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Telomere Lengths of Translocation-Associated and Nontranslocation-Associated Sarcomas Differ Dramatically

2004; Elsevier BV; Volume: 164; Issue: 5 Linguagem: Inglês

10.1016/s0002-9440(10)63710-8

1525-2191

Elizabeth A. Montgomery, Pedram Argani, Jessica Hicks, Angelo M. De Marzo, Alan K. Meeker,

Virus-based gene therapy research

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. 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. Telomeres play a key role in the maintenance of chromosomal stability. They are composed of specialized terminal DNA sequence repeats complexed with specific binding proteins and are located at the ends of every normal human chromosome.1Blackburn EH Structure and function of telomeres.Nature. 1991; 350: 569-573Crossref PubMed Scopus (3123) Google Scholar Telomere shortening is believed to be important in aging, cell senescence, cell replication, and malignant transformation. In contrast, pathological telomere elongation is found in a number of cancer types, either via telomerase enzymatic activity or by an alternative telomerase-independent pathway thought to involve homologous recombination, known as “alternative lengthening of telomeres” or ALT.2Bryan TM Englezou A Gupta J Bacchetti S Reddel RR Telomere elongation in immortal human cells without detectable telomerase activity.EMBO J. 1995; 14: 4240-4248Crossref PubMed Scopus (1125) Google Scholar, 3Henson JD Neumann AA Yeager TR Reddel RR Alternative lengthening of telomeres in mammalian cells.Oncogene. 2002; 21: 598-610Crossref PubMed Google Scholar, 4Reddel RR Bryan TM Colgin LM Perrem KT Yeager TR Alternative lengthening of telomeres in human cells.Radiat Res. 2001; 155: 194-200Crossref PubMed Scopus (92) Google Scholar A defining characteristic of the ALT phenotype is extreme telomere length heterogeneity with telomeres ranging from very short to very long. Studies of somatic cell hybrids between ALT-positive and telomerase-positive immortalized cells suggest that the two pathways are often mutually exclusive.5Perrem K Bryan TM Englezou A Hackl T Moy EL Reddel RR Repression of an alternative mechanism for lengthening of telomeres in somatic cell hybrids.Oncogene. 1999; 18: 3383-3390Crossref PubMed Scopus (96) Google Scholar When the ALT pathway is functional, nuclei in a subset of cells display aggregates of telomeric DNA that manifest themselves as medium-large brightly stained spots on hybridization with a fluorescently labeled probe specific for the telomeric DNA repeat sequence.6Yeager TR Neumann AA Englezou A Huschtscha LI Noble JR Reddel RR Telomerase-negative immortalized human cells contain a novel type of promyelocytic leukemia (PML) body.Cancer Res. 1999; 59: 4175-4179PubMed Google Scholar These brightly stained structures have previously been shown to reside in a variant form of the promyelocytic leukemia (PML) nuclear bodies normally present in all cells. In addition to the PML protein and telomeric DNA, these variant PML bodies have also been found to contain telomeric binding proteins (TRF-1, TRF-2), as well as proteins involved in DNA double-strand break repair and recombination (eg, RPA, Mre11/Nbs1/RAD50 complex, RAD51, RAD52). These structures are considered another hallmark of the ALT phenotype, and are referred to as ALT-associated PML bodies (APBs).3Henson JD Neumann AA Yeager TR Reddel RR Alternative lengthening of telomeres in mammalian cells.Oncogene. 2002; 21: 598-610Crossref PubMed Google Scholar, 4Reddel RR Bryan TM Colgin LM Perrem KT Yeager TR Alternative lengthening of telomeres in human cells.Radiat Res. 2001; 155: 194-200Crossref PubMed Scopus (92) Google Scholar As a generalization, sarcomas can be divided into those with simple karyotypes harboring specific translocations and those with complex karyotypes. The former display monotonous cytomorphology and lack anaphase bridges, a mitotic abnormality indicative of previous DNA double-strand breakage, whereas the latter often are pleomorphic and display anaphase bridges.7Montgomery E Wilentz RE Argani P Fisher C Hruban RH Kern SE Lengauer C Analysis of anaphase figures in routine histologic sections distinguishes chromosomally unstable from chromosomally stable malignancies.Cancer Biol Ther. 2003; 2: 248-252Crossref PubMed Scopus (54) Google Scholar Unlike epithelial cancers that typically possess abnormally short telomeres stabilized by telomerase, cancers of mesenchymal origin frequently lack telomerase and exhibit hallmarks of the ALT phenotype.8Bryan TM Englezou A Dalla-Pozza L Dunham MA Reddel RR Evidence for an alternative mechanism for maintaining telomere length in human tumors and tumor-derived cell lines.Nat Med. 1997; 3: 1271-1274Crossref PubMed Scopus (1057) Google Scholar, 9Ulaner GA Huang HY Otero J Zhao Z Ben-Porat L Satagopan JM Gorlick R Meyers P Healey JH Huvos AG Hoffman AR Ladanyi M Absence of a telomere maintenance mechanism as a favorable prognostic factor in patients with osteosarcoma.Cancer Res. 2003; 63: 1759-1763PubMed Google Scholar, 10Scheel C Schaefer KL Jauch A Keller M Wai D Brinkschmidt C van Valen F Boecker W Dockhorn-Dworniczak B Poremba C Alternative lengthening of telomeres is associated with chromosomal instability in osteosarcomas.Oncogene. 2001; 20: 3835-3844Crossref PubMed Scopus (142) Google Scholar Although the ALT pathway provides another mechanism for telomere length maintenance, it also appears to foster chromosomal instability, perhaps because of the presence of very short unstable telomeres on subsets of chromosomes in ALT-positive cells.10Scheel C Schaefer KL Jauch A Keller M Wai D Brinkschmidt C van Valen F Boecker W Dockhorn-Dworniczak B Poremba C Alternative lengthening of telomeres is associated with chromosomal instability in osteosarcomas.Oncogene. 2001; 20: 3835-3844Crossref PubMed Scopus (142) Google Scholar, 11Gisselsson D Bjork J Hoglund M Mertens F Dal Cin P Akerman M Mandahl N Abnormal nuclear shape in solid tumors reflects mitotic instability.Am J Pathol. 2001; 158: 199-206Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 12Gisselsson D Pettersson L Hoglund M Heidenblad M Gorunova L Wiegant J Mertens F Dal Cin P Mitelman F Mandahl N Chromosomal breakage-fusion-bridge events cause genetic intratumor heterogeneity.Proc Natl Acad Sci USA. 2000; 97: 5357-5362Crossref PubMed Scopus (313) Google Scholar The potential clinical consequences of the ALT phenotype remain primarily unknown as few studies to date have addressed this question. In glioblastoma multiforma, the presence of ALT was found to correlate with a more favorable outcome (survival).13Hakin-Smith V Jellinek DA Levy D Carroll T Teo M Timperley WR McKay MJ Reddel RR Royds JA Alternative lengthening of telomeres and survival in patients with glioblastoma multiforme.Lancet. 2003; 361: 836-838Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar However, Ulaner and colleagues9Ulaner GA Huang HY Otero J Zhao Z Ben-Porat L Satagopan JM Gorlick R Meyers P Healey JH Huvos AG Hoffman AR Ladanyi M Absence of a telomere maintenance mechanism as a favorable prognostic factor in patients with osteosarcoma.Cancer Res. 2003; 63: 1759-1763PubMed Google Scholar found no difference in survival for osteosarcoma patients when comparing telomerase-positive versus ALT cases. Interestingly, those osteosarcoma patients lacking both telomerase and ALT (no telomere maintenance mechanism evident) displayed an increased survival. A combined fluorescence in situ hybridization (FISH)/immunofluorescence method to assess telomere length in paraffin-embedded tissues has been established in our laboratory.14Meeker AK Gage WR Hicks JL Simon I Coffman JR Platz EA March GE De Marzo AM Telomere length assessment in human archival tissues: combined telomere fluorescence in situ hybridization and immunostaining.Am J Pathol. 2002; 160: 1259-1268Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar This in situ method allows for direct telomere length comparison between cells, because telomere length is proportional to fluorescent signal intensity in this assay. In addition, by providing identification of cell types via simultaneous immunohistochemistry, this method has the advantage of eliminating confounding contributions from contaminating normal cells.15Engelhardt M Albanell J Drullinsky P Han W Guillem J Scher HI Reuter V Moore MA Relative contribution of normal and neoplastic cells determines telomerase activity and telomere length in primary cancers of the prostate, colon, and sarcoma.Clin Cancer Res. 1997; 3: 1849-1857PubMed Google Scholar Because previous studies examining telomere lengths in human sarcomas have relied on pan-genomic Southern blot methodology using tumor homogenates, short-term in vitro cell cultures, or established cell lines, we have applied the in situ technique to directly assess and compare primary tumors of the two general types of sarcoma. Paraffin-embedded 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 retrieved from the archives of Johns Hopkins division of surgical pathology. The translocation-associated sarcomas had been previously shown to harbor their respective characteristic translocations, either via karyotyping or positive identification of the relevant fusion gene product.16Argani P Faria PA Epstein JI Reuter VE Perlman EJ Beckwith JB Ladanyi M Primary renal synovial sarcoma: molecular and morphologic delineation of an entity previously included among embryonal sarcomas of the kidney.Am J Surg Pathol. 2000; 24: 1087-1096Crossref PubMed Scopus (211) Google Scholar, 17Argani P Fritsch M Kadkol SS Schuster A Beckwith JB Perlman EJ Detection of the ETV6-NTRK3 chimeric RNA of infantile fibrosarcoma/cellular congenital mesoblastic nephroma in paraffin-embedded tissue: application to challenging pediatric renal stromal tumors.Mod Pathol. 2000; 13: 29-36Crossref PubMed Scopus (130) Google Scholar, 18Fritsch MK Bridge JA Schuster AE Perlman EJ Argani P Performance characteristics of a reverse transcriptase-polymerase chain reaction assay for the detection of tumor-specific fusion transcripts from archival tissue.Pediatr Dev Pathol. 2003; 6: 43-53Crossref PubMed Scopus (47) Google Scholar The other lesions were assumed to possess complex karyotypes, as these sarcoma types are known to display chromosome instability/complex karyotypes (Mitelman Database of Chromosome Aberrations in Cancer 2002, http://cgap.nci.nih/gov/chromosomes/mitelman).19Mertens F Fletcher CD Dal Cin P De Wever I Mandahl N Mitelman F Rosai J Rydholm A Sciot R Tallini G Van den Berghe H Vanni R Willen H Cytogenetic analysis of 46 pleomorphic soft tissue sarcomas and correlation with morphologic and clinical features: a report of the CHAMP Study Group. Chromosomes and Morphology.Genes Chromosom Cancer. 1998; 22: 16-25Crossref PubMed Scopus (159) Google Scholar, 20Mandahl N Fletcher CD Dal Cin P De Wever I Mertens F Mitelman F Rosai J Rydholm A Sciot R Tallini G Van Den Berghe H Vanni R Willen H Comparative cytogenetic study of spindle cell and pleomorphic leiomyosarcomas of soft tissues: a report from the CHAMP Study Group.Cancer Genet Cytogenet. 2000; 116: 66-73Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 21Fletcher CD Dal Cin P de Wever I Mandahl N Mertens F Mitelman F Rosai J Rydholm A Sciot R Tallini G van den Berghe H Vanni R Willen H Correlation between clinicopathological features and karyotype in spindle cell sarcomas. A report of 130 cases from the CHAMP study group.Am J Pathol. 1999; 154: 1841-1847Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar The method for in situ telomere staining and image analysis has been described in detail previously.14Meeker AK Gage WR Hicks JL Simon I Coffman JR Platz EA March GE De Marzo AM Telomere length assessment in human archival tissues: combined telomere fluorescence in situ hybridization and immunostaining.Am J Pathol. 2002; 160: 1259-1268Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar Briefly, deparaffinized slides were rehydrated, steam-treated in citrate buffer, and hybridized with a Cy3-labeled telomere-specific peptide nucleic acid (PNA) probe followed by DAPI (4′-6-diamidino-2-phenylindole) counterstaining and imaging, or were processed for indirect immunofluorescence before the DAPI staining using a primary antibody (anti-PML antibody, catalog no. PG-M3; DAKO Corp., Carpinteria, CA) incubated overnight at 4°C followed by application of fluorescent secondary antibody labeled with Alexa Fluor 488 (Molecular Probes, Eugene, OR). The PNA probe complementary to the mammalian telomere repeat sequence was obtained from Applied Biosystems (Framingham, MA) and has the sequence (N-terminus to C-terminus) CCCTAACCCTAACCCTAA with an N-terminal covalently linked Cy3 fluorescent dye. The analogous specificity control PNA, complementary to the Caenorhabditis elegans telomere repeat sequence that differs from the human repeat by a single nucleotide, was a generous gift from Dr. Carol Greider (Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD). Slides were imaged with a Zeiss Axioskop epifluorescence microscope equipped with short arc mercury lamp illumination (Carl Zeiss Inc., Thornwood, NY) and appropriate fluorescence filter sets (Omega Optical, Brattleboro, VT). Internal controls for telomere FISH staining in sarcomas consisted of endothelial cells, fibroblasts, and lymphocytes within the same tissue sample. Telomere lengths were evaluated by visual assessment of the fluorescent intensities of the telomeric signals, which are proportional to the length of telomeric TTAGGG DNA repeats.14Meeker AK Gage WR Hicks JL Simon I Coffman JR Platz EA March GE De Marzo AM Telomere length assessment in human archival tissues: combined telomere fluorescence in situ hybridization and immunostaining.Am J Pathol. 2002; 160: 1259-1268Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar Telomeres were directly compared to those of normal-appearing stromal cells within the same tissue section. Each case was scored by each of two authors (EM, AKM) using the following semiquantitative scoring system: lesions with normal telomere intensity had signals comparable to those of normal stromal fibroblasts and endothelial cells. Lesions with (moderately) short telomeres had telomere intensities appreciably dimmer than the normal stroma, but which were still readily detectable. Lesions were considered very short when telomeres demonstrated signals so dim that they were barely perceptible, dimmer than that of virtually any normal stromal cell we encountered in this study. Lesions with long telomeres demonstrated telomere signals that were appreciably brighter than that of stromal cells, and close to that of lymphocytes, which gave consistently bright signals. Lesions in which the signal varied between cells from bright to short, or very short to normal (ie, lesions with telomere signals spanning two scoring categories) were classified as heterogeneous for telomere length. Information on the presence or absence of anaphase bridges was obtained from previously published work on these sarcoma cases,7Montgomery E Wilentz RE Argani P Fisher C Hruban RH Kern SE Lengauer C Analysis of anaphase figures in routine histologic sections distinguishes chromosomally unstable from chromosomally stable malignancies.Cancer Biol Ther. 2003; 2: 248-252Crossref PubMed Scopus (54) Google Scholar as well as additional evaluation of mitotic figures in this study using both DAPI-stained nuclei and hematoxylin and eosin-stained adjacent sections. Criteria for anaphase bridge presence required a well-separated parallel anaphase plate displaying a perpendicularly aligned connecting chromatin filament. In all (nine of nine) cases with specific translocations, telomere length was similar to or reduced compared to that of surrounding nonneoplastic tissues (Figure 1, A and B). In contrast, malignant cells in seven of nine (78%) cases lacking specific translocations (four malignant fibrous histiocytomas, two leiomyosarcomas, and one pleomorphic rhabdomyosarcoma) displayed dramatically elongated telomeres as well as telomere length heterogeneity, compared to both surrounding normal cells and to tumors with known translocations (Figure 1; C to F), and this difference was statistically significant (P = 0.009, Fisher's test). However, in these sarcomas with long telomeres, telomere lengthening was highly variable from cell to cell in many of the cases (Table 1). In addition to markedly elongated telomeres, six of the nine complex cases exhibited brightly-stained intranuclear spots suggestive of APBs.6Yeager TR Neumann AA Englezou A Huschtscha LI Noble JR Reddel RR Telomerase-negative immortalized human cells contain a novel type of promyelocytic leukemia (PML) body.Cancer Res. 1999; 59: 4175-4179PubMed Google Scholar We performed immunohistochemical testing for co-localized PML protein, a component of APBs. Staining with specific antibodies for PML protein confirmed that these brightly staining foci were indeed APBs in all six cases (six of six, 100%) (Figure 1G). The proportion of cells displaying APB as well as the number and sizes of APB within a given cell were highly variable from case to case (Table 1), ranging from rare detectable APBs to 40% of cells staining positive. All six cases with APBs exhibited long telomeres. To confirm the specificity of the telomeric fluorescent signals we repeated the staining on directly adjacent tissue sections using a fluorescently labeled PNA probe targeted against the C. elegans telomere repeat sequence that differs from the human sequence by a single nucleotide (TTAGGC versus TTAGGG). Although C. elegans tissue controls exhibited positive staining with the C. elegans-specific probe, all human sarcomas were completely nonreactive indicating that the bright signals observed with the human-specific probe in pleomorphic sarcomas, as well as APBs, were indeed reflective of human telomeric DNA (data not shown).Table 1Summary of Results of Telomere AssessmentCaseSarcoma typeGradeTranslocation-associated?Gene fusion product identified?Status, intervalTelomere lengthsALT-associated PML bodies (% cells positive)Anaphase bridges found?1Malignant fibrous histiocytoma3NoNAANED, 1 yearShort-long, heterogeneous30%Yes2Malignant fibrous histiocytoma3NoNAANED, 1 yearShort-long, heterogeneous40%Yes3Leiomyosarcoma3NoNAAlive with lung metastases,3 yearsShort to long, heterogeneous10%Yes3aLeiomyosarcoma-Separate area from same tumor as case 33NoNAAlive with lung metastases,3 yearsLong to very long, moderate heterogeneity25%Yes4Leiomyosarcoma3NoNAAlive with lung metastases,2 yearsLong≤1%Not done5Malignant fibrous histiocytoma3NoNAAlive with lung metastases,5 yearsLong, Moderate heterogeneity≤1%Yes6Malignant fibrous histiocytoma2NoNAANED, 2 yearsLong≤1%Not done7Malignant peripheral nerve sheath tumor2NoNADead of metastatic disease, 3 yearsNormal long, heterogeneousNoYes8Pleomorphic rhabdomyosarcoma3NoNADead of metastatic disease, 2 yearsVery short to shortNoYes9Dedifferentiated chondrosarcoma3NoNAAlive with metastatic disease, 1 yearShort-normalNoNot done10Alveolar rhabdomyosarcoma3YesYes, PAX3/FKHRANED, 7 years after3 recurrencesShort-normalNoNo11Alveolar rhabdomyosarcoma3YesYes, PAX3/FKHRDead of metastaticShort-normalNoNodisease, 3 years12Desmoplastic small rounds cell tumor3YesYes, EWS/WT1NAShort-normalNoNo13Desmoplastic small round cell tumor3YesYes, EWS/WT1NAVery short-normalNoNo14Infantile fibrosarcoma1YesYes, ETV6/NTRK 3NANormalNoNo15Myxoid liposarcoma1YesYes, t(12; 16)Dead of metastatic disease, 4 yearsNormalNoNo16Cellular congenital mesoblastic nephroma1YesYes, ETV6/NTRK 3ANED, 5 yearsShort-normalNoNo17Synovial sarcoma2YesYes, SYT/SSXNAShort-normalNoNo18Synovial sarcoma3YesYes, SYT/SSXDead of metastatic disease, 3 yearsShort-normalNoNoAbbreviations: ANED, alive, no evidence of disease; NA, not available. Open table in a new tab Abbreviations: ANED, alive, no evidence of disease; NA, not available. The presence of telomere lengthening and PML bodies also correlated with the presence of anaphase bridges in pleomorphic sarcomas (Figure 2) (Table 1). However, two of the pleomorphic sarcomas lacked sufficient anaphases for evaluation (further details of the anaphase bridge results are published elsewhere7Montgomery E Wilentz RE Argani P Fisher C Hruban RH Kern SE Lengauer C Analysis of anaphase figures in routine histologic sections distinguishes chromosomally unstable from chromosomally stable malignancies.Cancer Biol Ther. 2003; 2: 248-252Crossref PubMed Scopus (54) Google Scholar). Sarcomas can be divided into two general categories, namely those with specific genetic alterations and those with nonspecific ones.22Borden EC Baker LH Bell RS Bramwell V Demetri GD Eisenberg BL Fletcher CD Fletcher JA Ladanyi M Meltzer P O'Sullivan B Parkinson DR Pisters PW Saxman S Singer S Sundaram M Van Oosterom AT Verweij J Waalen J Weiss SW Brennan MF Soft tissue sarcomas of adults: state of the translational science.Clin Cancer Res. 2003; 9: 1941-1956PubMed Google Scholar Those with specific alterations tend to have simple karyotypes with a specific reciprocal translocation, to occur in younger patients, to lack p53 alterations, and be rare in the Li-Fraumeni, bilateral retinoblastoma syndromes, or the setting after radiation. On routine microscopy, they generally display a homogeneous population of rounded or spindle cells. In contrast, those lacking specific alterations generally have complex karyotypes, no reproducible pattern of translocations, occur in older individuals, have p53 alterations, and are common in patients with Li-Fraumeni and bilateral retinoblastoma syndromes or after radiation. On routine microscopy, their appearances are highly variable but they often display pleomorphic spindle cells. Pathological telomere lengthening may potentially arise in sarcomas, either because of dysregulated telomerase activity, or by an alternative pathway known as ALT.3Henson JD Neumann AA Yeager TR Reddel RR Alternative lengthening of telomeres in mammalian cells.Oncogene. 2002; 21: 598-610Crossref PubMed Google Scholar, 4Reddel RR Bryan TM Colgin LM Perrem KT Yeager TR Alternative lengthening of telomeres in human cells.Radiat Res. 2001; 155: 194-200Crossref PubMed Scopus (92) Google Scholar When the ALT pathway is functional, nuclei of a subset of affected cells display a variant form of PML nuclear bodies, so named based on their disruption in neoplastic promyelocytic leukemia cells. These variant PML bodies, referred to as APBs,6Yeager TR Neumann AA Englezou A Huschtscha LI Noble JR Reddel RR Telomerase-negative immortalized human cells contain a novel type of promyelocytic leukemia (PML) body.Cancer Res. 1999; 59: 4175-4179PubMed Google Scholar are relatively large structures containing telomeric DNA, telomeric binding proteins (TRF1, TRF2), DNA replication protein (replication factor A), DNA recombination proteins (RAD51, RAD52), and PML protein. The telomere phenotype of sarcomas with complex karyotypes remains unclear but the ALT mechanism seems to be at work in at least some examples.10Scheel C Schaefer KL Jauch A Keller M Wai D Brinkschmidt C van Valen F Boecker W Dockhorn-Dworniczak B Poremba C Alternative lengthening of telomeres is associated with chromosomal instability in osteosarcomas.Oncogene. 2001; 20: 3835-3844Crossref PubMed Scopus (142) Google Scholar In this study, the first in situ telomere length assessment of these tumor types, we specifically explored the relationship between telomere length, the ALT phenotype, anaphase bridge formation, and sarcoma type. Although several methods for telomere length assessment have been developed, the high-resolution method used in this study14Meeker AK Gage WR Hicks JL Simon I Coffman JR Platz EA March GE De Marzo AM Telomere length assessment in human archival tissues: combined telomere fluorescence in situ hybridization and immunostaining.Am J Pathol. 2002; 160: 1259-1268Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar is applicable to standard formalin-fixed human tissue, and because the method is in situ, lengths in sarcomas can be directly compared to internal controls. Using TELI-FISH, we find that dramatic telomere lengthening corresponds with pleomorphic sarcomas that typically display complex karyotypes. In contrast, relatively normal, and/or shorter than normal, telomere lengths were observed in neoplasms with characteristic translocations. Furthermore, the proportion of cells displaying lengthened telomeres and ALT was highly variable in the complex cases. For example, in one portion of tumor 3, a leiomyosarcoma, telomeres were within the short-normal range, and APBs were common, whereas a separate focus of this tumor displayed widespread telomere elongation and fewer APBs. Regional variation in telomere length has been noted previously for liposarcomas by Schneider-Stock and colleagues.23Schneider-Stock R Boltze C Jager V Epplen J Landt O Peters B Rys J Roessner A Elevated telomerase activity, c-MYC-, and hTERT mRNA expression: association with tumour progression in malignant lipomatous tumours.J Pathol. 2003; 199: 517-525Crossref PubMed Scopus (36) Google Scholar Sarcomas with lengthened telomeres and APBs also displayed anaphase bridges. The details of this assessment are reported elsewhere,7Montgomery E Wilentz RE Argani P Fisher C Hruban RH Kern SE Lengauer C Analysis of anaphase figures in routine histologic sections distinguishes chromosomally unstable from chromosomally stable malignancies.Cancer Biol Ther. 2003; 2: 248-252Crossref PubMed Scopus (54) Google Scholar but anaphase bridges are known to occur when dicentric chromosomes are produced by chromosomal fusions after DNA double-strand breakage and are considered to be indicators of chromosomal instability. By routine light microscopy of hematoxylin and eosin-stained slides, they are seen as a perpendicularly aligned amphophilc filament connecting a well-separated parallel anaphase plate.7Montgomery E Wilentz RE Argani P Fisher C Hruban RH Kern SE Lengauer C Analysis of anaphase figures in routine histologic sections distinguishes chromosomally unstable from chromosomally stable malignancies.Cancer Biol Ther. 2003; 2: 248-252Crossref PubMed Scopus (54) Google Scholar Previously it has been shown that telomere lengths within ALT-positive cells are typically quite heterogeneous, ranging from abnormally long to very short. Because critically short telomeres become dysfunctional and lead to chromosome fusions, this seems a likely source of the anaphase bridges observed in these ALT-positive cases.24Gisselsson D Jonson T Petersen A Strombeck B Dal Cin P Hoglund M Mitelman F Mertens F Mandahl N Telomere dysfunction triggers extensive DNA fragmentation and evolution of complex chromosome abnormalities in human malignant tumors.Proc Natl Acad Sci USA. 2001; 98: 12683-12688Crossref PubMed Scopus (351) Google Scholar In the current study we find a correlation between complex karyotypes, abnormally long telomeres, APBs, and anaphase bridges. Of six complex cases in which both APB and anaphase bridges were evaluated, all showed the simultaneous presence of both features. APBs are considered a hallmark of the ALT telomere maintenance phenotype that is typically characterized by extreme telomere length deregulation resulting in widespread length heterogeneity. In sharp contrast, none of the 10 cases with simple karyotypes exhibited APB, anaphase bridges, abnormally elongated telomeres, or significant telomere length heterogeneity as assessed by in situ FISH analysis. Little is currently known regarding the potential prognostic significance of telomere maintenance status (eg, presence versus absence, type of maintenance mechanism) in sarcomas.9Ulaner GA Huang HY Otero J Zhao Z Ben-Porat L Satagopan JM Gorlick R Meyers P Healey JH Huvos AG Hoffman AR Ladanyi M Absence of a telomere maintenance mechanism as a favorable prognostic factor in patients with osteosarcoma.Cancer Res. 2003; 63: 1759-1763PubMed Google Scholar, 13Hakin-Smith V Jellinek DA Levy D Carroll T Teo M Timperley WR McKay MJ Reddel RR Royds JA Alternative lengthening of telomeres and survival in patients with glioblastoma multiforme.Lancet. 2003; 361: 836-838Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar In the current study we observed a tendency for more favorable outcomes in those sarcoma cases with evidence of the ALT mechanism among this set of predominantly high-grade sarcoma types. However, one caveat with respect to these findings is that the absolute number of cases examined in this study was small. More extensive research is clearly warranted. These findings are in contrast to those of epithelial neoplasms in which telomere shortening often accompanies neoplasia25deLange T Telomeres. 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The presence of ALT in pleomorphic sarcomas, plus the observed presence of elongated telomeres, may make them recalcitrant to emergent therapies directed against telomerase. In situ telomere length analysis allows for the rapid assessment of telomere lengths, as well as the presence of ALT, in human primary tumor samples and thus should facilitate further examination of the potential prognostic value of these characteristics in human sarcomas. We thank an anonymous reviewer for bringing to our attention the fact that the telomere length difference between the sarcoma groups (either with or without specific translocations) is statistically significant.