The alternative lengthening of telomeres pathway may operate in non-neoplastic human cells

The alternative lengthening of telomeres (ALT) mechanism represents an alternative to the enzyme telomerase in the maintenance of mammalian telomeres in 25-60% of sarcomas and a minority of carcinomas (about 5-15%). ALT-positive cells are distinguished by long and heterogeneous telomere length distributions by terminal restriction fragment (TRF) Southern blotting. Another diagnostic marker of ALT is discrete nuclear co-localized signals of telomeric DNA and the promyelocytic leukaemia protein (PML), referred to as ALT-associated PML bodies (APBs). Recently, we detected smaller sized co-localized PML and telomere DNA (APB-like) bodies in endothelial cells adjacent to astrocytoma tumour cells in situ. In this study, we examined a wide variety of non-neoplastic tissues, and report that co-localized signals of PML and telomere DNA are present in endothelial, stromal, and some epithelial cells. Co-localized signals of PML and telomere DNA showed an increased frequency in non-neoplastic cells with DNA damage. These results suggest that a mechanism similar to that in ALT-positive tumours also operates in non-neoplastic cells, which may be activated by DNA damage.     

Telomere maintenance by telomerase and by recombination can coexist in human cells

Immortal human cells maintain their telomeres by two independent mechanisms, a prevalent one dependent on de novo synthesis of telomeric DNA by telomerase, and a rarer one based on telomere recombination [alternative lengthening of telomeres (ALT)]. Studies with yeast have indicated that expression of telomerase inhibits telomere recombination. In the present study, we have investigated whether expression of telomerase in cells that use ALT would similarly reveal dominance of telomere elongation by telomerase over telomere recombination. Telomerase-negative WI38 VA13/2RA ALT cells were reconstituted for telomerase activity through ectopic expression of the enzyme subunits, hTERT and hTR, and the presence and function of telomerase and ALT were monitored during long term cell growth by enzymatic assays, detection of the ALT-associated PML bodies (APBs) and analysis of telomere dynamics. Our results indicate that telomerase activity and APBs persisted in the cells over at least 90 population doublings. The activity of both pathways on telomeres was determined by analysis of telomere length versus time by gel electrophoresis and in situ hybridization. ALT cells are characterized by very heterogeneous telomeres with a much longer average size than the telomeres of telomerase-positive cells. Telomere dynamics in our cells were compatible with both ALT and telomerase being biologically active since the long telomeres typical of ALT were maintained, while short telomeres, thought to be the preferential substrate of telomerase, were elongated. These findings, indicating that human cells may be capable of concomitantly utilizing both mechanisms of telomere maintenance without effects on their growth and viability, have implications for cancer therapy.     

Telomere maintenance in wilms tumors: First evidence for the presence of alternative lengthening of telomeres mechanism

Unlimited proliferative potential is a hallmark of cancer, and can be achieved through the activation of telomere maintenance mechanisms (TMMs). Most tumors activate telomerase, but a significant minority, mainly of mesenchymal origin, uses a recombination‐based, alternative lengthening of telomeres (ALT) mechanism. We investigated the presence of ALT in 34 Wilms tumor (WT) samples from 30 patients by using two approaches: (i) the detection of ALT‐associated promyelocytic leukemia (PML) nuclear bodies (APBs) by combined PML immunofluorescence and telomere fluorescence in situ hybridization and (ii) the assessment of terminal restriction fragment (TRF) length distribution by pulsed field gel electrophoresis. In parallel, telomerase activity (TA) was determined by the telomeric repeat amplification protocol (TRAP) assay. Based on APB expression, ALT was detectable in five samples as the sole TMM and in six samples in association with telomerase. Seventeen samples only expressed TA and in six cases no known TMM was appreciable. Results of TRF length distribution were available in 32 cases, and a concordance between APB and TRF data in defining the ALT phenotype was found in 26/32 cases (81%). The study provides the first evidence of the presence of ALT in WT, and indicates that in a small but defined fraction of cases (about 15%) ALT is the only TMM that supports the development of WT. © 2011 Wiley‐Liss, Inc.     

Alternative lengthening of telomeres--an enhanced chromosomal instability in aggressive non-MYCN amplified and telomere elongated neuroblastomas

Telomere length alterations are known to cause genomic instability and influence clinical course in several tumor types, but have been little investigated in neuroblastoma (NB), one of the most common childhood tumors. In the present study, telomere-dependent chromosomal instability and telomere length were determined in six NB cell lines and fifty tumor biopsies. The alternative lengthening of telomeres (ALT) pathway was assayed by scoring ALT-associated promyelocytic leukemia (PML) bodies (APBs). We found a reduced probability of overall survival for tumors with increased telomere length compared to cases with reduced or unchanged telomere length. In non-MYCN amplified tumors, a reduced or unchanged telomere length was associated with 100% overall survival. Tumor cells with increased telomere length had an elevated frequency of APBs, consistent with activation of the ALT pathway. The vast majority of tumor biopsies and cell lines exhibited an elevated rate of anaphase bridges, suggesting telomere-dependent chromosomal instability. This was more pronounced in tumors with increased telomere length. In cell lines, there was a close correlation between lack of telomere-protective TTAGGG-repeats, anaphase bridging, and remodeling of oncogene sequences. Thus, telomere-dependent chromosomal instability is highly prevalent in NB, and may contribute to the complexity of genomic alterations as well as therapy resistance in the absence of MYCN amplification and in this tumor type.     

A postulated role of p130 in telomere maintenance by human papillomavirus oncoprotein E7

High-risk human papillomaviruses (HR-HPVs) infections is highly associated with the development of cervical cancer. It is now recognized that telomere length maintenance or extension is indispensable for carcinogenesis. The early oncoproteins E6 and E7 are the main malignant transformation factors of HR-HPVs and they maintain telomeres by different mechanisms, of which E6 protein activating telomerase is well documented. Reports showed that E7 protein utilized an alternative lengthen of telomere (ALT) mechanism to restore telomere length, yet the underlying molecular basis remains largely unknown. We propose that degradation of tumor suppressor pRb family member p130 plays an essential role in E7-regulated telomere extension by ALT. ALT is a mechanism based on homologous recombination (HR) between telomere sister chromatids, and a number of proteins involved in the HR pathway, such as MRN [MRE11 (meiotic recombination 11)-Rad50-NBS1 (Nijmegen breakage syndrome 1)] complex are required for the ALT pathway. Rb family member p130 could inhibit ALT by interacting with Rad50, while HPV E7 could activate ALT by degrading p130. We will make E7 mutants which are defective in p130 degradation to test whether these cells have a limited life span. Besides, immunofluorescence assay will show an ALT-related promyelocytic leukemia (PML) body (APBs) in E7-expressing cells. Although cervical cancer usually has high telomerase activities since the expressing of HPV E6, the anti-telomerase therapy will be unavailable for cervical cancer since it may activate E7-induced ALT. Our hypothesis not only enrich the knowledge of the regulation of ALT, but also indicate that p130 may serve as a potential suppressor of ALT, and gene therapy of p130 may be used in cervical cancers.     

Human Sarcomas Are Mosaic for Telomerase-Dependent and Telomerase-Independent Telomere Maintenance Mechanisms: Implications for Telomere-Based Therapies

Telomere shortening necessitates that tumor cells activate a telomere maintenance mechanism (TMM) to support immortalization. Although most tumor cells activate expression of the enzyme telomerase, some cells elongate telomeres in a telomerase-independent manner, termed alternative lengthening of telomeres (ALT). Previous studies have evaluated the presence of telomerase or ALT mechanisms or both in a variety of tumor types. Our studies also show that TMMs are not mutually exclusive in some tumors. In contrast, our IHC analyses of human sarcomas identified a subset of tumors with some cells containing ALT-associated PML bodies, a hallmark of ALT, and separate cells expressing telomerase in the same tumor. By using a second set of human osteosarcomas, we merged IHC and biochemical analyses to characterize more fully the tumor TMM. The IHC data reveal the presence of both telomerase- and ALT-positive tumor cells in samples that demonstrate characteristics of both telomerase and ALT in biochemical assays. These assays, which measure telomere length and telomerase activity of tumor extracts, are conventionally used to classify tumor TMM. Our results suggest that TMM is not a single or perhaps static characteristic of some tumors and that TMM heterogeneity should be considered in tumor stratification. Furthermore, clinical interest in telomere-based therapies may necessitate accurate characterization of tumor TMM before treatment to maximize therapeutic efficacy.Activation of a telomere maintenance mechanism (TMM) is critical for tumor cell immortalization and cancer progression because telomeres shorten with each round of cellular division. Cells can use two known TMMs: catalytic activity of the enzyme telomerase or a telomerase-independent, recombination-based pathway termed alternative lengthening of telomeres (ALT). In contrast to cells with active telomerase, cells that use ALT are characterized by long, heterogeneous telomere lengths, ALT-associated PML bodies (APBs), and extrachromosomal telomeric repeats. Several studies have evaluated TMMs in human tumors^1,2 and demonstrated that ALT is overrepresented in mesenchymal tumors,^2–12 perhaps reflective of a lack of detectable telomerase expression in mesenchymal stem cells.¹³ More specifically, almost 60% of osteosarcomas exhibit ALT characteristics,^3–6 the highest incidence of any tumor yet evaluated. In contrast, epithelial tumors more often express telomerase,^1,2 suggesting there may be cell-specific preferences for TMM.Studies have demonstrated that either type of TMM can occur within the same cell, but only with experimental manipulation. Exogenous expression of functional telomerase within some ALT cell lines results in telomerase-mediated elongation of short telomeres, although cells still retain ALT characteristics^14–16; studies in other ALT cell lines have shown that telomerase expression inhibits ALT characteristics.¹⁷ Fusion experiments of telomerase-expressing and ALT cells have shown that the mechanisms governing telomere maintenance are complex. Some telomerase-expressing cells contain ALT inhibitors other than telomerase itself, because only some hybrid cells suppress ALT markers and maintain telomeres exclusively with telomerase.¹⁶ Other experiments with hybrid cells demonstrate suppression of telomerase activity and maintenance of telomeres by ALT, suggesting that some ALT cells contain telomerase inhibitors.¹⁸ Similar observations have not been made in vivo.Most tumors exhibit characteristics of one TMM. However, most published studies that have classified human tumors have also identified a subset of tumors not definitively ALT or telomerase positive.^{3–6,9–12,19–23} These tumors display ambiguous characteristics regarding telomere length, telomerase activity, or the presence of APBs. Some tumors have long, heterogeneous telomere lengths, suggestive of ALT, but exhibit telomerase activity; this ambiguity confounds accurate TMM characterization. Furthermore, although previous studies have identified tumors exhibiting both telomerase activity and the presence of APBs,^10,11 suggesting that both telomerase and ALT cells are present in the sample, it has not been demonstrated whether these results indicate separate cell populations, infiltrating normal cells/normal surrounding tissue, or the presence of multiple TMMs in one cell. Therefore, we asked whether human sarcomas could demonstrate mosaicism for TMM by evaluating tumors at a cellular level.     

Alternative lengthening of telomeres in human glioma stem cells

Cancer stem cells are increasingly recognized as major therapeutic targets. We report here the isolation of glioma stem cells (GSCs) maintaining telomere length through a telomerase-independent mechanism known as alternative lengthening of telomeres (ALTs). TG20 cells were isolated from a glioblastoma multiforme, which had the ALT phenotype. They have no detectable telomerase activity and extremely long and heterogeneous telomeres colocalizing with promyelocytic leukemia bodies. The cancer stem cell potential of TG20 cells was confirmed based on their expression of neural stem cell markers, their capacity of in vitro long-term proliferation and to form intracranial tumors in immune-deficient mice. Interestingly, we found that both in vitro and in vivo TG20 cells were significantly more resistant to ionizing radiation than GSCs with telomerase activity. Analysis of DNA damage foci, DNA double-strand breaks repair, and chromosome instability suggest that radiation resistance was related to interference of ALT pathway with DNA damage response. Therefore, our data show for the first time that the ALT pathway can confer to cancer stem cells the capacity to sustain long-term proliferation as telomerase activity and importantly may also affect treatment efficiency. TG20 cells are thus the first cellular model of GSCs displaying ALT and should prove to be useful for the development of specific treatment strategies.     

ALT-associated promyelocytic leukaemia body (APB) detection as a reproducible tool to assess alternative lengthening of telomere stability in liposarcomas

Most cancers maintain telomeres by activating telomerase, but a significant minority, mainly of mesenchymal origin, utilize an alternative lengthening of telomeres (ALT) mechanism. We previously showed the presence of ALT, as detected by ALT-associated promyelocytic leukaemia bodies (APBs) by combined promyelocytic leukaemia immunofluorescence and telomere fluorescence-in situ hybridization, in approximately 25% of frozen specimens obtained from adult patient liposarcomas and proved that ALT negatively affects patient prognosis. In the present study, we assessed the reproducibility of APB detection on frozen versus formalin-fixed, paraffin-embedded specimens from the same liposarcoma specimens and investigated the eventual stability of ALT in 103 different lesions from 40 adult patients followed during their disease. Irrespective of liposarcoma subtype, we (1) confirmed the presence of ALT in 21.4% of tumours; (2) demonstrated the reliability of ALT-associated promyelocytic leukaemia body detection in formalin-fixed, paraffin-embedded sections (with qualitative concordance between matched frozen and formalin-fixed, paraffin-embedded samples in 29/30 specimens, and high quantitative agreement, as indicated by a Spearman correlation coefficient of 0.85); and (3) suggested the stability of ALT status during disease evolution, since the ALT mechanism was never acquired in the 29 patients with initially ALT-negative lesions and lost over time in only two of 11 patients with initially ALT-positive liposarcomas. In conclusion, these results confirm the possibility of investigating the ALT mechanism in archival specimens to obtain biologically relevant information on liposarcoma progression, even when the primary lesion is not available.     

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

Two types of telomere maintenance mechanisms (TMMs) have been described in human tumors: telomerase activation and alternative lengthening of telomeres (ALT). Although the vast majority of epithelial tumors rely on telomerase activation, many mesenchymal tumors rely on ALT for telomere maintenance, but within this tumor group, the TMMs used by translocation-associated sarcomas have not been systematically studied. We studied telomere lengths and telomerase expression and activity in 30 uncultured tumor samples and in 10 cell lines of Ewing's sarcoma, a prototypical translocation-associated sarcoma, and compared the data to an identical analysis of 60 osteosarcomas, the most common type of sarcoma lacking a specific translocation. Telomerase activity was demonstrated in 21 Ewing's sarcoma tumor samples (70%) and in 9 of 10 Ewing's sarcoma cell lines. Evidence of ALT, indicated by the presence of long and heterogeneous telomeres, was observed only in the cell line without telomerase activity and in none of the 30 Ewing's sarcoma tumor samples. The 9 Ewing's sarcoma patients whose tumors lacked detectable telomerase activity did not differ significantly from the remaining patients in age, stage, EWSR1-FLI1 fusion type, prevalence of TP53 point mutations, or overall survival. The low prevalence of ALT in Ewing's sarcoma contrasted sharply with our data on TMMs in 60 osteosarcomas, which showed ALT in 38 of 60 cases (P<0.0001). The present study, together with emerging published data on other sarcoma types, suggests that a predominance of telomerase activation in the absence of ALT may characterize sarcomas with specific chromosomal translocations (such as Ewing's sarcoma), whereas a high prevalence of ALT appears typical of sarcomas with nonspecific complex karyotypes (such as osteosarcoma).     

The telomere lengthening mechanism in telomerase-negative immortal human cells does not involve the telomerase RNA subunit

According to the telomere hypothesis of senescence, the progressive shortening of telomeres that occurs upon division of normal somatic cells eventually leads to cellular senescence. The immortalisation of human cells is associated with the acquisition of a telomere maintenance mechanism which is usually dependent upon expression of the enzyme telomerase. About one third of in vitro immortalised human cell lines, however, have no detectable telomerase but contain telomeres that are abnormally long. The nature of the alternative telomere maintenance mechanism (referred to as ALT, for Alternative Lengthening of Telomeres) that must exist in these telomerase-negative cells has not been elucidated. It has previously been shown that abnormal lengthening of yeast telomeres may occur due to mutations in the yeast telomerase RNA gene. That this is not the mechanism of the abnormally long telomeres in ALT cell lines was demonstrated by the finding that seven of seven ALT lines have wild-type human telomerase RNA (hTR) sequence, including a novel polymorphism that is present in 30% of normal individuals. We found that two ALT cell lines have no detectable expression of the hTR gene. This shows that the ALT mechanism in these cell lines is not dependent on hTR. Expression of exogenous hTR via infection of these cells with a recombinant hTR-adenovirus vector did not result in telomerase activity, indicating that their lack of telomerase activity is not due to absence of hTR expression. We conclude that the ALT mechanism is not dependent on the expression of hTR, and does not involve mutations in the hTR sequence.      

Analysis of alternative lengthening of telomere markers in BRCA1 defective cells

Telomeres are specialized structures responsible for the chromosome end protection. Previous studies have revealed that defective BRCA1 may lead to elevated telomere fusions and accelerated telomere shortening. In addition, BRCA1 associates with promyelocytic leukemia (PML) bodies in alternative lengthening of telomeres (ALTs) positive cells. We report here elevated recombination rates at telomeres in cells from human BRCA1 mutation carriers and in mouse embryonic stem cells lacking both copies of functional Brca1. An increased recombination rate at telomeres is one of the signs of ALT. To investigate this possibility further we employed the C‐circle assay that identifies ALT unequivocally. Our results revealed elevated levels of ALT activity in Brca1 defective mouse cells. Similar results were obtained when the same cells were assayed for the presence of another ALT marker, namely the frequency of PML bodies. These results suggest that BRCA1 may act as a repressor of ALT. © 2016 The Authors Genes, Chromosomes & Cancer Published by Wiley Periodicals, Inc.     

Lack of correlation between telomere length and telomerase activity and expression in leukemic cells

The expression of three components of telomerase complex (hTR, hTERT, TP1) along with telomerase activity and telomere length in leukemic cells was investigated. Cells were isolated from peripheral blood and/or bone marrow of children with acute lymphoblastic (ALL) and non-lymphoblastic (ANLL) leukemia. Expression of three components of telomerase as well as telomerase activity was found in all leukemic cells. Chemiluminescent detection of terminal restriction fragments (TRF) from DNA isolated from ALL cells showed variable patterns expressing considerable heterogeneity of telomere length. The ALL cells appeared to have both long and short telomere lengths, in contrast to normal peripheral lymphocytes, which produced limited pattern of TRF. The ANLL cells produced predominantly short telomere pattern despite high telomerase activity and expression. It can be concluded that high telomerase activity and expression in leukemic cells is not always correlated with long telomeres (TRF pattern).     

Telomere lengths of translocation-associated and nontranslocation-associated sarcomas differ dramatically

Sarcomas can be divided into those with specific translocations displaying monotonous cytomorphology, and those with complex karyotypes and marked cellular pleomorphism. Telomeres contain terminal DNA sequence repeats that maintain chromosomal stability. Telomeres shorten with cell division and may become dysfunctional leading to chromosomal instability. Using a fluorescence in situ hybridization/immunofluorescence method to assess telomere lengths in archival tissues we analyzed these two types of sarcomas using paraffin-embedded primary tumor specimens. Tissues from nine sarcomas with characteristic translocations (two synovial sarcomas, two alveolar rhabdomyosarcomas, two desmoplastic round cell tumors, and one each of infantile fibrosarcoma, myxoid liposarcoma, cellular congenital mesoblastic nephroma) and nine without (four malignant fibrous histiocytomas, two leiomyosarcomas, one pleomorphic rhabdomyosarcoma, one dedifferentiated chondrosarcoma, and one malignant peripheral nerve sheath tumor) were analyzed. In all (nine of nine) cases with specific translocations, which generally have few karyotypic abnormalities, telomere lengths were similar to or reduced compared to surrounding nonneoplastic tissues. In contrast, telomeres in cases lacking specific translocations, which generally contain complex karyotypes, were often found to be dramatically lengthened and heterogeneous. In addition to markedly elongated telomeres, seven of nine (78%) complex cases exhibited large brightly stained regions corresponding to a specific type of promyelocytic leukemia nuclear body found in immortalized cells that maintain telomeres in a telomerase-independent manner [alternative lengthening of telomeres (ALT) pathway]. This phenotype is unlike that of epithelial neoplasms that typically display complex karyotypes with abnormally short telomeres maintained by the enzyme telomerase. The discovery of heterogeneous telomere lengths and evidence of the ALT pathway in the majority of sarcomas with complex karyotypes supports the existence of a telomere maintenance pathway incapable of full karyotypic stabilization in pleomorphic sarcomas. These findings provide additional molecular-genetic evidence supporting the dichotomous grouping of sarcomas into those with characteristic signature translocations without extensive additional karyotypic abnormalities, and those without such signature translocations that typically display very complex karyotypes, and point to telomere dysfunction as a plausible contributor to the chromosomal aberrations found in complex sarcomas.     

替代性端粒延长机制与端粒

端粒的维持除了可以通过端粒酶的作用外,还可以通过其他途径.这种不依赖端粒酶的延长途径被称为替代性端粒延长机制.替代性端粒延长机制与端粒酶途径之间存在着抑制效应.二者延长端粒的效果不一样,以致其后续效应对肿瘤演变产生不同的影响.对替代性端粒延长机制的理解有助于阐明肿瘤的发生,并对肿瘤的预防和治疗提出新的建议.     

Telomere biology in giant cell tumour of bone

Giant cell tumour of bone (GCTB) is a benign bone tumour known for the unpredictable clinical behaviour of recurrences and, in rare instances, distant metastases. It consists of uniformly distributed osteoclastic giant cells in a background of mononuclear rounded and spindle-shaped cells. Cytogenetically, telomeric associations are the most common chromosomal aberrations, which, however, are normally almost exclusively found in high-grade malignancies. GCTB has often been regarded as a polyclonal tumour, but more recently a recurrent specific aberration was reported, which suggests a possible role for disturbed telomere maintenance. Here we further investigate telomere maintenance in GCTB using 19 samples from 19 patients. A combination of immunofluorescence and FISH was performed, applying antibodies directed against promyelocytic leukaemia body-related antigen and hTERT and using telomere peptide nucleic acid probes. The TRAP assay and telomere restriction fragment length analysis were performed for functional detection of telomerase activity and alternative telomere lengthening. Both osteoclastic giant cells and mononuclear cells showed positivity for hTERT and promyelocytic leukaemia body-related antigen. In most mononuclear cells, co-expression was present. The TRAP assay demonstrated heterogeneous telomerase activity, while telomere restriction fragment length analysis showed non-heterogeneous telomere lengths, indicating the absence of alternative telomere lengthening. Confocal microscopy showed stereometric co-localization of nucleolin with promyelocytic leukaemia body-related antigen in association with telomeres in the spindle-shaped cells. hTERT was more diffusely distributed throughout the nucleus. Our results show that GCTB demonstrates remarkable telomere maintenance of activated telomerase and inactivated alternative telomere lengthening in the presence of normal mean telomere restriction fragment lengths. These findings strongly suggest that these aggregates, while activating telomerase, are part of a structural telomere protective-capping mechanism rather than of a telomere-lengthening mechanism. Telomere maintenance could be considered an important key factor in the pathogenesis of GCTB.     

Telomere uncapping and alternative lengthening of telomeres

A substantial number of human tumors utilize a telomerase-independent telomere length maintenance mechanism referred to as alternative lengthening of telomeres (ALT). Although it is known that ALT is a telomere-specific, loss of function phenotype, which involves lengthening of telomeres by homologous recombination-mediated replication of telomeric DNA, many of the details of these processes require elucidation. Here we discuss the current literature on ALT and telomere capping, specifically focusing on how alterations in telomere capping functions may permit activation of ALT and explain the phenotypic characteristics of cells in which this occurs.     

Different telomere maintenance mechanisms in alveolar and embryonal rhabdomyosarcoma

The activation of a telomere maintenance mechanism (TMM) is crucial for the immortalization of tumor cells. Most human cancers apply telomerase-dependent TMM but some use a mechanism called alternative lengthening of telomeres (ALT). The latter was suggested to be mainly characterizing sarcomas with nonspecific complex karyotypes, whereas telomerase activation is typical of sarcomas generated by specific translocations. In this study, we investigated the TMM and its association with survival in rhabdomyosarcoma (RMS), which is characterized by two major subtypes: one that is harboring a specific translocation (alveolar) and one that has a nonspecific karyotype (embryonal). Telomerase activity (TA), using telomerase repeat amplification protocol (TRAP) assay, and telomere length (TRF), using Southern blotting, were analyzed in tumor samples from 31 patients (16 embryonal and 15 alveolar). Alveolar RMS tumors exhibited no ALT phenotype and the majority presented TA. Some embryonal tumors exhibited an ALT or "ALT-like" phenotype which lacked TA, whereas others expressed telomerase-dependent TMM, and neither TA nor ALT correlated with outcome. The average TRF length of the embryonal tumors was significantly higher than that of the alveolar tumors (10.8 vs. 7.2 kb, P = 0.003). Interestingly, some tumors of both subtypes presented no TMM. These observations suggest that alveolar RMS predominantly use telomerase-dependent TMM, whereas in embryonal tumors both telomerase and ALT may play a role. These findings have important implications for understanding the role of TMM in the development of RMS tumors, and for future designing adapted treatment strategies.