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 role of telomere biology in bone marrow failure and other disorders

Telomeres, consisting of nucleotide repeats and a protein complex at chromosome ends, are essential in maintaining chromosomal integrity. Dyskeratosis congenita (DC) is the inherited bone marrow failure syndrome (IBMFS) that epitomizes the effects of abnormal telomere biology. Patients with DC have extremely short telomere lengths (<1st percentile) and many have mutations in telomere biology genes. Interpretation of telomere length in other IBMFSs is less straightforward. Abnormal telomere shortening has been reported in patients with apparently acquired hematologic disorders, including aplastic anemia, myeolodysplasia, paroxysmal nocturnal hemoglobinuria, and leukemia. In these disorders, the shortest-lived cells have the shortest telomeres, suggestive of increased hematopoietic stress. Telomeres are also markers of replicative and/or oxidative stress in other complex disease pathways, such as inflammation, stress, and carcinogenesis. The spectrum of related disorders caused by mutations in telomere biology genes extends beyond classical DC to include marrow failure that does not respond to immunosuppression, idiopathic pulmonary fibrosis, and possibly other syndromes. We suggest that such patients be categorized as having an inherited disorder of telomere biology. Longitudinal studies of patients with very short telomeres but without classical DC are necessary to further understand the long-term sequelae, such as malignancy, osteonecrosis/osteoporosis, and pulmonary and liver disease.     

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.     

Definitive molecular cytogenetic characterization of 15 colorectal cancer cell lines

In defining the genetic profiles in cancer, cytogenetically aberrant cell lines derived from primary tumors are important tools for the study of carcinogenesis. Here, we present the results of a comprehensive investigation of 15 established colorectal cancer cell lines using spectral karyotyping (SKY), fluorescence in situ hybridization, and comparative genomic hybridization (CGH). Detailed karyotypic analysis by SKY on five of the lines (P53HCT116, T84, NCI‐H508, NCI‐H716, and SK‐CO‐1) is described here for the first time. The five lines with karyotypes in the diploid range and that are characterized by defects in DNA mismatch repair had a mean of 4.8 chromosomal abnormalities per line, whereas the 10 aneuploid lines exhibited complex karyotypes and a mean of 30 chromosomal abnormalities. Of the 150 clonal translocations, only eight were balanced and none were recurrent among the lines. We also reviewed the karyotypes of 345 cases of adenocarcinoma of the large intestine listed in the Mitelman Database of Chromosome Aberrations in Cancer. The types of abnormalities observed in the cell lines reflected those seen in primary tumors: there were no recurrent translocations in either tumors or cell lines; isochromosomes were the most common recurrent abnormalities; and breakpoints occurred most frequently at the centromeric/pericentromeric and telomere regions. Of the genomic imbalances detected by array CGH, 87% correlated with chromosome aberrations observed in the SKY studies. The fact that chromosome abnormalities predominantly result in copy number changes rather than specific chromosome or gene fusions suggests that this may be the major mechanism leading to carcinogenesis in colorectal cancer. Published 2009 Wiley‐Liss, Inc.     

Telomere length and telomerase activity: effect of ageing on human NK cells

Telomeres are repeats of TTAGGG sequences located at the end of eukaryotic chromosomes. They are essential for stabilisation and protection of chromosomal ends and for the regulation of cell replicative capacity. Due to the end-replication defect of DNA polymerase, telomeres shorten progressively with each cell division and telomere length may be an indicator of the replicative history of a cell. Compensatory mechanisms for the telomere loss have been identified. The most widely studied one is mediated by telomerase a ribonuclear protein-enzyme complex that synthesise telomeric repeats. In this study we have investigated whether NK cells, derived from a group of old healthy subjects, underwent the modifications of telomere length and telomerase activity observed in other sub-populations of lymphocytes with advancing age. We demonstrated that: (a) telomere shortening occurred and telomerase activity decreased in human NK cells with ageing; (b) the rate of telomere loss was different under and over 80 years of age; (c) similarly to telomere shortening, the modification of telomerase activity was particularly evident in octogenarians; (d) subjects with the most evident modifications of telomeres and telomerase were the oldest and those with increased NK cell numbers.     

Cytogenetic analysis of 46 pleomorphic soft tissue sarcomas and correlation with morphologic and clinical features: A report of the CHAMP study group

With the aim of identifying objective cytogenetic-morphologic correlations, we evaluated 46 pleomorphic soft tissue sarcomas (mainly diagnosed originally as malignant fibrous histiocytomas) with clonal chromosome aberrations both cytogenetically and morphologically as part of an international collaborative study. By detailed histopathologic examination, most cases could be categorized into specific tumor types. Eight sarcomas were diagnosed as lipogenic (4 pleomorphic, 1 combined pleomorphic and myxoid/round cell, and 3 dedifferentiated liposarcomas), 19 as myogenic [11 leiomyosarcomas, 1 rhabdomyosarcoma, 4 myosarcomas not otherwise specified (NOS), and 3 probable myosarcomas NOS], 8 as myxofibrosarcomas, 1 as a malignant peripheral nerve sheath tumor, 1 as malignant mesenchymoma, 1 as extraskeletal osteosarcoma, 1 as sarcoma resembling proliferative fasciitis, and 7 as pleomorphic sarcomas NOS. In a three-grade system, 10 tumors were grade 2 and 36 were grade 3. The majority had highly complex karyotypes. A total of 24 recurrent abnormalities (defined by their presence in at least five cases) were detected: ring chromosomes, homogeneously staining regions (hsr) and/or double minute chromosomes (dmin), and structural rearrangement of 22 different chromosome bands or regions. The frequency and distribution of the recurrent karyotypic features were uneven. Grade 3 tumors displayed, on average, more aberrations per case than did grade 2 tumors. Nine of the selected abnormalities, including hsr/dmin and rearrangements of 19p13 and 19q13, were found only among the high-grade tumors. When the tumors were subdivided according to lineage of differentiation, the highest frequency of aberrations was seen in pleomorphic sarcomas NOS, followed by myxofibrosarcomas, myogenic sarcomas, and lipogenic sarcomas. None of the selected rearrangements was, however, specific for any of these subgroups. The sole consistent cytogenetic-morphologic association was that all three dedifferentiated liposarcomas had multiple abnormal clones, at least one of which contained supernumerary ring chromosomes. Due mainly to karyotype complexity, it therefore seems unlikely that cytogenetic analysis can assist in the differential diagnostic subclassification of pleomorphic sarcomas, nor was there any clear-cut indication that the karyotypic picture could be used to predict clinical outcome. Although the mean number of recurrent chromosome aberrations was almost twice as high in sarcomas that gave rise to metastases as among those that did not, no particular aberration was restricted to either of the two subgroups. Genes Chromosomes Cancer 22:16–25, 1998. © 1998 Wiley-Liss, Inc.     

Inside the mammalian telomere interactome: regulation and regulatory activities of telomeres

Work in model organisms, such as mouse, yeast, Tetrahymena, ciliates, and plants, has led to a deeper understanding of telomere biology. Telomeres together with telomere-binding proteins have evolved to protect chromosomal ends and maintain chromosomal length and integrity. Over the last two decades, biochemical, molecular, cellular, and genetic studies have greatly enhanced our knowledge of the unique function and structure of telomeres and telomere-associated factors. In this review, we focus on the important advances, in terms of our knowledge and the methods used, in understanding mammalian telomere regulation by telomeric proteins. Recently, the 6 telomeric proteins (TRF1, TRF2, POT1, TIN2, RAP1, and TPP1) were found to form a high-order complex. This complex and its associated partners provide the basis for constructing an interaction map of telomere regulators in mammalian cells, which we named the Telomere Interactome. The Telomere Interactome incorporates the various telomere signaling pathways and represents the molecular machinery that regulates mammalian telomeres. The establishment of the Telomere Interactome will also enable the integration of the intricate circuitries that regulate telomeres with other cellular interactomes in vertebrates.     

Telomere-specific fluorescence in situ hybridization analysis of couples with five or more recurrent miscarriages

Fluorescence in situ hybridization analysis using telomere specific probes has been used to detect cryptic translocations in the chromosomal telomeric regions. This study was performed in five clinically normal couples who have had five or more spontaneous abortions and whose karyotypes were found to be normal using conventional cytogenetic techniques. Using the telomere specific probes, in one couple we determined a cryptic translocation between chromosome 3 and 10, and, in another couple, the signal in chromosome 20 was detected in another chromosome, which was probably a D group chromosome. Additionally, in the latter and also in two other couples, we observed a polymorphism. The approach will be helpful for screening cryptic translocations using telomere specific multiple probe sets in couples with recurrent miscarriages. As prenatal diagnosis will be available for these couples for future pregnancies, it will be possible to help these families to have healthy fetuses.     

Telomere‐associated proteins: cross‐talk between telomere maintenance and telomere‐lengthening mechanisms

Telomeres, the ends of eukaryotic chromosomes, have been the subject of intense investigation over the last decade. As telomere dysfunction has been associated with ageing and developing cancer, understanding the exact mechanisms regulating telomere structure and function is essential for the prevention and treatment of human cancers and age‐related diseases. The mechanisms by which cells maintain telomere lengthening involve either telomerase or the alternative lengthening of the telomere pathway, although specific mechanisms of the latter and the relationship between the two are as yet unknown. Many cellular factors directly (TRF1/TRF2) and indirectly (shelterin‐complex, PinX, Apollo and tankyrase) interact with telomeres, and their interplay influences telomere structure and function. One challenge comes from the observation that many DNA damage response proteins are stably associated with telomeres and contribute to several other aspects of telomere function. This review focuses on the different components involved in telomere maintenance and their role in telomere length homeostasis. Special attention is paid to understanding how these telomere‐associated factors, and mainly those involved in double‐strand break repair, perform their activities at the telomere ends. Copyright © 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

The role of genetic testing in soft tissue sarcoma

Soft tissue tumours represent a heterogeneous group of mesenchymal lesions and their classification continues to evolve as a result of incorporating advances in cytogenetic and molecular techniques. In the last decade traditional diagnostic approaches were supplemented with a significant number of reliable molecular diagnostic tools, detecting tumour type-specific genetic alterations. In addition, the successful application of some of these techniques to formalin-fixed paraffin-embedded tissue made it possible to subject a broader range of clinical material to molecular analysis. Thus, molecular genetics has already become an integral part of the work-up in some tumours, such as paediatric small blue round cell tumours, which demonstrate characteristic translocations. Several lines of evidence suggest that sarcomas can be divided into two major genetic groups: (i) sarcomas with specific genetic alterations and usually simple karyotypes, such as reciprocal chromosomal translocations (e.g. FUS-DDIT3 in myxoid liposarcoma) and specific oncogenic mutations (e.g. KIT mutation in gastrointestinal stromal tumours); and (i) sarcomas with non-specific genetic alterations and complex unbalanced karyotypes. Some of these genetic abnormalities, including chromosomal numerical changes, translocations, gene amplifications or large deletions can be apparent at the cytogenetic level (karyotyping, fluorescence in situ hybridization), while others, such as small deletions, insertions or point mutations, require molecular genetic techniques (polymerase chain reaction and sequence analysis). This review focuses on the applicability of genetic testing in the diagnosis and prognosis of soft tissue sarcomas, and gives a realistic appraisal of the ancillary role of molecular techniques, including its advantages and limitations.     

Telomere biology in healthy aging and disease

Aging is a biological process that affects most cells, organisms and species. Telomeres have been postulated as a universal biological clock that shortens in parallel with aging in cells. Telomeres are located at the end of the chromosomes and consist of an evolutionary conserved repetitive nucleotide sequence ranging in length from a few hundred base pairs in yeast till several kilo base pairs in vertebrates. Telomeres associate with shelterin proteins and form a complex protecting the chromosomal deoxyribonucleic acid (DNA) from recognition by the DNA damage-repair system. Due to the “end-replication problem” telomeres shorten with each mitotic cycle resulting in cumulative telomere attrition during aging. When telomeres reach a critical length the cell will not further undergo cell divisions and become senescent or otherwise dysfunctional. Telomere shortening has not only been linked to aging but also to several age associated diseases, including tumorigenesis, coronary artery disease, and heart failure. In the current review, we will discuss the role of telomere biology in relation to aging and aging associated diseases.     

Alcoholics show reduced telomere length in the oesophagus

Telomeres are repetitive G-rich DNA sequences located at the ends of chromosomes. Chromosomal and genomic instability due to telomere dysfunction plays an important role in carcinogenesis. To study telomere shortening in the oesophageal epithelium of alcoholics, we measured the telomere lengths of basal and parabasal cells in comparison with those of non-alcoholics using Q-FISH and our original software, Tissue Telo, and also assessed histological inflammation. Telomeres in basal cells were significantly shorter in alcoholics than in age-matched normal controls. Prominent histological findings of chronic inflammation were not evident in either alcoholics or non-alcoholics. Our finding that telomeres in the oesophageal epithelium are shorter in alcoholics than in non-alcoholics indicates that telomere shortening may be associated with the frequent occurrence of squamous cell carcinoma in alcoholics. Further studies to clarify the reason for the large annual loss of telomere length with rapid turnover or lower telomerase activity in the oesophageal epithelium of alcoholics will be necessary.     

The structural basis of molecular genetic deletions. An integration of classical cytogenetic and molecular analyses in pancreatic adenocarcinoma.

Molecular genetic alterations are known to be important in human carcinoma, but the structural basis of these changes is largely unknown. To examine the basis of these changes, we compared the karyotypic chromosomal abnormalities of primary pancreatic adenocarcinomas with the molecular changes identified in these same cancers. In 14 cancers with abnormal karyotypes, 65% (123 of 188) of the chromosomal arms with molecular loss of heterozygosity (LOH) were associated with karyotypic structural anomalies. Karyotypic changes accounting for these molecular allelic losses included 83 chromosome losses, 18 partial deletions, nine isochromosomes, eight additions, and five translocations. Eight bomozygous deletions were also identified by molecular analyses. Of the three homozygous deletions identified at 9p21, the only karyotypic change was a single case in which one entire copy of chromosome 9 was deleted. Of the four homozygous deletions identified at 18q21.1, one showed a loss of both copies of chromosome 18, two showed a loss of one copy of chromosome 18, and the fourth had two structurally normal copies of chromosome 18. One homozygous deletion was identified at 13q12.3, and the karyotype revealed the loss of one entire copy of chromosome 13. The second copy of chromosome 13 in this carcinoma was structurally normal. These results indicate that chromosomal structural anomalies can account for two-thirds of the LOH in pancreatic adenocarcinomas and that most homozygous deletions are likely to be interstitial chromosomal deletions that are below the detection limit of conventional karyotypic analyses. Some of the molecular deletions detected as LOH on chromosomes with karyotypically normal structure can be explained by chromosomal loss with reduplication of the remaining chromosome.     

Telomere distribution pattern and synapsis initiation during spermatogenesis in zebrafish

Background: Telomeres are located at ends of eukaryotic chromosomes and can affect proper chromosomal positioning. During spermatogenesis, the appropriate dynamics and behavior of chromosomes is crucial to generate haploid cells through meiosis. Here, we describe telomere distribution patterns during spermatogenesis in zebrafish, especially during meiotic prophase I, using fluorescence in situ hybridization. This was combined with synaptonemal complex protein 3 immunostaining, which allows the staging of spermatocytes. Results: During spermatogonial proliferation and the preleptotene stage, telomeres were dispersed throughout the nucleus. During the leptotene stage, telomeres temporarily moved to one pole of the nucleus at which γ‐tubulin was located, forming the telomere bouquet. The cluster lasted until the onset of zygotene where it coincided with terminal synapsis initiation. They then spread around the periphery of the nucleus during the zygotene to pachytene stages. During postmeiotic stages, telomeres in spermatids and sperm were again dispersed throughout the nuclei. Application of this procedure in meiotic mutants confirmed that meiotic telomere clustering is independent of axial element formation of the synaptonemal complex. Conclusions: These data clearly showed the clustering and distributions of telomeres throughout spermatogenesis in zebrafish. This procedure could be used to screen for mutants that have primary defects in telomere clustering. Developmental Dynamics 243:1448–1456, 2014. © 2014 Wiley Periodicals, Inc.     

Critically short telomeres in acute myeloid leukemia with loss or gain of parts of chromosomes

Telomeres, nucleoprotein complexes at chromosome ends, protect chromosomes against end-to-end fusion. Previous in vitro studies in human fibroblast models indicated that telomere dysfunction results in chromosome instability. Loss of telomere function can result either from critical shortening of telomeric DNA or from loss of distinct telomere-capping proteins. It is less clear whether telomere dysfunction has an important role in human cancer development in vivo. Acute myeloid leukemia (AML) is a good model to study mechanisms that generate chromosome instability in human cancer development because distinct groups of AML are characterized either by aberrations that theoretically could result from telomere dysfunction (terminal deletions, gains/losses of chromosome parts, nonreciprocal translocations), or aberrations that are unlikely to result from telomere dysfunction (e.g., reciprocal translocations or inversions). Here we demonstrate that AML with multiple chromosome aberrations that theoretically could result from telomere dysfunction is invariably characterized by critically short telomeres. Short telomeres in this group are not associated with low telomerase activity or decreased expression of essential telomeric capping proteins TRF2 and POT1. In contrast, telomerase activity levels are significantly higher in AML with short telomeres. Notably, short telomeres in the presence of high telomerase may relate to significantly higher expression of TRF1, a negative regulator of telomere length. Our observations suggest that, consistent with previous in vitro fibroblast models, age-related critical telomere shortening may have a role in generating chromosome instability in human AML development.     

Telomere shortening and chromosomal instability in myelodysplastic syndromes

Telomere shortening and chromosomal instability are believed to play an important role in the development of myeloid neoplasia. So far, published data are only available on the average telomere length in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), but not on the telomere length of individual chromosomes. We used a new technique, telomere/centromere‐fluorescence in situ hybridization (T/C‐FISH), which combines fluorescence R‐banding and FISH using a probe against the telomere repeats to measure the telomere length of each chromosome arm in 78 patients with MDS. In line with the previous results, patients with MDS showed significantly shorter telomeres than those of healthy controls. Telomere lengths did not differ significantly between distinct morphological subtypes of MDS. However, there was a significant difference in telomere length between patients with an isolated monosomy 7 and patients with a normal karyotype (P < 0.05). Notably, patients with an isolated monosomy 7 showed significantly longer telomeres than patients with a normal karyotype in many chromosome arms, among them 7p and 7q. Neo‐telomeres were found in two patients with a complex karyotype, in one case at the fusion site of a dic(14;20). Normal and aberrant metaphases of the same patient did not differ in telomere length, thus indicating to telomere shortening as a basic mechanism affecting all hematopoietic cells in patients with MDS. In some MDS subtypes, like MDS with isolated monosomy 7, telomeres may be stabilized and even increase in length because of the activation of telomerase or alternative mechanisms. © 2009 Wiley‐Liss, Inc.     

Q‐FISH analysis of telomere and chromosome instability in the oesophagus with and without squamous cell carcinoma in situ

Chromosomal and genomic instability due to telomere dysfunction is known to play an important role in carcinogenesis. To study telomere dysfunction in the surrounding background epithelium of squamous cell carcinoma in situ (CIS) of the oesophagus, we measured telomere lengths of basal and parabasal cells of epithelia with and without CIS using quantitative fluorescence in situ hybridization (Q‐FISH) and our original software, Tissue Telo. Additionally, we assessed histological inflammation and the anaphase bridge index. In non‐cancerous epithelium, telomeres in basal cells were significantly longer than those in parabasal cells, whereas CIS showed a homogeneous telomere pattern in the basal and parabasal cells. Telomeres in basal and parabasal cells were significantly shorter in the background with CIS than in epithelium from age‐matched normal controls. Significant negative correlation was observed between the normalized telomere : centromere ratio (reflected telomere length) and the anaphase bridge index in non‐cancerous epithelia from both normal controls and the CIS background with no histological inflammation. These findings indicate that tissue stem cells may be located among basal cells, and that telomere length distribution in component cell types differs between CIS and non‐cancerous epithelium. We have demonstrated conclusively that oesophageal CIS arises from epithelium with short telomeres and chromosomal instability in the absence of histological inflammation. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

The nature of telomere fusion and a definition of the critical telomere length in human cells

The loss of telomere function can result in telomeric fusion events that lead to the types of genomic rearrangements, such as nonreciprocal translocations, that typify early-stage carcinogenesis. By using single-molecule approaches to characterize fusion events, we provide a functional definition of fusogenic telomeres in human cells. We show that approximately half of the fusion events contained no canonical telomere repeats at the fusion point; of those that did, the longest was 12.8 repeats. Furthermore, in addition to end-replication losses, human telomeres are subjected to large-scale deletion events that occur in the presence or absence of telomerase. Here we show that these telomeres are fusogenic, and thus despite the majority of telomeres being maintained at a stable length in normal human cells, a subset of stochastically shortened telomeres can potentially cause chromosomal instability. Telomere fusion was accompanied by the deletion of one or both telomeres extending several kilobases into the telomere-adjacent DNA, and microhomology was observed at the fusion points. This contrasted with telomere fusion that was observed following the experimental disruption of TRF2. The distinct error-prone mutational profile of fusion between critically shortened telomeres in human cells was reminiscent of Ku-independent microhomology-mediated end-joining.