Identification of genetic variants in pharmacokinetic genes associated with Ewing Sarcoma treatment outcome.

BACKGROUND: Despite the effectiveness of current treatment protocols for Ewing sarcoma (ES), many patients still experience relapse, and survival following recurrence is <15%. We aimed to identify genetic variants that predict treatment outcome in children diagnosed with ES. PATIENTS AND METHODS: We carried out a pharmacogenetic study of 384 single-nucleotide polymorphisms (SNPs) in 24 key transport or metabolism genes relevant to drugs used to treat in pediatric patients (<30 years) with histologically confirmed ES. We studied the association of genotypes with tumor response and overall survival (OS) in a discovery cohort of 106 Spanish children, with replication in a second cohort of 389 pediatric patients from across Europe. RESULTS: We identified associations with OS (P < 0.05) for three SNPs in the Spanish cohort that were replicated in the European cohort. The strongest association observed was with rs7190447, located in the ATP-binding cassette subfamily C member 6 (ABCC6) gene [discovery: hazard ratio (HR) = 14.30, 95% confidence interval (CI) = 1.53-134, P = 0.020; replication: HR = 9.28, 95% CI = 2.20-39.2, P = 0.0024] and its correlated SNP rs7192303, which was predicted to have a plausible regulatory function. We also replicated associations with rs4148737 in the ATP-binding cassette subfamily B member 1 (ABCB1) gene (discovery: HR = 2.96, 95% CI = 1.08-8.10, P = 0.034; replication: HR = 1.60, 95% CI = 1.05-2.44, P = 0.029), which we have previously found to be associated with poorer OS in pediatric osteosarcoma patients, and rs11188147 in cytochrome P450 family 2 subfamily C member 8 gene (CYP2C8) (discovery : HR = 2.49, 95% CI = 1.06-5.87, P = 0.037; replication: HR = 1.77, 95% CI = 1.06-2.96, P = 0.030), an enzyme involved in the oxidative metabolism of the ES chemotherapeutic agents cyclophosphamide and ifosfamide. None of the associations with tumor response were replicated. CONCLUSION: Using an integrated pathway-based approach, we identified polymorphisms in ABCC6, ABCB1 and CYP2C8 associated with OS. These associations were replicated in a large independent cohort, highlighting the importance of pharmacokinetic genes as prognostic markers in ES.

Development of curative therapies for Ewing sarcomas by interdisciplinary cooperative groups in Europe.

Curative therapies for Ewing sarcoma have been developed within cooperative groups. Consecutive clinical trials have systematically assessed the impact and timing of local therapy and the activity of cytotoxic drugs and their combinations. They have led to an increase of long-term disease-free survival to around 70% in patients with localized disease. Translational research in ES remains an area in which interdisciplinary and international cooperation is essential for future progress. This article reviews current state-of-the art therapy, with a focus on trials performed in Europe, and summarizes novel strategies to further advance both the cure rates and quality of survival.

Variability in functional p53 reactivation by PRIMA-1(Met)/APR-246 in Ewing sarcoma.

BACKGROUND: Though p53 mutations are rare in ES, there is a strong indication that p53 mutant tumours form a particularly bad prognostic group. As such, novel treatment strategies are warranted that would specifically target and eradicate tumour cells containing mutant p53 in this subset of ES patients. METHODS: PRIMA-1(Met), also known as APR-246, is a small organic molecule that has been shown to restore tumour-suppressor function primarily to mutant p53 and also to induce cell death in various cancer types. In this study, we interrogated the ability of APR-246 to induce apoptosis and inhibit tumour growth in ES cells with different p53 mutations. RESULTS: APR-246 variably induced apoptosis, associated with Noxa, Puma or p21(WAF1) upregulation, in both mutant and wild-type p53 harbouring cells. The apoptosis-inducing capability of APR-246 was markedly reduced in ES cell lines transfected with p53 siRNA. Three ES cell lines established from the same patient at different stages of the disease and two cell lines of different patients with identical p53 mutations all exhibited different sensitivities to APR-246, indicating cellular context dependency. Comparative transcriptome analysis on the three cell lines established from the same patient identified differential expression levels of several TP53 and apoptosis-associated genes such as APOL6, PENK, PCDH7 and MST4 in the APR-246-sensitive cell line relative to the less APR-246-sensitive cell lines. CONCLUSION: This is the first study reporting the biological response of Ewing sarcoma cells to APR-246 exposure and shows gross variability in responses. Our study also proposes candidate genes whose expression might be associated with ES cells' sensitivity to APR-246. With APR-246 currently in early-phase clinical trials, our findings call for caution in considering it as a potential adjuvant to conventional ES-specific chemotherapeutics.

Suppression of KCMF1 by constitutive high CD99 expression is involved in the migratory ability of Ewing's sarcoma cells.

High CD99 expression levels and rearrangements of the EWS gene with ETS transcription factor genes characterize the Ewing's sarcoma family of tumors (ESFT). CD99 is a cell surface glycoprotein whose engagement has been implicated in cell proliferation as well as upregulation and transport of several transmembrane proteins in hematopoietic cells. In ESFT, antibody ligation of CD99 induces fast homotypic cell aggregation and cell death although its functional role in these processes remains largely unknown. Here, using an RNAi approach, we studied for the first time the consequences of modulated CD99 expression in six different ESFT cell lines, representing the most frequent variant forms of EWS gene rearrangement. CD99 suppression resulted in growth inhibition and reduced migration of ESFT cells. Among genes whose expression changes in response to CD99 modulation, the potassium-channel modulatory factor KCMF1 was consistently upregulated. In a series of 22 primary ESFT, KCMF1 expression levels inversely correlated with CD99 abundancy. Cells forced to express ectopic KCMF1 showed a similar reduction in migratory ability as CD99 silenced ESFT cells. Our results suggest that in ESFT, high CD99 expression levels contribute to the malignant properties of ESFT by promoting growth and migration of tumor cells and identify KCMF1 as a potential metastasis suppressor gene downregulated by high constitutive CD99 expression in ESFT.

EWS-FLI1 perturbs MRTFB/YAP-1/TEAD target gene regulation inhibiting cytoskeletal autoregulatory feedback in Ewing sarcoma.

Ewing sarcoma (EWS) is a paediatric bone cancer with high metastatic potential. Cellular plasticity resulting from dynamic cytoskeletal reorganization, typically regulated via the Rho pathway, is a prerequisite for metastasis initiation. Here, we interrogated the role of the Ewing sarcoma driver oncogene EWS-FLI1 in cytoskeletal reprogramming. We report that EWS-FLI1 strongly represses the activity of the Rho-F-actin signal pathway transcriptional effector MRTFB, affecting the expression of a large number of EWS-FLI1-anticorrelated genes including structural and regulatory cytoskeletal genes. Consistent with this finding, chromatin immunoprecipitation sequencing (ChIP-seq) revealed strong overlaps in myocardin-related transcription factor B (MRTFB) and EWS-FLI1 chromatin occupation, especially for EWS-FLI1-anticorrelated genes. Binding of the transcriptional co-activator Yes-associated protein (YAP)-1, enrichment of TEAD-binding motifs in these shared genomic binding regions and overlapping transcriptional footprints of MRTFB and TEAD factors led us to propose synergy between MRTFB and the YAP/TEAD complex in the regulation of EWS-FLI1-anticorrelated genes. We propose that EWS-FLI1 suppresses the Rho-actin pathway by perturbation of a MRTFB/YAP-1/TEAD transcriptional module, which directly affects the actin-autoregulatory feedback loop. As spontaneous fluctuations in EWS-FLI1 levels of Ewing sarcoma cells in vitro and in vivo, associated with a switch between a proliferative, non-migratory EWS-FLI1-high and a non-proliferative highly migratory EWS-FLI1-low state, were recently described, our data provide a mechanistic basis for the underlying EWS-FLI1-dependent reversible cytoskeletal reprogramming of Ewing sarcoma cells.

A small nuclear RNA, hdm365, is the major processing product of the human mdm2 gene.

mdm2 encodes for an E3 ubiquitin ligase targeting constitutively expressed p53 for proteasomal degradation. Several protein isoforms have been described for human MDM2 (HDM2), some of which may correspond to splicing variants detectable by RT-PCR in many tumors. Upon cellular stress, p53 becomes resistant to MDM2 and, in a feedback loop, up-regulates mdm2 transcription. The physiological relevance of stress-induced mdm2 gene activity is not well understood. We describe a small nuclear RNA of 365 bases comprised of the first five hdm2 exons and lacking polyadenylation. hdm365 precedes full-length hdm2 RNA expression after induction by p53 and accumulates to significant levels in the nucleus, detectable at the site of hdm2 transcription and processing only. Considering a 10-fold lower stability and high steady-state levels of the novel RNA species, hdm365 appears to be the major processing product of hdm2 transcripts. hdm365 induction was observed after ectopic expression of p53 and after DNA damaging treatment of tumor cell lines, primary fibroblasts and lymphocytes, and was not related to apoptosis. Corresponding truncated transcripts were observed in hdm2 amplified cells. High stress-inducible expression levels, absence of a corresponding protein, and nuclear localisation of hdm365 suggest a novel RNA-based function for hdm2.

Adenovirus E1A does not induce the Ewing tumor-associated gene fusion EWS-FLI1.

Rearrangement of the EWS gene with FLI1 is thought to occur early in the pathogenesis of Ewing's sarcoma family tumors (EFTs) because the chromosomal aberration is pathognomonic for this disease. Recently, adenovirus (Ad) 5 E1A protein has been reported to induce this gene rearrangement in a variety of cell types. This finding, if generally substantiated, not only suggests an etiological role for viral agents in the generation of oncogenic chromosomal aberrations but would also significantly impact the use of adenoviral vectors for gene therapy. In contrast, we now report on the absence of EWS-FLI1 chimeric products from short- and long-term cultures of stably Ad-transformed cells lines and from transiently E1A-expressing cell lines. In addition, we demonstrate the absence of E1A from EFTs. We conclude that there is no role for Ads in EFT pathogenesis. Consequently, evidence for a viral genesis of tumor-specific gene rearrangements is not available.

The Ewing tumor family of peripheral primitive neuroectodermal tumors expresses human gastrin-releasing peptide.

The Ewing tumor family of peripheral primitive neuroectodermal tumors (pPNETs) are characterized by chromosomal translocations leading to EWS-ETS gene fusions. These hybrid genes express chimeric proteins that are thought to act as aberrant transcription factors. We therefore used differential display-PCR to compare gene expression patterns in pPNET cell lines with those of other small round cell tumors (SRCTs) of childhood. This technique detected differential expression of sequences corresponding to human gastrin-releasing peptide (GRP) in pPNET cell lines but not in other SRCT cell lines. Subsequent Northern and reverse transcription-PCR analysis of SRCT cell lines confirmed GRP positivity in all pPNET lines tested. Of primary tumors tested by reverse transcription-PCR, GRP expression was found in 7 (44%) of 16 pPNETs but in no other primary SRCTs examined. Expression of the GRP receptor gene was demonstrable in 55% of pPNET cell lines and 25% of primary pPNET tumors but also in several other SRCTs. Radioimmunoassays and immunohistochemistry confirmed expression of bioactive GRP peptide in pPNET cell lines and primary tumors, respectively. Moreover, in vitro growth of a pPNET cell line was slowed by treatment with a GRP receptor antagonist and accelerated by a GRP receptor agonist. GRP is a known autocrine growth factor in small cell lung cancer and other neuroendocrine tumors. Its expression in pPNETs provides further evidence for a neuroectodermal histogenesis of these tumors and suggests that autocrine growth of this family of tumors may be at least partially regulated by GRP.

The EWS protein is dispensable for Ewing tumor growth.

EWS encodes a ubiquitously expressed RNA binding protein with largely unknown function. In Ewing sarcoma family tumors (EFT), one allele is rearranged with an ETS gene. This is the first description of an EFT with a complete EWS deficiency in the presence of two copies of a rearranged chromosome 22 carrying an interstitial EWS-FLI1 translocation. Absence of EWS protein suggested that it is dispensable for EFT growth. By sequencing of EWS cDNA from unrelated EFTs, we excluded inactivation of EWS as a general mechanism in EFT pathogenesis. Rather, EWS was found to be uniformly expressed in two splicing variants of similar abundancy, EWSalpha and EWSbeta, which differ in a single amino acid. Three EWS negative cell lines were established, which will serve as valuable models to study normal and aberrant EWS function upon reintroduction into the tumor cells.

CD99 regulates neural differentiation of Ewing sarcoma cells through miR-34a-Notch-mediated control of NF-κB signaling.

Sarcomas are mesenchymal tumors characterized by blocked differentiation process. In Ewing sarcoma (EWS) both CD99 and EWS-FLI1 concur to oncogenesis and inhibition of differentiation. Here, we demonstrate that uncoupling CD99 from EWS-FLI1 by silencing the former, nuclear factor-κB (NF-κB) signaling is inhibited and the neural differentiation program is re-established. NF-κB inhibition passes through miR-34a-mediated repression of Notch pathway. CD99 counteracts EWS-FLI1 in controlling NF-κB signaling through the miR-34a, which is increased and secreted into exosomes released by CD99-silenced EWS cells. Delivery of exosomes from CD99-silenced cells was sufficient to induce neural differentiation in recipient EWS cells through miR-34a inhibition of Notch-NF-κB signaling. Notably, even the partial delivery of CD99 small interfering RNA may have a broad effect on the entire tumor cell population owing to the spread operated by their miR-34a-enriched exosomes, a feature opening to a new therapeutic option.

p53 mosaicism with an exon 8 germline mutation in the founder of a cancer-prone pedigree.

Changes in the tumor-suppressor gene p53 are frequently acquired during the course of malignant development of human tumors. Recently, constitutional heterozygous mutations in p53 exon 7 have been identified as the primary cause of cancer predisposition in cases of the familial Li-Fraumeni cancer syndrome. These findings underline the need for extensive mutation screening in families with high cancer incidence. This report describes the detection and follow-up by two-dimensional single-strand conformation polymorphism analysis (2DSSCP) of a new germline mutation of p53 exon 8 in a case of suspected Li-Fraumeni syndrome. Although a high cancer incidence had been reported in the family history of the father of siblings suffering from brain tumor and rhabdomyosarcoma, a constitutional heterozygous p53 mutation was identified only in the affected children. Retrospective analysis of archival tissue of a half-sister who died several years ago from a tumor of previously uncertain diagnosis revealed the same mutation. The mutation had therefore occurred in the germ cells of the mother, who thus appears to be a mosaic. The cancer predisposition of the paternal ancestors must have been due to other factors.

Characterization of distinct consecutive phases in non-genotoxic p53-induced apoptosis of Ewing tumor cells and the rate-limiting role of caspase 8.

To dissect the p53-dependent apoptotic pathway, events following induction of temperature sensitive (ts) p53val138 were studied in a Ewing tumor cell line. Transcriptional deregulation of p53 targets first observable after 1 h at 32 degrees C preceded activation of caspases and the break-down of mitochondrial respiratory activity. Activation of caspases was first observed 4 h after p53 induction. Using peptide inhibitors we identified activation of caspase 8 upstream of caspases-9 and -3. Although the caspase 8 specific inhibitor z-IETD.fmk did not affect translocation of BAX to the mitochondrial membrane and cytochrome C release it almost completely blocked cleavage of the prototype caspase substrate PARP and DNA fragmentation while enforcing mitochondrial depolarization and production of reactive oxygene species (ROS). Activation of caspase 8 did not involve death-domain receptor signaling. Expression of BCL2 only partially suppressed caspase activation but blocked apoptosis. Replacement of the N-terminus of p53val138 by the related VP16 transactivation domain created a ts p53 with a tanscriptional activity indistinguishable from p53val138 until the time of caspase activation. However, the VP16 - p53 fusion failed to trigger caspases and subsequent induction of the ROS producing gene pig3 paralleled by complete loss of apoptotic activity. These results indicate that p53-dependent transcriptional deregulation, triggering of the caspase cascade and the mitochondrial break-down occur in a timely ordered sequence coordinated by the genuine p53 amino terminus and suggest caspase 8 and PIG3 as key regulatory elements in this process. Oncogene (2000) 19, 4096 - 4107

Narrow spectrum of infrequent p53 mutations and absence of MDM2 amplification in Ewing tumours.

The p53 and MDM2 genes are part of a physiological pathway frequently impaired in human cancer. With the exception of tumours occasionally associated with hereditary predisposition, childhood malignancies have not been studied in detail yet. This is the first report on the analysis of p53 and MDM2 in a group of non-hereditary paediatric neoplasms referred to as the Ewing tumours (ETs). Thirty-seven primary tumours and cell lines from 19 patients were screened for the presence of p53 mutations. Only 5% of the primary tumour specimens were found to carry an alteration within this gene. However, p53 mutations were 10-fold enriched in ET cell lines, thus indicating a selective growth advantage in vitro. Strikingly, five out of nine alterations detected were missense mutations within codon 273, which were previously reported to impair only partially the normal p53 function. Two single-base substitutions occurred at codons 277 and 176, and two alterations were loss-of-function mutations. Investigation of the MDM2 gene revealed neither gene amplification in the primary tumours and cell lines nor significant overexpression in any of the cell lines. Our data therefore suggest that impairment of cellular mechanisms involving p53 is rare in a distinct group of childhood malignancies.

Among genes involved in the RB dependent cell cycle regulatory cascade, the p16 tumor suppressor gene is frequently lost in the Ewing family of tumors.

The pRB cell cycle regulatory cascade is frequently perturbed in neoplasia by overexpression of a component of the pRB-phosphorylating cyclin D1/CDK4 complex or by inactivation of pRB or the CDK4 inhibitors p16 and p15. We investigated the status and expression of p16, p15, CCND1, CDK4 and RB genes in the Ewing family of tumors. P16 loss was observed in 8 of 27 tumors (30%) and in 12 of 23 (52%) tumor cell lines from unrelated patients. There were no discrepancies in the p16 status between primary tumors and the corresponding cell lines and between cell lines established from consecutive tumor samples. p15 was codeleted in most cases but p15 mRNA was absent also in cell lines retaining the gene. In addition, posttranscriptional p16 inactivation was observed in two cases. Although no evidence for CDK4 or CCND1 amplification was obtained, expression of these genes varied considerably in the cell lines in a case specific manner. In wild-type p16 cell lines, pRB expression was lost in one case. Our data indicate that, despite the absence of cytogenetically detectable 9p21 chromosomal aberrations, p16 deletions constitute the most frequent secondary molecular aberration in Ewing tumors so far. These results are discussed in the context of the stage of disease and the clinical outcome of the patients. The potential prognostic impact of these findings remains to be further evaluated.

Use of transcription reporters with novel p53 binding sites to target tumour cells expressing endogenous or virally transduced p53 mutants with altered sequence-specificity.

p53 triple mutants (120N/121G/277H, 120H/121G/ 277H, 120S/121G/277H and 120H/121G/277Y) have altered sequence specificity in bandshift assays in vitro and transcription assays in vivo. These mutants activate transcription from the site TTT CATG AAA but not from wild type sites. The triple mutants activate more strongly than p53 with a single 277Y mutation. The TTT site matches the wild type p53 consensus at only 4/10 positions and is not recognised by wild type p53. 277Y mutations have been described in human tumours, and Ewing tumour cells expressing this mutant from the endogenous p53 locus selectively activate transcription from transfected luciferase reporters regulated by TTT-mutant p53 binding sites. p53 mutants with altered sequence specificity have potential advantages for cancer gene therapy: if used to activate transcription of conditionally toxic genes they would allow tumour-targeting by p53, which acts as a sensor for the malignant state, but place control over cell killing in the hands of the clinician. Rare tumours expressing such mutants from the endogenous p53 locus could be targeted directly with p53-regulated suicide vectors, but for most tumours both the p53 mutant and the reporter would need to be encoded by the virus.

Oncogenic EWS-Fli1 interacts with hsRPB7, a subunit of human RNA polymerase II.

As a result of the t(11;22)(q24;q12) chromosomal translocation characterizing the Ewing family of tumors (ET), the amino terminal portion of EWS, an RNA binding protein of unknown function, is fused to the DNA-binding domain of the ets transcription factor Fli1. The hybrid EWS-Fli1 protein acts as a strong transcriptional activator and, in contrast to wildtype Fli1, is a potent transforming agent. Similar rearrangements involving EWS or the highly homologous TLS with various transcription factors have been found in several types of human tumors. Employing yeast two-hybrid cloning we isolated the seventh largest subunit of human RNA polymerase II (hsRPB7) as a protein that specifically interacts with the amino terminus of EWS. This association was confirmed by in vitro immunocoprecipitation. In nuclear extracts, hsRPB7 was found to copurify with EWS-Fli1 but not with Fli1. Overexpression of recombinant hsRPB7 specifically increased gene activation by EWS-chimeric transcription factors. Replacement of the EWS portion by hsRPB7 in the oncogenic fusion protein restored the transactivating potential of the chimera. Our results suggest that the interaction of the amino terminus of EWS with hsRPB7 contributes to the transactivation function of EWS-Fli1 and, since hsRPB7 has characteristics of a regulatory subunit of RNA polymerase II, may influence promoter selectivity.

Overexpression of the pseudoautosomal gene MIC2 in Ewing's sarcoma and peripheral primitive neuroectodermal tumor.

Ewing's Sarcoma (ES), the second most frequent bone tumor in childhood and adolescence, and the probably closely related peripheral primitive neuroectodermal tumor (pPNET) share a unique cytogenetic translocation between chromosomes 11 and 22. Both of them expose high amounts of a glycoprotein on their cell surface, which can be specifically detected by the mAb HBA-71. The cDNA coding for the HBA-71 antigen was isolated by screening a cDNA expression library constructed from a pPNET-derived cell line. Nucleotide sequencing revealed the HBA-71 antigen to be the product of the pseudoautosomal gene MIC2 previously identified by the mAb 12E7 in haematopoietic cells. This antigen is a glycoprotein with a molecular weight of about 29,000 and is expressed in low amounts in most human cell lines and probably normal tissues and tumors with only a few exceptions. In T-cells the antigen is involved in cell adhesion processes. In ES- and pPNET-derived cell lines MIC2 expression is significantly enhanced. No gross changes in posttranslational modification could be observed. The high expression results in easy and specific detection of the antigen in immunocytochemical analysis of paraffin embedded tissue sections making HBA-71 a useful tool in tumor diagnosis.

Co-amplification of a novel gene, NAG, with the N-myc gene in neuroblastoma.

Substantial evidence implicates amplification of the N-myc gene with aggressive tumor growth and poor outcome in neuroblastoma. However some evidence suggests that this gene alone is not the sole determinant of outcome in N-myc amplified tumors. We have searched for genes that co-amplify with N-myc in neuroblastoma by means of two-dimensional analysis of genomic restriction digests. Using this approach, we have identified and cloned a novel genomic fragment which is co-amplified with N-myc in neuroblastomas. This fragment was mapped in close vicinity to N-myc on chromosome arm 2p24. It was amplified in 5/8 N-myc amplified neuroblastoma cell lines and in 9/13 N-myc amplified tumors. Using a PCR-based approach we isolated a 4.5 kb c-DNA sequence that is partly contained in the genomic fragment. The open reading frame of the cDNA encodes a predicted protein of 1353 amino acids (aa). The homology of the predicted protein, which we designated NAG (neuroblastoma amplified gene), to a C. elegans protein of as yet unknown function, and its ubiquitous expression suggest that NAG may serve an essential function. By Northern blot analysis we showed that amplification of the cloned gene correlates with over-expression in neuroblastoma cell lines. Amplification and consequent over-expression of NAG may, therefore, contribute to the phenotype of a subset of neuroblastomas.

Does expression of different EWS chimeric transcripts define clinically distinct risk groups of Ewing tumor patients?

PURPOSE: Because of the high heterogeneity of EWS gene fusions with FLI1 and ERG genes due to variable chromosomal breakpoint locations in Ewing tumors (ET) (14 different chimeric transcripts identified so far), we evaluated the clinical impact of the expression of diverse fusion transcripts in ET patients. PATIENTS AND METHODS: In a European multicenter study, 147 ET were analyzed by reverse-transcriptase polymerase chain reaction (RT-PCR) and the molecular data statistically compared with all clinical data available. RESULTS: Most tumors expressed chimeric transcripts with fusion of EWS exon 7 to FLI1 exon 6 (75 of 147) (type I) or five (39 of 147) and EWS exon 10 to FLI1 exon 5 (eight of 147) or 6 (five of 147). In five cases, chimerism between EWS exon 9 and FLI1 exons 4 and EWS exon 7 and FLI1 exon 7 or 8 was observed. Fifteen cases of EWS-ERG rearrangement were identified. In 85 of these patients treated in the European Cooperative Ewing Sarcoma Studies, molecular results were analyzed in comparison to age, sex, tumor localization, tumor volume, and disease extension. No significant correlation between the various fusion types and these features were observed. Relapse-free survival (RFS) for the 31 patients with localized disease and fusion type I tended to be longer compared with the 24 patients with localized tumors bearing other chimeric transcripts (P = .04). CONCLUSION: Results suggest a possible advantage in PFS for patients with localized disease and fusion type I transcripts, although this will require prospective validation with a larger number of patients and longer follow-up periods.