 63.6%). Two patients were treated only with chemotherapy and they died. Patients with embryonal histology had a significantly better 5-year EFS (87.5%) than patients with alveolar histology (39.1%; P = 0.013). Some patients reported symptoms of fecal incontinence. The median Wexner fecal incontinence score was 9 (possible range: 0-20), and the median QOL score was 90.5 (applicable range: 0-144).
The outcome of these patients remains unsatisfactory. Prognostic factors for a favorable outcome are tumor size of smaller than 5 cm, negative locoregional lymph nodes, age less than 10 years, low IRS group, and embryonal histology. Fecal incontinence seems to be a problem.
Ewing sarcoma (ES) is the second most frequent bone cancer in childhood and is characterized by the presence of the balanced translocation t(11;22)(q24;q12) in more than 85% of cases, generating a ...dysregulated transcription factor EWS/FLI1. This fusion protein is an essential oncogenic component of ES development which is necessary for tumor cell maintenance and represents an attractive therapeutic target. To search for modulators of EWS/FLI1 activity we screened a library of 153 targeted compounds and identified inhibitors of the PI3K pathway to directly modulate EWS/FLI1 transcription. Surprisingly, treatment of four different ES cell lines with BEZ235 resulted in down regulation of EWS/FLI1 mRNA and protein by ~50% with subsequent modulation of target gene expression. Analysis of the EWS/FLI1 promoter region (-2239/+67) using various deletion constructs identified two 14 bp minimal elements as being important for EWS/FLI1 transcription. We identified SP1 as modulator of EWS/FLI1 gene expression and demonstrated direct binding to one of these regions in the EWS/FLI1 promoter by EMSA and ChIP experiments. These results provide the first insights on the transcriptional regulation of EWS/FLI1, an area that has not been investigated so far, and offer an additional molecular explanation for the known sensitivity of ES cell lines to PI3K inhibition.
Rhabdomyosarcoma is a pediatric tumor type, which is classified based on histological criteria into two major subgroups, namely embryonal rhabdomyosarcoma and alveolar rhabdomyosarcoma. The majority, ...but not all, alveolar rhabdomyosarcoma carry the specific PAX3(7)/FKHR-translocation, whereas there is no consistent genetic abnormality recognized in embryonal rhabdomyosarcoma. To gain additional insight into the genetic characteristics of these subtypes, we used oligonucleotide microarrays to measure the expression profiles of a group of 29 rhabdomyosarcoma biopsy samples (15 embryonal rhabdomyosarcoma, and 10 translocation-positive and 4 translocation-negative alveolar rhabdomyosarcoma). Hierarchical clustering revealed expression signatures clearly discriminating all three of the subgroups. Differentially expressed genes included several tyrosine kinases and G protein-coupled receptors, which might be amenable to pharmacological intervention. In addition, the alveolar rhabdomyosarcoma signature was used to classify an additional alveolar rhabdomyosarcoma case lacking any known PAX3 or PAX7 fusion as belonging to the translocation-positive group, leading to the identification of a novel translocation t(2;2)(q35;p23), which generates a fusion protein composed of PAX3 and the nuclear receptor coactivator NCOA1, having similar transactivation properties as PAX3/FKHR. These experiments demonstrate for the first time that gene expression profiling is capable of identifying novel chromosomal translocations.
To develop a score predicting the risk of bacteremia in cancer patients with fever and neutropenia (FN), and to evaluate its performance.
Pediatric patients with cancer presenting with FN induced by ...nonmyeloablative chemotherapy were observed in a prospective multicenter study. A score predicting the risk of bacteremia was developed from a multivariate mixed logistic regression model. Its cross-validated predictive performance was compared with that of published risk prediction rules.
Bacteremia was reported in 67 (16%) of 423 FN episodes. In 34 episodes (8%), bacteremia became known only after reassessment after 8 to 24 hours of inpatient management. Predicting bacteremia at reassessment was better than prediction at presentation with FN. A differential leukocyte count did not increase the predictive performance. The reassessment score predicting future bacteremia in 390 episodes without known bacteremia used the following 4 variables: hemoglobin ≥ 90 g/L at presentation (weight 3), platelet count <50 G/L (3), shaking chills (5), and other need for inpatient treatment or observation according to the treating physician (3). Applying a threshold ≥ 3, the score--simplified into a low-risk checklist--predicted bacteremia with 100% sensitivity, with 54 episodes (13%) classified as low-risk, and a specificity of 15%.
This reassessment score, simplified into a low-risk checklist of 4 routinely accessible characteristics, identifies pediatric patients with FN at risk for bacteremia. It has the potential to contribute to the reduction of use of antimicrobials in, and to shorten the length of hospital stays of pediatric patients with cancer and FN.
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