Andrew Proctor

Samantha C Brownhill

Susan Ann Burchill
email: s.a.burchill@leeds.ac.uk

The promise of telomere length, telomerase activity and its regulation in the translocation dependent cancer ESFT; clinical challenges and utility

Keywords: Telomerase, EWS-ETS fusion transcripts, Translocation-dependent cancer, Clinical utility, Standardisation

The Ewing's sarcoma family of tumours (ESFT) are diagnosed by EWS-ETS gene translocations. The resulting fusion proteins play a role in both the initiation and maintenance of these solid aggressive malignant tumours, suppressing cellular senescence and increasing cell proliferation and survival. EWS-ETS fusion proteins have altered transcriptional activity, inducing expression of a number of different target genes including telomerase. Up-regulation of hTERT is most likely responsible for the high levels of telomerase activity in primary ESFT, although telomerase activity and expression of hTERT are not predictive of outcome. However levels of telomerase activity in peripheral blood may be useful to monitor response to some therapeutics.

Despite high levels of telomerase activity, telomeres in ESFT are frequently shorter than those of matched normal cells. Uncertainty about the role that telomerase and regulators of its activity play in maintenance of telomere length in normal and cancer cells, and lack of studies examining the relationship between telomerase activity, regulators of its activity and their clinical significance in patient samples has limited their introduction into clinical practice. Studies in clinical samples using standardised assays are critical to establish how telomerase and regulators of its activity might best be exploited for patient benefit.

The Ewing's sarcoma family of tumours (ESFT) include Ewing's sarcoma, peripheral primitive neuroectodermal tumours (pPNET) and Askin tumour. These solid tumours share similar histology, under the light microscope appearing as small round cells with regular nuclei surrounded by a narrow rim of clear or pale cytoplasm (Figure 1). Some tumours contain rosettes suggestive of a tendency towards neural differentiation, although these tumours are largely poorly differentiated. Ultrastructurally the tumour cells are primitive, with scant organelles and cytoplasmic glycogen pools or aggregates [START_REF] Burchill | Primitive Neuroectodermal Tumours[END_REF], 2]. The tumours arise in a number of different sites, most frequently in the bone (Ewing's sarcoma) or soft tissues (pPNET or Askin tumour). The ESFT of bone are thought to originate in the intra-medullary cavity rapidly progressing onto the cortex to produce a soft tissue component, whereas soft tissue ESFT often occur adjacent to the bone with no apparent intraosseous component or in isolated soft tissues. There is a peak incidence of diagnosis in young people between the ages of 10 and 24 years, affecting approximately 0.6 per million total population with a male to female ratio of 1.5:1. ESFT is not associated with familial cancer syndromes, although caucasians are more frequently affected than afro-caribbean and chinese populations [START_REF] Glass | Epidemiology of bone cancer in children[END_REF][START_REF] Li | Rarity of Ewing's sarcoma in China[END_REF][START_REF] Parkin | The international incidence of childhood cancer[END_REF]. The identification of non-random chromosome rearrangements between the EWS gene on chromosome 22q12 and a member of the ETS gene family is strong evidence for the common histogenesis of these tumours, and provides a valuable characteristic for their differential diagnosis from other small round cell tumours of childhood [START_REF] Burchill | Molecular abnormalities in Ewing's sarcoma[END_REF].

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Multi-agent chemotherapy, surgery, and radiation therapy have improved the 5 year progression-free survival (PFS) of newly diagnosed patients with localised disease to 70%. However, the 5 year PFS for patients who present with pulmonary metastases is 40% and for those with disease in the bone and/or bone marrow is <20%. Despite aggressive treatment regimens and improved local control therapy, recurrence and metastasis continue to pose the most difficult challenge for management and curative treatment of these solid cancers. Of the patients who relapse only 49-60% achieve partial or complete remission to second line treatment [START_REF] Barker | Survival after recurrence of Ewing's sarcoma family of tumors[END_REF][START_REF] Hunold | Topotecan and cyclophosphamide in patients with refractory or relapsed Ewing tumors[END_REF][START_REF] Shankar | Outcome after relapse in an unselected cohort of children and adolescents with Ewing sarcoma[END_REF][START_REF] Wagner | Temozolomide and intravenous irinotecan for treatment of advanced Ewing sarcoma[END_REF]. These figures emphasise the urgent need for greater understanding of the molecular events that contribute to the development and maintenance of the ESFT malignant phenotype, to identify both biomarkers to improve selection and stratification of patients for therapy and targets and pathways for the design of more effective cancer therapeutics.

EWS-ETS translocations and the transformed phenotype

ESFT is one of the few solid tumours for which the underlying molecular genetic abnormality has been described, that is rearrangement of the EWS gene on chromosome 22q12 with an ETS gene family member. These rearrangements have not been reported in normal cells. The most frequent translocation is the t(11;22)(q24;q12) which is described in 85% of tumours, resulting in the chimeric EWS-FLI1 fusion. Other EWS-ETS fusions have been described in the remaining 15% of tumours (Table 1).

These diagnostic translocations can be identified by conventional G-banding, although diagnostic accuracy is often improved using reverse transcriptase polymerase chain reaction and/or fluorescent in situ hybridisation (FISH) [START_REF] Desmaze | Unicolor and bicolor in situ hybridization in the diagnosis of peripheral neuroepithelioma and related tumors[END_REF][START_REF] Peter | A multiplex real-time pcr assay for the detection of gene fusions observed in solid tumors[END_REF][START_REF] Taylor | Diagnosis of Ewing's sarcoma and peripheral neuroectodermal tumour based on the detection of t(11;22) using fluorescence in situ hybridisation[END_REF] (Figure 1).

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The EWS-ETS fusion proteins contain the transactivating domain of EWS and the DNA binding domain of the corresponding ETS fusion partner (Table 1). Consistent with the hypothesis that the EWS-ETS fusions are important for the development and maintenance of the ESFT malignant phenotype, expression of EWS-FLI1 in a proportion of NIH3T3 cells increases the ability of these cells to form tumours in soft agar or in immunocompromised mice [START_REF] May | Ewing sarcoma 11;22 translocation produces a chimeric transcription factor that requires the DNA-binding domain encoded by FLI1 for transformation[END_REF][START_REF] Thompson | Divergent Ewing's sarcoma EWS/ETS fusions confer a common tumorigenic phenotype on NIH3T3 cells[END_REF], whereas decreased expression of EWS-FLI1 in ESFT cells in vitro appears to decrease proliferation [START_REF] Kovar | EWS/FLI-1 antagonists induce growth inhibition of Ewing tumor cells in vitro[END_REF][START_REF] Prieur | EWS/FLI-1 silencing and gene profiling of Ewing cells reveal downstream oncogenic pathways and a crucial role for repression of insulin-like growth factor binding protein 3[END_REF][START_REF] Tanaka | EWS-Fli1 antisense oligodeoxynucleotide inhibits proliferation of human Ewing's sarcoma and primitive neuroectodermal tumor cells[END_REF] and reduce tumour growth and/or metastases in preclinical mouse models [START_REF] Prieur | EWS/FLI-1 silencing and gene profiling of Ewing cells reveal downstream oncogenic pathways and a crucial role for repression of insulin-like growth factor binding protein 3[END_REF][START_REF] Chansky | Targeting of EWS/FLI-1 by RNA interference attenuates the tumor phenotype of Ewing's sarcoma cells in vitro[END_REF][START_REF] Hu-Lieskovan | Sequencespecific knockdown of EWS-FLI1 by targeted, nonviral delivery of small interfering RNA inhibits tumor growth in a murine model of metastatic Ewing's sarcoma[END_REF][START_REF] Lambert | EWS fli-1 antisense nanocapsules inhibits ewing sarcomarelated tumor in mice[END_REF][START_REF] Ouchida | Loss of tumorigenicity of Ewing's sarcoma cells expressing antisense RNA to EWS-fusion transcripts[END_REF]. More recently EWS-FLI1 has been shown to suppress cellular senescence [START_REF] Hu | EWS/FLI1 suppresses retinoblastoma protein function and senescence in Ewing's sarcoma cells[END_REF][START_REF] Matsunobu | The possible role of EWS-Fli1 in evasion of senescence in Ewing family tumors[END_REF]. The transforming activity of EWS-ETS fusions has largely been attributed to the altered transcriptional activity and targets of the EWS-ETS gene fusion proteins compared to those of the wild-type genes alone [START_REF] May | Ewing sarcoma 11;22 translocation produces a chimeric transcription factor that requires the DNA-binding domain encoded by FLI1 for transformation[END_REF][START_REF] Bailly | DNA-binding and transcriptional activation properties of the EWS-FLI-1 fusion protein resulting from the t(11;22) translocation in Ewing sarcoma[END_REF][START_REF] Ladanyi | EWS-FLI1 and Ewing's sarcoma: recent molecular data and new insights[END_REF][START_REF] May | The Ewing's sarcoma EWS/FLI-1 fusion gene encodes a more potent transcriptional activator and is a more powerful transforming gene than FLI-1[END_REF][START_REF] Ohno | EWS/Fli-1 chimeric protein is a transcriptional activator[END_REF][START_REF] Siligan | EWS-FLI1 target genes recovered from Ewing's sarcoma chromatin[END_REF]. A number of target genes are differentially induced or repressed during EWS-FLI1 dependent transformation [START_REF] Burchill | Molecular abnormalities in Ewing's sarcoma[END_REF], although the detailed mechanism of transformation remains elusive. Since no single or group of gene(s) has yet been identified that reproduces the ESFT malignant phenotype it has been suggested that transcriptional activity may not be the only mechanism of EWS-ETS fusion-induced transformation. Consistent with this hypothesis, introduction of point mutations in the FLI1 DNA binding domain (preventing DNA binding) do not necessarily inhibit the transforming activity of EWS-FLI1 [START_REF] Jaishankar | Transforming activity of EWS/FLI is not strictly dependent upon DNA-binding activity[END_REF]. Furthermore some studies have suggested that the transforming activity of EWS-FLI1 might be attributed to modulation of RNA processing [START_REF] Chansky | Oncogenic TLS/ERG and EWS/Fli-1 fusion proteins inhibit RNA splicing mediated by YB-1 protein[END_REF][START_REF] Knoop | EWS/FLI alters 5'-splice site selection[END_REF][START_REF] Yang | EWS.Fli-1 fusion protein interacts with hyperphosphorylated RNA polymerase II and interferes with serine-arginine proteinmediated RNA splicing[END_REF]. These observations are consistent with the hypothesis that the fusion gene alone is insufficient to cause overt ESFT, and that accumulation of secondary genetic events is essential for clinical manifestation of the transformed phenotype.

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Secondary abnormalities

Although the EWS-ETS fusions are important for the development and maintenance of the ESFT malignant phenotype, secondary genetic abnormalities have been described in over 80% of tumours. Various numerical and structural aberrations have been identified, some of which are reported to be prognostically significant. Chromosome gain is more frequently described than chromosome loss, those patients presenting with more than 50 chromosomes having a significantly worse over-all survival rate [START_REF] Roberts | Ploidy and karyotype complexity are powerful prognostic indicators in the Ewing's sarcoma family of tumors: a study by the United Kingdom Cancer Cytogenetics and the Children's Cancer and Leukaemia Group[END_REF]. A more complex karyotype has also been associated with a worse outcome [START_REF] Roberts | Ploidy and karyotype complexity are powerful prognostic indicators in the Ewing's sarcoma family of tumors: a study by the United Kingdom Cancer Cytogenetics and the Children's Cancer and Leukaemia Group[END_REF][START_REF] Zielenska | Acquisition of secondary structural chromosomal changes in pediatric ewing sarcoma is a probable prognostic factor for tumor response and clinical outcome[END_REF]. Although high-ploidy has been described in a small subset of tumours [START_REF] Betts | Cytogenetic characterization of Ewing tumors with high-ploidy[END_REF], ESFT are usually diploid or near triploid [START_REF] Niggli | DNA ploidy and proliferative activity (S-phase) in childhood soft-tissue sarcomas: their value as prognostic indicators[END_REF]. Trisomies of chromosome 8 [START_REF] Armengol | Recurrent gains of 1q, 8 and 12 in the Ewing family of tumours by comparative genomic hybridization[END_REF][START_REF] Hattinger | Prognostic impact of deletions at 1p36 and numerical aberrations in Ewing tumors[END_REF][START_REF] Maurici | Frequency and implications of chromosome 8 and 12 gains in Ewing sarcoma[END_REF][START_REF] Tarkkanen | Clinical correlations of genetic changes by comparative genomic hybridization in Ewing sarcoma and related tumors[END_REF] and 12 [START_REF] Hattinger | Prognostic impact of deletions at 1p36 and numerical aberrations in Ewing tumors[END_REF] are frequently described and associated (alone or in combination) with poor disease course and clinical outcome, although this has been disputed [START_REF] Roberts | Ploidy and karyotype complexity are powerful prognostic indicators in the Ewing's sarcoma family of tumors: a study by the United Kingdom Cancer Cytogenetics and the Children's Cancer and Leukaemia Group[END_REF][START_REF] Brisset | CGH analysis of secondary genetic changes in Ewing tumors: correlation with metastatic disease in a series of 43 cases[END_REF]. Several other trisomies including 2, 5, 7, 14 and 21 have been reported, although there is no compelling evidence that these are of prognostic value. A number of structural imbalances have also been described, including the unbalanced translocation t(1;16) [START_REF] Zielenska | Acquisition of secondary structural chromosomal changes in pediatric ewing sarcoma is a probable prognostic factor for tumor response and clinical outcome[END_REF][START_REF] Douglass | A second nonrandom translocation, der(16)t(1;16)(q21;q13), in Ewing sarcoma and peripheral neuroectodermal tumor[END_REF][START_REF] Kovar | 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[END_REF][START_REF] Kullendorff | Cytogenetic aberrations in Ewing sarcoma: are secondary changes associated with clinical outcome?[END_REF][START_REF] Mugneret | Chromosomes in Ewing's sarcoma. II. Nonrandom additional changes, trisomy 8 and der(16)t(1;16)[END_REF], deletions of 1p36 [START_REF] Zielenska | Acquisition of secondary structural chromosomal changes in pediatric ewing sarcoma is a probable prognostic factor for tumor response and clinical outcome[END_REF][START_REF] Hattinger | Prognostic impact of deletions at 1p36 and numerical aberrations in Ewing tumors[END_REF][START_REF] Kovar | 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[END_REF], 9p21 [START_REF] Roberts | Ploidy and karyotype complexity are powerful prognostic indicators in the Ewing's sarcoma family of tumors: a study by the United Kingdom Cancer Cytogenetics and the Children's Cancer and Leukaemia Group[END_REF][START_REF] Kovar | 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[END_REF][START_REF] Huang | Ewing sarcomas with p53 mutation or p16/p14ARF homozygous deletion: a highly lethal subset associated with poor chemoresponse[END_REF][START_REF] Lopez-Guerrero | Molecular analysis of the 9p21 locus and p53 genes in Ewing family tumors[END_REF][START_REF] Maitra | Aberrant expression of tumor suppressor proteins in the Ewing family of tumors[END_REF][START_REF] Wei | Prognostic impact of INK4A deletion in Ewing sarcoma[END_REF] and gain of 1q [START_REF] Roberts | Ploidy and karyotype complexity are powerful prognostic indicators in the Ewing's sarcoma family of tumors: a study by the United Kingdom Cancer Cytogenetics and the Children's Cancer and Leukaemia Group[END_REF][START_REF] Hattinger | Prognostic impact of chromosomal aberrations in Ewing tumours[END_REF][START_REF] Ozaki | Genetic imbalances revealed by comparative genomic hybridization in Ewing tumors[END_REF]. Interestingly, the number of imbalances is generally reported to be more common in localised than metastatic tumours [START_REF] Hattinger | Prognostic impact of deletions at 1p36 and numerical aberrations in Ewing tumors[END_REF][START_REF] Brisset | CGH analysis of secondary genetic changes in Ewing tumors: correlation with metastatic disease in a series of 43 cases[END_REF].

However the clinical significance of many of these secondary abnormalities remains unclear, reflecting in part the small number of patients in which they have been systematically evaluated.

The most frequently described secondary genetic aberrations in ESFT are deletions of the CDKN2A locus on chromosome 9p, and abnormalities of genes that regulate the G1 cell A C C E P T E D M A N U S C R I P T Linking powered by eXtyles cycle checkpoint. Although loss of the retinoblastoma gene product pRb is rarely described in primary ESFT [START_REF] Kovar | 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[END_REF][START_REF] Maitra | Aberrant expression of tumor suppressor proteins in the Ewing family of tumors[END_REF], loss of the cyclin-dependent kinase (CDK) inhibitors p16, p21 WAF and p27 KIP1 (that bind to the CDK-cyclin D1 complexes preventing inactivation of pRb and consequently stopping G1/S transition) have been reported. Of these, deletion of CDKN2A exon 1α (which codes for p16) is the most frequently described, occurring in between 13 and 39% of primary ESFT [START_REF] Kovar | 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[END_REF][START_REF] Huang | Ewing sarcomas with p53 mutation or p16/p14ARF homozygous deletion: a highly lethal subset associated with poor chemoresponse[END_REF][START_REF] Maitra | Aberrant expression of tumor suppressor proteins in the Ewing family of tumors[END_REF][START_REF] Wei | Prognostic impact of INK4A deletion in Ewing sarcoma[END_REF][START_REF] Brownhill | Chromosome 9p21 gene copy number and prognostic significance of p16 in ESFT[END_REF][START_REF] Tsuchiya | Analysis of the p16INK4, p14ARF, p15, TP53, and MDM2 genes and their prognostic implications in osteosarcoma and Ewing sarcoma[END_REF].

Although mutations in p21 WAF have not been described loss of expression is reported in 55-60% of tumours [START_REF] Maitra | Aberrant expression of tumor suppressor proteins in the Ewing family of tumors[END_REF][START_REF] De Alava | Prognostic impact of P53 status in Ewing sarcoma[END_REF]; whether this is of prognostic significance remains to be seen. Loss or low expression of the cyclin dependent kinase inhibitor p27 KIP1 has however been related to poor event-free survival [START_REF] Matsunobu | The prognostic and therapeutic relevance of p27kip1 in Ewing's family tumors[END_REF]. Aberrations of p53 have also been described [START_REF] Kovar | 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[END_REF][START_REF] Patino-Garcia | Analysis of the p16INK4 and TP53 tumor suppressor genes in bone sarcoma pediatric patients[END_REF][START_REF] Toguchida | Mutation spectrum of the p53 gene in bone and soft tissue sarcomas[END_REF], and although mutations of p53 are reported in only a small subset of primary ESFT (<10%) in these patients they are associated with a poor prognosis [START_REF] Lopez-Guerrero | Molecular analysis of the 9p21 locus and p53 genes in Ewing family tumors[END_REF][START_REF] De Alava | Prognostic impact of P53 status in Ewing sarcoma[END_REF][START_REF] Kovar | Narrow spectrum of infrequent p53 mutations and absence of MDM2 amplification in Ewing tumours[END_REF]]. However, most tumours express wild-type p53, high levels of which in primary tumours appear to be an indicator of poor outcome [START_REF] Abudu | Overexpression of p53 protein in primary Ewing's sarcoma of bone: relationship to tumour stage, response and prognosis[END_REF][START_REF] Amir | Expression of p53 Linking powered by eXtyles gene product and cell proliferation marker Ki-67 in Ewing's sarcoma: correlation with clinical outcome[END_REF].

ESFT signatures and cell of origin

Genome-wide analysis of primary ESFT has identified tumour specific gene signatures that can distinguish ESFT from other bone and soft tissue cancers, [START_REF] Baer | Profiling and functional annotation of mRNA gene expression in pediatric rhabdomyosarcoma and Ewing's sarcoma[END_REF][START_REF] Bandres | Gene expression profile of ewing sarcoma cell lines differing in their EWS-FLI1 fusion type[END_REF][START_REF] Davicioni | Diagnostic and prognostic sarcoma signatures[END_REF][START_REF] Ferreira | Array CGH and gene-expression profiling reveals distinct genomic instability patterns associated with DNA repair and cell-cycle checkpoint pathways in Ewing's sarcoma[END_REF][START_REF] Ohali | Prediction of high risk Ewing's sarcoma by gene expression profiling[END_REF]. It is anticipated that these profiles will elucidate common molecular pathways that are responsible for the ESFT phenotype [START_REF] Schaefer | Microarray analysis of Ewing's sarcoma family of tumours reveals characteristic gene expression signatures associated with metastasis and resistance to chemotherapy[END_REF], and may identify common targets for the development of novel therapeutics.

Recent profiles of tumours [START_REF] Staege | DNA microarrays reveal relationship of Ewing family tumors to both endothelial and fetal neural crest-derived cells and define novel targets[END_REF] and model systems of ESFT [START_REF] Bandres | Gene expression profile of ewing sarcoma cell lines differing in their EWS-FLI1 fusion type[END_REF] suggest that ESFT may arise in as yet poorly defined mesenchymal progenitor cells. Consistent with this hypothesis, knock-down of EWS-FLI1 in ESFT cells results in a gene expression profile A C C E P T E D M A N U S C R I P T Linking powered by eXtyles that more closely resembles the mesenchymal stem cell than the original ESFT cell [START_REF] Tirode | Mesenchymal stem cell features of Ewing tumors[END_REF].

Furthermore expression of EWS-FLI1 in human bone marrow-derived mesenchymal progenitor cells induces transformation to generate cells that grow tumours that resemble ESFT histologically in nude mice [START_REF] Miyagawa | Inducible expression of chimeric EWS/ETS proteins confers Ewing's family tumor-like phenotypes to human mesenchymal progenitor cells[END_REF][START_REF] Riggi | EWS-FLI-1 expression triggers a Ewing's sarcoma initiation program in primary human mesenchymal stem cells[END_REF].

Early studies favour a primitive neural crest cell as the putative cell of origin, based largely on immature tumour neural phenotype and the potential of some cells for neural differentiation [START_REF] Cavazzana | Experimental evidence for a neural origin of Ewing's sarcoma of bone[END_REF][START_REF] Noguera | Dynamic model of differentiation in Ewing's sarcoma cells. Comparative analysis of morphologic, immunocytochemical, and oncogene expression parameters[END_REF]. As these tumours are predominantly cholinergic [START_REF] O'regan | A Ewing's sarcoma cell line showing some, but not all, of the traits of a cholinergic neuron[END_REF][START_REF] Thiele | Pediatric peripheral neuroectodermal tumors, oncogenes, and differentiation[END_REF], they may arise from postganglionic parasympathetic primordial cells which are located throughout the parasympathetic autonomic nervous system. The wide distribution of these pluripotent stem cells is consistent with the variety of soft tissue and bony locations in which ESFT are found. More recently the expression of EWS-FLI1 in neural crest stem cells has been shown to disrupt normal neural crest development [START_REF] Coles | EWS-FLI1 causes neuroepithelial defects and abrogates emigration of neural crest stem cells[END_REF], providing further support that neural crest stem cells may be the cells of origin for this aggressive malignant tumour. However expression of the EWS-ETS fusions can also induce transformation of fibroblasts [START_REF] May | Ewing sarcoma 11;22 translocation produces a chimeric transcription factor that requires the DNA-binding domain encoded by FLI1 for transformation[END_REF], haemopoietic cells [START_REF] Codrington | The Ews-ERG fusion protein can initiate neoplasia from lineage-committed haematopoietic cells[END_REF] and cells derived from primary bone [START_REF] Castillero-Trejo | Expression of the EWS/FLI-1 oncogene in murine primary bone-derived cells Results in EWS/FLI-1-dependent, ewing sarcoma-like tumors[END_REF], demonstrating that although the transforming activity of the EWS-ETS fusion may be dependent on cellular context this is not strictly limited to a primitive mesenchymal or neural cell milieu. Furthermore the capacity of the EWS-FLI1 fusion to induce a neurosecretory-phenotype in mesenchymal-derived NIH 3T3 cells [START_REF] Teitell | EWS/ETS fusion genes induce epithelial and neuroectodermal differentiation in NIH 3T3 fibroblasts[END_REF] makes the identification of the cell in which ESFT arise even more challenging, since the limited neural phenotype associated with some ESFT might not reflect the cell of origin but rather be a consequence of the transforming EWS-FLI1 fusion. The cell, or indeed cells, in which ESFT arise therefore currently remain(s) elusive.

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Telomerase and regulation of its activity in ESFT

Telomerase activity is up-regulated by the transcriptional activator EWS-FLI1 [START_REF] Takahashi | EWS/ETS fusions activate telomerase in Ewing's tumors[END_REF], the most frequently described primary gene alteration that characterises ESFT. It is therefore unsurprising that telomerase activity has been detected in both primary ESFT samples [START_REF] Ohali | Association between telomerase activity and outcome in patients with nonmetastatic Ewing family of tumors[END_REF][START_REF] Sotillo-Pineiro | Telomerase activity and telomere length in primary and metastatic tumors from pediatric bone cancer patients[END_REF][START_REF] Ulaner | Divergent patterns of telomere maintenance mechanisms among human sarcomas: sharply contrasting prevalence of the alternative lengthening of telomeres mechanism in Ewing's sarcomas and osteosarcomas[END_REF] and ESFT cell lines [START_REF] Takahashi | EWS/ETS fusions activate telomerase in Ewing's tumors[END_REF][START_REF] Ulaner | Divergent patterns of telomere maintenance mechanisms among human sarcomas: sharply contrasting prevalence of the alternative lengthening of telomeres mechanism in Ewing's sarcomas and osteosarcomas[END_REF][START_REF] Kleideiter | Telomerase activity in cell lines of pediatric soft tissue sarcomas[END_REF][START_REF] Lanvers-Kaminsky | Doxorubicin modulates telomerase activity in Ewing's sarcoma in vitro and in vivo[END_REF]. The detection of the human telomerase reverse transcriptase (hTERT) catalytic subunit of telomerase in primary ESFT by immunohistochemistry is consistent with these observations [START_REF] Fuchs | hTERT Is highly expressed in Ewing's sarcoma and activated by EWS-ETS oncoproteins[END_REF]. However, neither telomerase activity nor expression of hTERT have been reported to predict outcome in patients with ESFT [START_REF] Ohali | Association between telomerase activity and outcome in patients with nonmetastatic Ewing family of tumors[END_REF][START_REF] Fuchs | hTERT Is highly expressed in Ewing's sarcoma and activated by EWS-ETS oncoproteins[END_REF]. One study has described higher levels of telomerase activity in metastatic lesions of ESFT compared to levels in tumours at diagnosis [START_REF] Sotillo-Pineiro | Telomerase activity and telomere length in primary and metastatic tumors from pediatric bone cancer patients[END_REF], though the proportion of positive primary tumours in this study was much lower (12%) than that reported in other studies (70% [START_REF] Ulaner | Divergent patterns of telomere maintenance mechanisms among human sarcomas: sharply contrasting prevalence of the alternative lengthening of telomeres mechanism in Ewing's sarcomas and osteosarcomas[END_REF] and 84% [START_REF] Ohali | Association between telomerase activity and outcome in patients with nonmetastatic Ewing family of tumors[END_REF]). Although this requires further investigation, increased telomerase activity in metastatic secondary lesions compared to that in tumour at diagnosis would be consistent with the hypothesis that tumours continually select for incrementally higher levels of telomerase activity as they proliferate and acquire genetic changes associated with invasive cancer [START_REF] Ghaffari | Telomerase activity and telomere length in patients with acute promyelocytic leukemia: indicative of proliferative activity, disease progression, and overall survival[END_REF][START_REF] Kolquist | Expression of TERT in early premalignant lesions and a subset of cells in normal tissues[END_REF].

Although telomerase activity is not prognostic in primary tumour, in a small group of patients consistently high telomerase activity in peripheral blood taken during follow-up correlated with a reduced progression free survival compared to that in patients where telomerase activity was low or became low after treatment [START_REF] Ohali | Association between telomerase activity and outcome in patients with nonmetastatic Ewing family of tumors[END_REF]. Telomerase activity measured in peripheral blood from patients with ESFT may arise from disseminating ESFT cells. Indeed Ohali et al [START_REF] Ohali | Association between telomerase activity and outcome in patients with nonmetastatic Ewing family of tumors[END_REF] reported a good correlation between telomerase activity and the presence of disease detected by RT-PCR for EWS-ETS fusion

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ACCEPTED MANUSCRIPT 10 Linking powered by eXtyles transcripts, suggesting that telomerase may be a useful marker of minimal residual disease. However the reduced sensitivity of ESFT cell detection using telomerase activity (1 ESFT cell in 1 x 10 4 normal cells) compared to that of RT-PCR for EWS-ETS fusions (1 cell in 1 x 10 6 normal cells) and discordance between TA and RT-PCR for EWS-ETS fusions in peripheral blood from some patients [START_REF] Ohali | Association between telomerase activity and outcome in patients with nonmetastatic Ewing family of tumors[END_REF], does not support this hypothesis. Nevertheless high telomerase activity, arising from ESFT cells in peripheral blood taken from patients on follow up, may be a useful marker of response and clinical course independent of minimal disease status. In vitro studies have shown that the chemotherapeutics doxorubicin [START_REF] Lanvers-Kaminsky | Doxorubicin modulates telomerase activity in Ewing's sarcoma in vitro and in vivo[END_REF] and imatinib [START_REF] Uziel | Imatinib mesylate (Gleevec) downregulates telomerase activity and inhibits proliferation in telomeraseexpressing cell lines[END_REF] down-regulate telomerase activity in ESFT cell lines, consistent with the hypothesis that changes in telomerase activity and/or factors regulating this activity may be useful biomarkers of response to therapy during treatment and follow up. The imatinib-induced decrease in telomerase activity is associated with induction of cell cycle arrest, although the mechanism of action is unclear [START_REF] Uziel | Imatinib mesylate (Gleevec) downregulates telomerase activity and inhibits proliferation in telomeraseexpressing cell lines[END_REF].The hypothesis that changes in telomerase activity within haemopoietic compartments might reflect tumour burden or be a useful biomarker of response measured in peripheral blood mononuclear cells is not new (Table 2). However these possibilities require further investigation in prospective quality controlled clinical trials, with relevant controls to account for the variability in telomerase activity within peripheral blood mononuclear cells from non-treated agematched controls.

Telomerase activity in ESFT cells is most likely increased through EWS-FLI1 dependent up-regulation of hTERT, either directly [START_REF] Takahashi | EWS/ETS fusions activate telomerase in Ewing's tumors[END_REF] or indirectly through a target gene such as MYC-C which activates the hTERT promoter [START_REF] Wang | Myc activates telomerase[END_REF][START_REF] Wu | Direct activation of TERT transcription by c-MYC[END_REF] and is itself up-regulated by EWS-

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M A N U S C R I P T Linking powered by eXtyles FLI1 [START_REF] Bailly | DNA-binding and transcriptional activation properties of the EWS-FLI-1 fusion protein resulting from the t(11;22) translocation in Ewing sarcoma[END_REF][START_REF] Dauphinot | Analysis of the expression of cell cycle regulators in Ewing cell lines: EWS-FLI-1 modulates p57KIP2and c-Myc expression[END_REF] (Figure 2). However increased copy number of the genes encoding components of the telomerase pathway, such as the regulatory human telomerase RNA (hTR) and hTERT genes located on chromosomes 3q26.3 and 5p15.33 respectively, might also be responsible for the up-regulation of telomerase activity in ESFT. Consistent with this idea trisomy of chromosome 5 has been reported in 15% [START_REF] Roberts | Ploidy and karyotype complexity are powerful prognostic indicators in the Ewing's sarcoma family of tumors: a study by the United Kingdom Cancer Cytogenetics and the Children's Cancer and Leukaemia Group[END_REF] and gain of 5p in 20% [START_REF] Ferreira | Array CGH and gene-expression profiling reveals distinct genomic instability patterns associated with DNA repair and cell-cycle checkpoint pathways in Ewing's sarcoma[END_REF] of primary tumours, although one study has failed to identify gain of 3q or 5p in 20 primary ESFT [START_REF] Armengol | Recurrent gains of 1q, 8 and 12 in the Ewing family of tumours by comparative genomic hybridization[END_REF]. Chromosomal gains and gene amplifications involving chromosome arms 5p and 3q are among the most frequently described in human tumours, and increased gene dosage of the telomerase regulatory genes hTERT and hTR has been described and associated with clonal evolution in a variety of cancers, suggesting this provides a growth advantage for these cells [START_REF] Cao | Increased copy number of the TERT and TERC telomerase subunit genes in cancer cells[END_REF].

Tumour microenvironment might also influence the activity of telomerase in ESFT, since like most solid tumours the ESFT microenvironment is heterogeneous with areas of hypoxia reflecting the abnormal vasculature network and high proliferation rate of the cells [START_REF] Dunst | Prognostic impact of tumor perfusion in MR-imaging studies in Ewing tumors[END_REF]. Hypoxia-induced stabilisation of hypoxia-inducible factor-1 (HIF-1) is reported to increase telomerase activity through several mechanisms including upregulation [START_REF] Nishi | Hypoxia-inducible factor 1 mediates upregulation of telomerase (hTERT)[END_REF][START_REF] Yatabe | HIF-1-mediated activation of telomerase in cervical cancer cells[END_REF] or alternative splicing [START_REF] Anderson | Hypoxic regulation of telomerase gene expression by transcriptional and post-transcriptional mechanisms[END_REF] of hTERT mRNA, up-regulation of hTERT protein [START_REF] Yatabe | HIF-1-mediated activation of telomerase in cervical cancer cells[END_REF][START_REF] Lou | The opposing effect of hypoxia-inducible factor-2alpha on expression of telomerase reverse transcriptase[END_REF] or up-regulation of hTR [START_REF] Anderson | Hypoxic regulation of telomerase gene expression by transcriptional and post-transcriptional mechanisms[END_REF]. Although it is not known whether HIF-1 directly influences the transcriptional activity of the EWS-ETS protein, the CREBbinding protein p300 promotes HIF-1 transcriptional activity [START_REF] Bardos | Negative and positive regulation of HIF-1: a complex network[END_REF] and also cooperates with EWS-ETS in the initiation complex of TERT transcription [START_REF] Takahashi | EWS/ETS fusions activate telomerase in Ewing's tumors[END_REF], implying that hypoxia may influence telomerase activity in ESFT. Furthermore the alternative splice variants (wild-type, α-, β-, α-β-) of hTERT are differentially expressed in ESFT cell lines

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in hypoxia and normoxia, suggesting that alternative splicing of the hTERT gene may regulate telomerase activity in ESFT cells as described in other systems [START_REF] Colgin | The hTERTalpha splice variant is a dominant negative inhibitor of telomerase activity[END_REF][START_REF] Kilian | Isolation of a candidate human telomerase catalytic subunit gene, which reveals complex splicing patterns in different cell types[END_REF][START_REF] Saeboe-Larssen | Characterization of novel alternative splicing sites in human telomerase reverse transcriptase (hTERT): analysis of expression and mutual correlation in mRNA isoforms from normal and tumour tissues[END_REF][START_REF] Ulaner | Regulation of telomerase by alternate splicing of human telomerase reverse transcriptase (hTERT) in normal and neoplastic ovary, endometrium and myometrium[END_REF][START_REF] Yi | Quantitation of telomerase components and hTERT mRNA splicing patterns in immortal human cells[END_REF] (Figure 2).

Telomere maintenance in ESFT

ESFT are reported to express and utilise telomerase to maintain telomere length [START_REF] Ulaner | Divergent patterns of telomere maintenance mechanisms among human sarcomas: sharply contrasting prevalence of the alternative lengthening of telomeres mechanism in Ewing's sarcomas and osteosarcomas[END_REF].

However despite the presence of functional telomerase, telomeres in ESFT are frequently shorter than those of matched normal cells [START_REF] Ulaner | Divergent patterns of telomere maintenance mechanisms among human sarcomas: sharply contrasting prevalence of the alternative lengthening of telomeres mechanism in Ewing's sarcomas and osteosarcomas[END_REF][START_REF] Avigad | Short Telomeres: A Novel Potential Predictor of Relapse in Ewing Sarcoma[END_REF][START_REF] Montgomery | Telomere lengths of translocation-associated and nontranslocation-associated sarcomas differ dramatically[END_REF]. Therefore, although telomere stabilisation is a requisite step in tumorigenesis the shortening of telomeres must occur at some point in ESFT development. When this shortening takes place and whether it occurs in telomerase positive or negative cells is not clear. Since critically shortened telomeres have been described in precancerous lesions [START_REF] Kolquist | Expression of TERT in early premalignant lesions and a subset of cells in normal tissues[END_REF][START_REF] Meeker | Telomere length abnormalities occur early in the initiation of epithelial carcinogenesis[END_REF][START_REF] Wu | Telomere dysfunction: a potential cancer predisposition factor[END_REF] and in patients with Fanconi anaemia that have an increased susceptibility to develop cancer [START_REF] Hanson | Telomere shortening in Fanconi anaemia demonstrated by a direct FISH approach[END_REF], it seems likely that telomere shortening is an early event driving the development of ESFT. Therefore the short telomeres may be a consequence of rapid proliferation prior to activation of telomerase. In this model ESFT may arise in telomerase negative cells by generation of the EWS-ETS translocations, which drive proliferation resulting in the development of shortened telomeres leading to chromosome instability, crisis, and subsequently activation of telomerase to stabilise the telomere and suppress cellular senescence (Figure 3). Consistent with the idea that EWS-ETS play a role in the maintenance of the ESFT malignant phenotype through maintenance of telomere length, EWS-FLI1 is reported to up-regulate telomerase activity [START_REF] Takahashi | EWS/ETS fusions activate telomerase in Ewing's tumors[END_REF] and prevent cellular senescence [START_REF] Hu | EWS/FLI1 suppresses retinoblastoma protein function and senescence in Ewing's sarcoma cells[END_REF][START_REF] Matsunobu | The possible role of EWS-Fli1 in evasion of senescence in Ewing family tumors[END_REF]. This hypothesis is consistent with the concept that chromosomal instability underlies the Those patients that present with ESFT in which the telomeres are shorter compared to those with unchanged or longer telomeres are reported to have a 5-fold increased risk of relapse [START_REF] Avigad | Short Telomeres: A Novel Potential Predictor of Relapse in Ewing Sarcoma[END_REF]. Whether abnormal shortened telomeres and accumulation of G1 cell cycle checkpoint defects in ESFT predict a worse outcome is not know, although it is interesting to speculate that together these may predispose cells to unbalanced chromosome segregation at cell division and development of a more complex genotype.

However, levels of telomerase activity in ESFT at diagnosis are elevated but do not

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ACCEPTED MANUSCRIPT 14 Linking powered by eXtyles correlate with telomere length and are not predictive of outcome [START_REF] Ohali | Association between telomerase activity and outcome in patients with nonmetastatic Ewing family of tumors[END_REF][START_REF] Avigad | Short Telomeres: A Novel Potential Predictor of Relapse in Ewing Sarcoma[END_REF]. These data might be taken to suggest that telomere length is not dependent on telomerase activity in ESFT. However it is most likely that telomerase has a fundamental telomere-dependent role in the initiation of tumorigenesis (described above), and subsequently plays both telomere-dependent and -independent roles in the progression and maintenance of the malignant phenotype [START_REF] Cong | Actions of human telomerase beyond telomeres[END_REF].

Clinical utility; potential, challenges and reporting.

Increasing evidence that dysfunctional telomeres play a causal role in the process of malignant transformation has made them attractive as potential biomarkers for early cancer diagnosis and prognostication. That abnormally short telomeres are prevalent at the premalignant stage also raises the interesting possibility that avoidance or reversal of the malignant phenotype by interfering with factors that regulate telomere length could be a potential strategy for cancer chemoprevention. Since the majority of tumours appear to rely on telomerase to maintain telomere length for immortalisation and genomic stabilisation, this enzyme is an attractive target for the design of novel cancer therapeutics [START_REF] Harley | Telomerase and cancer therapeutics[END_REF][START_REF] Kelland | Targeting the limitless replicative potential of cancer: the telomerase/telomere pathway[END_REF][START_REF] Shay | Targeting telomerase for cancer therapeutics[END_REF]. Recent studies demonstrating telomere independent tumour-enhancing activities of telomerase [START_REF] Bollmann | The many faces of telomerase: emerging extratelomeric effects[END_REF] make it even more appealing as a target. Although the role of telomerase and telomere dynamics in normal and cancer cells is not fully understood, it is anticipated that increased understanding of these processes will allow for their more precise exploitation for therapeutic advantage.

Even though much about telomere biology and the plethora of proteins that play roles in maintenance of telomere length remain unclear, several proof of principal studies have demonstrated that telomere length, telomerase activity and in some cases the potential

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15 Linking powered by eXtyles regulators of its activity hTR and hTERT may be of predictive and/or prognostic value (Table 2). The key advantage of these as biomarkers is that they are features of most cancer cells (including putative cancer stem cells), and critical for the development and progression of these malignancies. However uncertainty about the significance of telomere length and of low or transient expression of telomerase and regulators of its activity in normal biology compared to cancer patho-physiology has slowed their introduction into clinical practice. This highlights the importance of investigating these factors in paired tumour and normal samples, and evaluating the impact of tumour heterogeneity on their potential clinical significance. These studies will benefit from analyses in specific cancer and normal cell populations, and an evaluation of the impact that proliferation and cell cycle dependent regulation of telomerase activity will have.

The use of in situ assays to detect telomerase activity [START_REF] Ohyashiki | Cytological detection of telomerase activity using an in situ telomeric repeat amplification protocol assay[END_REF][START_REF] Uehara | In situ telomerase activity in pancreatic juice may discriminate pancreatic cancer from other pancreatic diseases[END_REF] and hTERT mRNA [START_REF] Cheng | Human telomerase reverse transcriptase activated by E6 oncoprotein is required for human papillomavirus-16/18-infected lung tumorigenesis[END_REF][START_REF] Chou | In situ detection of hTERT mRNA relates to Ki-67 labeling index in papillary thyroid carcinoma[END_REF][START_REF] Kumaki | Telomerase activity and expression of human telomerase RNA component and human telomerase reverse transcriptase in lung carcinomas[END_REF], or technologies such as laser capture micro-dissection [START_REF] Shammas | Telomere maintenance in laser capture microdissection-purified Barrett's adenocarcinoma cells and effect of telomerase inhibition in vivo[END_REF][START_REF] Wu | Limitations on the quantitative Linking powered by eXtyles determination of telomerase activity by the electrophoretic and ELISA based TRAP assays[END_REF] or cell sorting [START_REF] Baerlocher | Telomere length measurement by fluorescence in situ hybridization and flow cytometry: tips and pitfalls[END_REF][START_REF] Hultdin | Telomere analysis by fluorescence in situ hybridization and flow cytometry[END_REF][START_REF] Rufer | Telomere fluorescence measurements in granulocytes and T lymphocyte subsets point to a high turnover of hematopoietic stem cells and memory T cells in early childhood[END_REF][START_REF] Rufer | Telomere length dynamics in human lymphocyte subpopulations measured by flow cytometry[END_REF] to isolate specific cell populations prior to assays for telomerase or regulators of its activity will facilitate these studies. Failure to define the clinical utility of these markers also reflects the small patient numbers in individual studies and lack of standardisation of assays and reporting, emphasising the importance of prospective studies utilising standardised assays. Such studies demand that an assay is robust, reproducible, sensitive, and specific (Figure 4), and that standardisation is established from sample collection, transfer and storage in the laboratory, through to analysis and data reporting. Linking powered by eXtyles In this final section we have discussed some of the fundamental practical and technical challenges of evaluating telomere length, hTR, hTERT and telomerase activity in clinical samples.

Telomere shortening in tumour compared to adjacent non-tumour or peripheral blood is attractive as a prognostic marker as the assays are performed on DNA which is relatively stable and readily isolated from tissues. This may also be informative in different cancer types (Table 2), regardless of the mechanism of telomere maintenance.

Telomere length is traditionally measured by determination of a mean telomere restriction fragment (TRF) length. This requires restriction enzyme digest of genomic DNA (~1μg) from tumour and a paired normal tissue, Southern blot and hybridisation to a probe for the telomeric repeats (TTAGGG) n . This method is most informative when DNA is extracted from fresh frozen tissue comprising purely tumour or normal cells, overcoming the heterogeneity of tumours and providing cancer cell specific estimations of telomere length and not an average telomere length from a mix of normal and cancer cells. However sufficient amounts of this precious resource are not always available, especially when cancers are diagnosed by small needle biopsies and in rare cancers such as ESFT. Therefore alternative methods for the measurement of telomere length have been developed (Table 3). Most useful has been the development of a quantitative assay utilising the polymerase chain reaction (PCR), which has the advantage that it can be performed in amounts of DNA (~200ng) that can be isolated from small numbers of pure cell populations. This assay is also robust, quantitative, rapid and amenable to high throughput [START_REF] Cawthon | Telomere measurement by quantitative PCR[END_REF][START_REF] Svenson | Breast cancer survival is associated with telomere length in peripheral blood cells[END_REF][START_REF] Zhang | Telomere length in hepatocellular carcinoma and paired adjacent non-tumor tissues by quantitative PCR[END_REF].

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Most studies of telomere length have focussed on the measurement of average telomere length in tumour compared to normal cells. However this may not be as informative as analysing telomere length of individual chromosomes, since it may be that attrition of specific chromosomes (and dysfunction of the genes which they carry) will initiate the malignant process. Single telomere length analysis (STELA) has identified large-scale differences in telomere length throughout development [START_REF] Baird | Extensive allelic variation and ultrashort telomeres in senescent human cells[END_REF], as well as a class of extremely short telomeres in human cancer cell lines [START_REF] Xu | Human cancer cells harbor T-stumps, a distinct class of extremely short telomeres[END_REF]. However, the biological and clinical significance of specific telomere shortening in both normal and cancer cells requires further investigation. Currently STELA has only been developed to analyse telomere length of a limited number of chromosomes including chromosomes 2p, 11q, 12q, 17p and the sex chromosomes [START_REF] Baird | Extensive allelic variation and ultrashort telomeres in senescent human cells[END_REF][START_REF] Britt-Compton | Structural stability and chromosome-specific telomere length is governed by cisacting determinants in humans[END_REF], although there is the potential to develop the technique to examine telomere length in all chromosomes.

Telomerase is the enzyme, which together with telomere-binding proteins, appears to be essential for the immortalisation of most human cancer cells. However the initial expectation that telomerase activity would be a universal specific marker of malignancy has not been realised and its potential clinical utility remains unresolved.

Telomerase activity is most widely measured in clinical samples using the PCR based telomerase repeat amplification protocol (TRAP) assay [START_REF] Kim | Specific association of human telomerase activity with immortal cells and cancer[END_REF], or adaptations of this protocol. The assay measures the telomerase dependent addition of (TTAGGG) n repeats to the end of a synthetic primer with a telomere-like sequence (TS primer). The extended oligonucleotide products are then amplified by PCR and the amplified DNA band products (6bp apart) separated by polyacrylamide gel electrophoresis and typically visualised by autoradiography. Semi-quantitative evaluation relative to an internal Linking powered by eXtyles standard is possible using densitometry; the method of detection depending on the label used e.g. phosphoimager [START_REF] Piatyszek | Detection of telomerase activity in human cells and tumors by a telomeric repeat amplification protocol (TRAP)[END_REF], fluoroimaging [START_REF] Aldous | A fluorescent method for detection of telomerase activity[END_REF] or in-gel staining with SYBR Green™ [START_REF] Fajkus | Plant cells express telomerase activity upon transfer to callus culture, without extensively changing telomere lengths[END_REF][START_REF] Riha | Developmental control of telomere lengths and telomerase activity in plants[END_REF]. Each assay includes a series of controls to validate the data including a negative control (pretreatment of the samples with heat or RNase A) and a positive control cell line. This assay is highly sensitive, reportedly detecting telomerase activity in as few as 10 immortal cancer cells [START_REF] Wright | Modifications of a telomeric repeat amplification protocol (TRAP) result in increased reliability, linearity and sensitivity[END_REF]. With this sensitivity telomerase activity has been detected in some normal somatic cells [START_REF] Hiyama | Activation of telomerase in human lymphocytes and hematopoietic progenitor cells[END_REF][START_REF] Hiyama | Telomerase activity in small-cell and non-small-cell lung cancers[END_REF][START_REF] Hiyama | Telomerase activity in human breast tumors[END_REF] and in several benign tumours [START_REF] Hiyama | Telomerase activity in human breast tumors[END_REF][START_REF] Matthews | Telomerase activity and telomere length in thyroid neoplasia: biological and clinical implications[END_REF]. Usually telomerase activity is higher in immortalised cancer cells than in these normal somatic or benign cancer cells. However to evaluate and compare telomerase activity between normal and cancer cells a reliable method of quantification, with a common reporting standard is needed. More recently telomerase activity has been measured using a real-time quantitative TRAP assay which requires smaller amounts of clinical material, important when analysing small numbers of isolated tumour, tumour stem cells or normal cells from valuable clinical material (Table 3). Real-time TRAP also enables a more accurate quantification of telomerase activity, and with the inclusion of standardised telomerase reference materials may be useful to define the threshold of telomerase activity attributed to non-cancerous cells and so better evaluate clinical relevance [START_REF] Jakupciak | Preparation and characterization of candidate reference materials for telomerase assays[END_REF].

hTERT and hTR are two major components of the active telomerase complex, and have been investigated as surrogate markers of telomerase activity. Expression of hTERT mRNA and protein appears to be more restricted than expression of hTR RNA which is detected in all cell types; a protein product of hTR has not been described. All are frequently over expressed in tumours compared to normal cells, and may be prognostic or

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predictive of outcome in some cancer patients (Table 2). Furthermore hTERT profiles may be predictive of response to some therapeutics [START_REF] Buttitta | Human telomerase reverse transcriptase mRNA expression assessed by real-time reverse transcription polymerase chain reaction predicts chemosensitivity in patients with ovarian carcinoma[END_REF][START_REF] Sun | The telomerase activity and expression of hTERT gene can serve as indicators in the anti-cancer treatment of human ovarian cancer[END_REF]. Measurement of these regulators might therefore predict patient response to a drug, assess efficacy of treatment or monitor disease status to predict relapse. hTERT has been widely described as a major regulator of telomerase activity [START_REF] Cong | Human telomerase and its regulation[END_REF][START_REF] Janknecht | On the road to immortality: hTERT upregulation in cancer cells[END_REF], and a correlation between hTERT and telomerase activity has been reported in several different cancer types including ESFT [START_REF] Ohali | Association between telomerase activity and outcome in patients with nonmetastatic Ewing family of tumors[END_REF]. This has made hTERT the most widely used surrogate marker for telomerase activity. However, hTERT mRNA has several spice variants some of which are inactive and competitive inhibitors of hTERT driven activation of telomerase activity (Figure 2), these are also expressed in cells that do not express telomerase activity suggestive of a telomerase-independent activity [START_REF] Ulaner | Telomerase activity in human development is regulated by human telomerase reverse transcriptase Linking powered by eXtyles (hTERT) transcription and by alternate splicing of hTERT transcripts[END_REF]. There is increasing evidence that hTR is also over expressed in tumour compared to normal cells where it may play a role in telomere maintenance and regulation of telomerase activity [START_REF] Avilion | Human telomerase RNA and telomerase activity in immortal cell lines and tumor tissues[END_REF][START_REF] Blasco | Telomere shortening and tumor formation by mouse cells lacking telomerase RNA[END_REF][START_REF] Feng | The RNA component of human telomerase[END_REF][START_REF] Soder | Tumour specific regulation of telomerase RNA gene expression visualized by in situ hybridization[END_REF] and predict prognosis [START_REF] Poremba | Telomerase activity and telomerase subunits gene expression patterns in neuroblastoma: a molecular and immunohistochemical study establishing prognostic tools for fresh-frozen and paraffin-embedded tissues[END_REF].

High levels of hTR have also been described in tumours that utilise ALT to maintain telomere length [START_REF] Cairney | High level of telomerase RNA gene expression is associated with chromatin modification, the ALT phenotype and poor prognosis in liposarcoma[END_REF], suggesting that hTR, like hTERT, has telomerase-independent activity and so may be an informative biomarker regardless of the mechanism of telomere maintenance.

As biomarkers hTERT and hTR are attractive as they can be measured using the high through put sensitive quantitative reverse transcriptase (RT)-PCR, and may be informative in readily accessible clinical materials such as washings, brushings, fine needle aspirations, peripheral blood and urine. Using quantitative RT-PCR the sensitivity of hTERT detection is 9-10 malignant cells [START_REF] Jakupciak | Analytical validation of telomerase activity for cancer early detection: TRAP/PCR-CE and hTERT mRNA quantification assay for high-throughput screening of tumor cells[END_REF]; (Brownhill, unpublished observations), whereas hTR can be detected in a single cell (Brownhill, unpublished observations) Linking powered by eXtyles consistent with its higher expression levels. However, defining their clinical utility has been hampered by their expression in normal cells, low copy number per cell (hTERT) and lack of consensus about their relationship with telomerase activity and the role they play in tumorigenesis. As with TRAP assays [START_REF] Jakupciak | Real-time telomerase activity measurements for detection of cancer[END_REF] within a clinical setting, quantitative RT-PCR may be useful to determine a threshold of normal expression and so define levels relevant to disease status and outcome. Studies in patient samples have not systematically examined the relationship between telomerase activity, hTERT and its isoforms and hTR (Figure 2) and their clinical significance. These studies are critically important to establish how these factors might best be used to inform management of patients with cancer.

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21 Linking powered by eXtyles

Figure 2. Potential sites of telomerase regulation in ESFT

The hTERT gene is regulated by numerous transcription factors acting on the promoter. EWS-ETS fusion transcripts can complex with CBP/p300 leading to up-regulation of both hTERT transcription and telomerase activity (* it is not clear if it has the same effect on hTR). There is also an ETS binding site in the hTERT promoter which may be the target for some fusion proteins (**). Different ETS family members may also confer altered interactions with the hTERT promoter on EWS-ETS fusions [START_REF] Takahashi | EWS/ETS fusions activate telomerase in Ewing's tumors[END_REF], which may include adjacent binding sites like AP-1 although this possibility requires investigation .

The up-regulation of MYC-C by the EWS-ETS fusions may also in turn promote hTERT and hTR transcription through the promoter E-box sites [START_REF] Bailly | DNA-binding and transcriptional activation properties of the EWS-FLI-1 fusion protein resulting from the t(11;22) translocation in Ewing sarcoma[END_REF][START_REF] Dauphinot | Analysis of the expression of cell cycle regulators in Ewing cell lines: EWS-FLI-1 modulates p57KIP2and c-Myc expression[END_REF]. HIF1 can also act at the hTERT and hTR promoters, relevant in areas of hypoxia within ESFT [START_REF] Dunst | Prognostic impact of tumor perfusion in MR-imaging studies in Ewing tumors[END_REF]. Alternative splicing is an important regulatory mechanism for the hTERT gene and all of the major The alpha splice variant has a dominant negative action on telomerase [START_REF] Colgin | The hTERTalpha splice variant is a dominant negative inhibitor of telomerase activity[END_REF][START_REF] Yi | An alternate splicing variant of the human telomerase catalytic subunit inhibits telomerase activity[END_REF] although it is not clear whether regulation by splicing takes place at the RNA or protein level ( †).

Tumorigenesis :

Metastasis Recurrence The kit provides a standard that can be used in assays therefore allowing standardised reporting of results. Reported to have an increased specificity compared to the incorporation of SYBR green. Improved reliability, assay time and accuracy of quantification compared to the standard TRAP assay. Suitable for analysis of tumour, BM, PB, and body fluids including urine and bile. For BM, PB and fluids white blood cells must be purified prior to protein extraction. Analysis is possible with only 1000 cells or 500 ng protein. Tumour specific telomere length possible if tumour cells are isolated by laser capture microdissection.

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Real-time PCR system needed It is suggested that an internal control is used to normalise TA during amplification however it has been reported that the internal control reduced rather than improved the correlation coefficient. [START_REF] Hou | Real-time quantitative telomeric repeat amplification protocol assay for the detection of telomerase activity[END_REF][START_REF] Shim | Quantitative detection of telomerase activity by real-time TRAP assay in the body fluids of cancer patients[END_REF] A C C E P T E D M A N U S C R I P T

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Table 3. Summary of the techniques used to determine telomere length and telomerase activity A number of different techniques have been used to determine telomere length and telomerase activity. This table identifies these techniques and details their advantages and disadvantages including their suitability for the analysis of clinical samples

  eXtyles generation of changes in the chromosome and generation of genomic heterogeneity, to maintain proliferation and drive development of the malignant phenotype during tumorigenesis. Of course it may be that ESFT arise in cells that express telomerase but that this is insufficient to maintain telomere length in the rapidly dividing transformed cells, resulting in EWS-ETS dependent up-regulation of telomerase to stabilise the telomere at an albeit shortened length. Both of these possibilities suggest that telomerasemediated stabilisation of telomeres is not an early event in tumorigenesis, and are supported by observations that EWS-ETS fusion-induced tumorigenesis is dependent on permissive cellular environment. Whether the ESFT precursor cell has shortened telomeres resulting in genetic instability leading to translocation of the EWS gene, or whether the EWS-ETS translocations arise in precursor cells inducing genetic instability resulting in shortened telomeres, up-regulation of telomerase activity and accumulation of secondary genetic abnormalities remains to be seen. Increased understanding of the relationship between secondary genetic changes, genome instability, tumour progression and the identification and characterisation of the cell(s) in which ESFT arise will inform the development of these hypotheses.

Figure 1 .

 1 Figure 1. Molecular characterisation of ESFT

  eXtyles mRNA isoforms are present in ESFT cells in vitro (Proctor, unpublished observations).

Figure 3 .

 3 Figure 3. Telomere biology and telomerase in ESFT ESFT contain EWS-ETS fusions, have shortened telomeres and increased levels of telomerase activity. It is unclear whether telomere shortening leads to increased genetic instability to facilitate the formation of the EWS-ETS translocation or whether the EWS-ETS fusion is the initiating event that drives cell proliferation resulting in telomere shortening. What is clearer is that the EWS-ETS fusions and factors like hypoxia and MYC-C are important in the reactivation of telomerase, which along with acquired abnormalities in cell cycle regulators allow ESFT cells to avoid senescence and stabilise the shortened telomeres. Continued cell division in the presence of shortened telomeres is permissive to the acquisition of further oncogenic changes, which contribute to the development and maintenance of the cancer phenotype.

Figure 4 .

 4 Figure 4. Considerations prior to introduction of assays in clinical trialsSummary of the considerations that need to be addressed to evaluate the potential clinical relevance of biomarkers such as telomerase in clinical samples

Table 1 Non-random EWS gene rearrangements identified in bone and soft tissue sarcomas a.

 1 Translocation of the EWS gene on chromosome 22q12 with a member of the ETS family of transcription factors is diagnostic of ESFT (shown in bold). Additional rearrangements of the EWS gene and alternative translocations described in other sarcomas are shown.

	b. The EWS breakpoint on chromosome 22q12 is usually constant, but the breakpoint within the partner ETS gene family
	member can vary. The most frequently described abnormality in ESFT is the t(11;22)(q24;q12) leading to the EWS-FLI1
	type 1 gene fusion transcript ; this is generated from the first seven exons of EWS joined to exons 6-9 of Fli1.
	a.
	FUSION PROTEIN	TRANSLOCATION	TUMOUR TYPE
	EWS-FLI1	t(11;22)(q24;q12)	ESFT
	EWS-ERG	t(21;22)(q22;q12)
	EWS-ETV1	t(7;22)(p22;q12)
	EWS-FEV	t(2;22)(q33;q12)
	EWS-ETV4	t(17;22)(q12;q12)
	EWS-WT1	t(11;22)(p13;q12)	Desmoplastic small round cell tumour
	EWS-CHOP	t(12;22)(q13;q12)	Myxoid liposarcoma
	FUS-CHOP	t(12;16)(q13;p11)
	EWS-ATF1	t(12;22)(q13;q12)	Clear cell sarcoma
	EWS-NR4A3	t(9;22)(q22;q12)	Extraskeletal myxoid chondrosarcoma
	TAF1168-NR4A3	t(9;17)(q22;q11)
	TCF12-NR4A3	t(9;15)(q22;q21)
	EWS-ATF1	t(12;22)(q13;q12)	Angiomatoid fibrous histocytoma
	FUS-ATF1	t(12;16)(q13;p11)

Table 1

 1

	Fusion Transcripts

Acknowledgments.

Thank you to Mr Paul Roberts, Department of Cytogentics, SJUH, Leeds for providing image of G-banding and FISH analysis used in Figure 1.

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Specificity

Tumour

Short telomeres= Poor prognosis [START_REF] Ulaner | Regulation of telomerase by alternate splicing of human telomerase reverse transcriptase (hTERT) in normal and neoplastic ovary, endometrium and myometrium[END_REF] Detectable Telomerase activity = Not significant [START_REF] Takahashi | EWS/ETS fusions activate telomerase in Ewing's tumors[END_REF][START_REF] Jakupciak | Analytical validation of telomerase activity for cancer early detection: TRAP/PCR-CE and hTERT mRNA quantification assay for high-throughput screening of tumor cells[END_REF] Detectable hTERT protein levels = Not significant [START_REF] Lanvers-Kaminsky | Doxorubicin modulates telomerase activity in Ewing's sarcoma in vitro and in vivo[END_REF] High telomerase activity = Not significant [START_REF] Jakupciak | Real-time telomerase activity measurements for detection of cancer[END_REF] Detectable telomerase activity in 12.5% of primary tumours and 100% metastases; High telomerase activity = Poor prognosis [START_REF] Ohali | Association between telomerase activity and outcome in patients with nonmetastatic Ewing family of tumors[END_REF] Peripheral Blood High telomerase activity and High hTERT expression in follow-up = Poor prognosis [START_REF] Takahashi | EWS/ETS fusions activate telomerase in Ewing's tumors[END_REF] Neuroblastoma No Telomerase

Tumour

Short telomeres = Good prognosis [START_REF] Amiel | Molecular cytogenetic parameters in Ewing sarcoma[END_REF] High Telomerase activity = Poor prognosis [START_REF] Feng | The RNA component of human telomerase[END_REF][START_REF] Amiel | Molecular cytogenetic parameters in Ewing sarcoma[END_REF][START_REF] Sangiorgi | Presence of telomerase activity in different musculoskeletal tumor histotypes and correlation with aggressiveness[END_REF][START_REF] Ohali | Telomere length is a prognostic factor in neuroblastoma[END_REF] High hTERT and hTR levels = Poor prognosis [START_REF] Feng | The RNA component of human telomerase[END_REF] High WT hTERT levels = Poor prognosis [START_REF] Hiyama | Correlating telomerase activity levels with human neuroblastoma outcomes[END_REF] Low hTR levels and Low telomerase activity = Good prognosis [START_REF] Poremba | Telomerase activity distinguishes between neuroblastomas with good and poor prognosis[END_REF] Detectable

Tumour

Long telomeres = High tumour grade [START_REF] Wurl | Co-expression of survivin and TERT and risk of tumour-related death in patients with soft-tissue sarcoma[END_REF] High telomerase activity = Tumour progression/ High grade [START_REF] Wurl | Co-expression of survivin and TERT and risk of tumour-related death in patients with soft-tissue sarcoma[END_REF][START_REF] Ohali | Different telomere maintenance mechanisms in alveolar and embryonal rhabdomyosarcoma[END_REF] High hTERT = Tumour progression/ High grade [START_REF] Wurl | Co-expression of survivin and TERT and risk of tumour-related death in patients with soft-tissue sarcoma[END_REF][START_REF] Ohali | Different telomere maintenance mechanisms in alveolar and embryonal rhabdomyosarcoma[END_REF] High hTR = Poor prognosis [START_REF] Soder | Tumour specific regulation of telomerase RNA gene expression visualized by in situ hybridization[END_REF] Melanoma No Telomerase

Tumour

High telomerase activity = Early metastatic spread [START_REF] Schneider-Stock | Elevated telomerase activity, c-MYC-, and hTERT mRNA expression: association with tumour progression in malignant lipomatous tumours[END_REF] High telomerase activity = Poor pathological features [START_REF] Schneider-Stock | High telomerase activity and high HTRT mRNA expression differentiate pure myxoid and myxoid/round-cell liposarcomas[END_REF] Bladder Carcinoma No Telomerase

Tumour

High hTERT level = Worse tumour grade and stage [START_REF] Rudolph | Telomerase activity in melanocytic lesions: A potential marker of tumor biology[END_REF][START_REF] Carvalho | Telomerase activity in prognostic histopathologic features of melanoma[END_REF] Detectable hTR = No correlation with prognosis [START_REF] Carvalho | Telomerase activity in prognostic histopathologic features of melanoma[END_REF] Detectable telomerase activity = No correlation with prognosis [START_REF] De Kok | Quantitative measurement of telomerase reverse transcriptase (hTERT) mRNA in urothelial cell carcinomas[END_REF] Colorectal Carcinoma No Telomerase

Tumour

High telomerase activity = Poor prognosis [START_REF] Takihana | Realtime quantitative analysis for human telomerase reverse transcriptase mRNA and human telomerase RNA component mRNA expressions as markers for clinicopathologic parameters in urinary bladder cancer[END_REF][START_REF] Sanchini | Relevance of urine telomerase in the diagnosis of bladder cancer[END_REF] High hTERT level = Poor prognosis [START_REF] Bautista | Telomerase activity is a prognostic factor for recurrence and survival in rectal cancer[END_REF] Gastric Carcinoma No Telomerase

Tumour

High telomerase activity (distal cancers) = Poor prognosis [START_REF] Tatsumoto | High telomerase activity is an independent prognostic indicator of poor outcome in colorectal cancer[END_REF] Telomerase activity detected = Poor prognosis [START_REF] Smith | Human telomerase reverse transcriptase (hTERT) and Ki-67 are better predictors of survival than established clinical indicators in patients Linking powered by eXtyles undergoing curative hepatic resection for colorectal metastases[END_REF] High telomerase activity = Higher grade [START_REF] Usselmann | Telomerase activity and patient survival after surgery for gastric and oesophageal cancer[END_REF] A C C E P T E D M A N U S C R I P T

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Breast Carcinoma No Telomerase

Tumour

Telomerase activity = No correlation with prognosis [START_REF] Hiyama | Telomerase activity in gastric cancer[END_REF][START_REF] Kakeji | Gastric cancer with high telomerase activity shows rapid development and invasiveness[END_REF] High telomerase activity = Poor prognosis [START_REF] Carey | Telomerase activity and prognosis in primary breast cancers[END_REF] High telomerase activity = Recurrence rate [START_REF] Mokbel | Telomerase activity and prognosis in breast cancer[END_REF] hTERT detected = Poor prognosis [START_REF] Clark | Telomerase activity and survival of patients with node-positive breast cancer[END_REF] High hTERT = Poor prognosis [START_REF] Kimura | A study on telomerase activity and prognosis in breast cancer[END_REF] Non small cell Lung carcinoma (NSCLC)

No Telomerase

Tumour

Telomerase activity = Poor prognosis [START_REF] Bieche | Quantitation of hTERT gene expression in sporadic breast tumors with a real-time reverse transcription-polymerase chain reaction assay[END_REF][START_REF] Poremba | Telomerase as a prognostic marker in breast cancer: high-throughput tissue microarray analysis of hTERT and hTR[END_REF] Telomerase activity and high hTERT expression = Poor prognosis [START_REF] Gonzalez-Quevedo | Cooperative role of telomerase activity and p16 expression in the prognosis of non-small-cell lung cancer[END_REF] Short telomeres = Poor prognosis [START_REF] Taga | Prognostic impact of telomerase activity in non-small cell lung cancers[END_REF] Gliomas No

Telomerase + ALT (16-25%) [START_REF] Marchetti | Prediction of survival in stage I lung carcinoma patients by telomerase function evaluation[END_REF][START_REF] Frias | Telomere shortening is associated with poor prognosis and telomerase activity correlates with DNA repair impairment in non-small cell lung cancer[END_REF] Tumour High telomerase activity = Poor prognosis [START_REF] Chen | Association of mutant TP53 with alternative lengthening of telomeres and favorable prognosis in glioma[END_REF] Focal hTERT = Poor prognosis [START_REF] Hakin-Smith | Alternative lengthening of telomeres and survival in patients with glioblastoma multiforme[END_REF] hTERT MNS16A genotype = No correlation with prognosis [START_REF] Huang | Correlation of clinical features and telomerase activity in human gliomas[END_REF] hTERT MNS16A SS genotype = Better prognosis [START_REF] Falchetti | In situ detection of telomerase catalytic subunit mRNA in glioblastoma multiforme[END_REF] ALT phenotype = Better prognosis [START_REF] Marchetti | Prediction of survival in stage I lung carcinoma patients by telomerase function evaluation[END_REF][START_REF] Frias | Telomere shortening is associated with poor prognosis and telomerase activity correlates with DNA repair impairment in non-small cell lung cancer[END_REF]

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT

Telomerase activity = No correlation with remission rate [START_REF] Verstovsek | Increased telomerase activity is associated with shorter survival in patients with chronic phase chronic myeloid leukemia[END_REF] hTERT level = no correlation with remission rate in childhood leukaemia [START_REF] Ohyashiki | Clinical implications of telomerase activity levels in acute leukemia[END_REF] Telomerase activity = No correlation with remission rate in adult AML [START_REF] Verstovsek | Telomerase activity is prognostic in pediatric patients with acute myeloid leukemia: comparison with adult acute myeloid leukemia[END_REF] High Telomerase activity = Better remission rate [START_REF] Cogulu | The evaluation of hTERT mRNA expression in acute leukemia children and 2 years follow-up of 40 cases[END_REF] BM High telomerase activity = Poor prognosis in pediatric patients [START_REF] Verstovsek | Increased telomerase activity is associated with shorter survival in patients with chronic phase chronic myeloid leukemia[END_REF] High hTERT = Good prognosis in childhood ALL [START_REF] Ohyashiki | Clinical implications of telomerase activity levels in acute leukemia[END_REF] Chronic lymphocytic leukaemia (CLL)

No Telomerase A C C E P T E D M A N U S C R I P T

PBMC