Telomeric associations correlate with telomere length reduction and clonal chromosome aberrations in giant cell tumor of bone.

Giant cell tumor of bone (GCTB) is characterized cytogenetically by frequent telomeric associations (tas). To explore the mechanisms behind the formation of tas in GCTB and to investigate their karyotypic consequences, the frequencies of tas and clonal aberrations other than tas in 20 GCTBs were compared to telomere length and status, as assessed by quantitative PCR, fluorescence in situ hybridization (FISH), and expression levels of four genes involved in telomere maintenance. Based on the G-banding results, the tumors were divided into two groups, one with a high frequency of tas and one with a low frequency. Clonal aberrations were found to be restricted to the group with a high level of tas, and the same group showed a significantly larger reduction in telomere length in tumor cells compared to peripheral blood cells. Furthermore, 65 out of 66 tas analyzed by FISH were negative for telomeric sequences. The expression levels of TERT, TERF1, TERF2, and POT1 did not correlate with telomere length or the frequency of tas. Thus, the present findings provide strong support for the notion that decreased telomere length is a prerequisite for tas in GCTBs and that the clonal changes occurring in GCTBs are derived from tas.

POU5F1, encoding a key regulator of stem cell pluripotency, is fused to EWSR1 in hidradenoma of the skin and mucoepidermoid carcinoma of the salivary glands.

The EWSR1 gene is known to play a crucial role in the development of a number of different bone and soft tissue tumours, notably Ewing's sarcoma. POU5F1 is expressed during early development to maintain the totipotent status of embryonic stem and germ cells. In the present study, we report the fusion of EWSR1 and POU5F1 in two types of epithelial tumours: hidradenoma of the skin and mucoepidermoid carcinoma of the salivary glands. This finding not only broadens considerably the spectrum of neoplasms associated with EWSR1 fusion genes but also strengthens the evidence for shared pathogenetic mechanisms in the development of adnexal and salivary gland tumours. Reminiscent of the previously reported fusion genes involving EWSR1, the identified transcript is predicted to encode a chimeric protein consisting of the EWSR1 amino-terminal domain and the POU5F1 carboxy-terminal domain. We assessed the transcriptional activation potential of the chimera compared to the wild-type proteins, as well as activation of transcription through the oct/sox composite element known to bind POU5F1. Among other POU5F1 target genes, this element is present in the promoter of NANOG and in the distal enhancer of POU5F1 itself. Our results show that although the chimera is capable of significant transcriptional activation, it may in fact convey a negative regulatory effect on target genes.

Frequent deletion of the CDKN2A locus in chordoma: analysis of chromosomal imbalances using array comparative genomic hybridisation.

The initiating somatic genetic events in chordoma development have not yet been identified. Most cytogenetically investigated chordomas have displayed near-diploid or moderately hypodiploid karyotypes, with several numerical and structural rearrangements. However, no consistent structural chromosome aberration has been reported. This is the first array-based study characterising DNA copy number changes in chordoma. Array comparative genomic hybridisation (aCGH) identified copy number alterations in all samples and imbalances affecting 5 or more out of the 21 investigated tumours were seen on all chromosomes. In general, deletions were more common than gains and no high-level amplification was found, supporting previous findings of primarily losses of large chromosomal regions as an important mechanism in chordoma development. Although small imbalances were commonly found, the vast majority of these were detected in single cases; no small deletion affecting all tumours could be discerned. However, the CDKN2A and CDKN2B loci in 9p21 were homo- or heterozygously lost in 70% of the tumours, a finding corroborated by fluorescence in situ hybridisation, suggesting that inactivation of these genes constitute an important step in chordoma development.

Genetic intratumour heterogeneity in high-grade brain tumours is associated with telomere-dependent mitotic instability.

Glioblastoma multiforme (GBM) and other high-grade brain tumours are typically characterized by complex chromosome abnormalities and extensive intratumour cytogenetic heterogeneity. The mechanisms behind this diversity have been little explored. In this study, we analysed the pattern of chromosome segregation at mitosis in 20 brain tumours. We found an abnormal segregation of chromatids at mitosis through anaphase bridging (10-25% of anaphase cells) in all 10 GBMs. Anaphase bridging was also found in two medulloblastomas (7-15%), one anaplastic astrocytoma (17%) and one oligodendroglioma (6%). These tumours showed a relatively high degree of cytogenetic complexity and heterogeneity. In contrast, cell division abnormalities were not found in low-grade brain tumours with less complex karyotypes, including two pilocytic astrocytomas and two ependymomas. Further analysis of two GBMs by fluorescence in situ hybridization with telomeric repeat probes revealed excessive shortening of TTAGGG repeats, indicating dysfunctional protection of chromosome ends. In xenografts established from these GBMs, there was a gradual reduction in cytogenetic heterogeneity through successive passages as the proportion of abnormally short telomeres was reduced and the frequency of anaphase bridges decreased from >25% to 0. However, bridging could be reintroduced in late-passage xenograft cells by pharmacological induction of telomere shortening, using a small-molecule telomerase inhibitor. Telomere-dependent abnormal segregation of chromosomes at mitosis is thus a common phenomenon in high-grade brain tumours and may be one important factor behind cytogenetic intratumour diversity in GBM.

Generation of human cortical neurons from a new immortal fetal neural stem cell line.

Isolation and expansion of neural stem cells (NSCs) of human origin are crucial for successful development of cell therapy approaches in neurodegenerative diseases. Different epigenetic and genetic immortalization strategies have been established for long-term maintenance and expansion of these cells in vitro. Here we report the generation of a new, clonal NSC (hc-NSC) line, derived from human fetal cortical tissue, based on v-myc immortalization. Using immunocytochemistry, we show that these cells retain the characteristics of NSCs after more than 50 passages. Under proliferation conditions, when supplemented with epidermal and basic fibroblast growth factors, the hc-NSCs expressed neural stem/progenitor cell markers like nestin, vimentin and Sox2. When growth factors were withdrawn, proliferation and expression of v-myc and telomerase were dramatically reduced, and the hc-NSCs differentiated into glia and neurons (mostly glutamatergic and GABAergic, as well as tyrosine hydroxylase-positive, presumably dopaminergic neurons). RT-PCR analysis showed that the hc-NSCs retained expression of Pax6, Emx2 and Neurogenin2, which are genes associated with regionalization and cell commitment in cortical precursors during brain development. Our data indicate that this hc-NSC line could be useful for exploring the potential of human NSCs to replace dead or damaged cortical cells in animal models of acute and chronic neurodegenerative diseases. Taking advantage of its clonality and homogeneity, this cell line will also be a valuable experimental tool to study the regulatory role of intrinsic and extrinsic factors in human NSC biology.

Gene copy number changes in dermatofibrosarcoma protuberans - a fine-resolution study using array comparative genomic hybridization.

Dermatofibrosarcoma protuberans (DFSP) is a rare, slow-growing, low-grade dermal tumor. Cytogenetic and FISH studies have revealed that the chromosomal rearrangements characteristic of DFSP tumors involve both translocations and the formation of a supernumerary ring derived from chromosomes 17 and 22. The t(17;22) (q22;q13.1) translocation generates a gene fusion between COL1A1 and PDGFB, which serves as a diagnostic marker of DFSP. In the present study we performed array-CGH (aCGH) analysis on ten DFSP tumors. The COL1A1 region at 17q was gained in 71% (5/7) of the samples and the PDGFB region at 22q was gained in 43% (3/7) of the individual samples. In addition to the 17q and 22q gains, altogether 17 minimal common regions of gain and one region of loss were detected.

FISH mapping of i(7q) in acute leukemias and myxoid liposarcoma reveals clustered breakpoints in 7p11.2: implications for formation and pathogenetic outcome of the idic(7)(p11.2).

Isochromosome 7q - i(7q) - is seen in a wide variety of hematologic malignancies and solid tumors, often as a secondary change to a characteristic primary translocation. Despite its high frequency, nothing is known about the formation and the pathogenetic outcome of this abnormality. To address these issues, we performed a detailed fluorescence in situ hybridization (FISH) investigation of four acute lymphoblastic leukemias, one acute myeloid leukemia, and two myxoid liposarcomas with i(7q). Using FISH with bacterial artificial chromosomes (BACs) mapping between 7p12.2 and 7q11.2, the breakpoints (BPs) in all seven cases were shown to cluster to an approximately 340 kb segment at 7p11.2, covered by the overlapping BAC probes RP11-760D2 and RP11-10F11. Thus, the i(7q) should formally be designated idic(7) (p11.2). In one of the cases, FISH with fosmids could narrow down the BP further to an 80-kb sequence delineated by G248P81983A10 and G248P8793H7. No known genes are located in the 340-kb BP cluster region, indicating that the idic(7)(p11.2) does not result in a fusion or deregulation of genes in this segment. The pathogenetically important outcome is thus likely to be an altered gene expression because of copy number changes. The clustering of breakpoints might be due to frequent intrachromosomal duplicons in the BP region.

Identification of a novel amplicon at distal 17q containing the BIRC5/SURVIVIN gene in malignant peripheral nerve sheath tumours.

Previous studies have suggested that amplification of genes, notably the TOP2A gene, on chromosome arm 17q may be important for the development of malignant peripheral nerve sheath tumour (MPNST). In order to study the frequency, distribution, and chromosomal organization of rearrangements at 17q, interphase and metaphase fluorescence in situ hybridization (FISH) were used to evaluate copy number changes at 17q in 28 MPNSTs. Increased copy numbers were seen for the ERBB2 and TOP2A genes in eight and nine cases, respectively, supporting a potential role for these two genes in MPNST tumourigenesis. Net gain of distal 17q material was observed in 16 of the 28 MPNSTs, with high-level gain in three cases, and was associated with poor outcome. Among the 26 patients for whom follow-up data were available, gain of distal 17q was present in 11 of 12 tumours that had metastasized, compared with 4 of 14 of those that had not metastasized. Detailed FISH mapping analysis of metaphase spreads identified a 2 Mb commonly gained/amplified region at 17q25. Among the genes mapping to this region, BIRC5, which encodes the baculoviral IAP repeat-containing protein 5/survivin protein, is a strong candidate target gene for amplification, as it has been previously shown to be overexpressed in neurofibromatosis type 1-associated MPNST. Three other genes that co-amplified with BIRC5 represent other potential candidate genes: PTDSR involved in apoptosis; SEPT9 overexpressed in human malignant brain tumours; and SOCS3 involved in cell survival and differentiation of neurons.

Genomic profiling of bone and soft tissue tumors with supernumerary ring chromosomes using tiling resolution bacterial artificial chromosome microarrays.

Ring chromosomes and/or giant marker chromosomes have been observed in a variety of human tumor types, but they are particularly common in a subgroup of mesenchymal tumors of low-grade or borderline malignancy. These rings and markers have been shown to contain amplified material predominantly from 12q13-15, but also sequences from other chromosomes. Such amplified sequences were mapped in detail by genome-wide array comparative genomic hybridization in ring-containing tumor samples from soft tissue (n = 15) and bone (n = 6), using tiling resolution microarrays, encompassing 32 433 bacterial artificial chromosome clones. The DNA copy number profiles revealed multiple amplification targets, in many cases highly discontinuous, leading to delineation of large numbers of very small amplicons. A total number of 356 (median size: 0.64 Mb) amplicons were seen in the soft tissue tumors and 90 (median size: 1.19 Mb) in the bone tumors. Notably, more than 40% of all amplicons in both soft tissue and bone tumors were mapped to chromosome 12, and at least one of the previously reported recurrent amplifications in 12q13.3-14.1 and 12q15.1, including SAS and CDK4, and MDM2, respectively, were present in 85% of the soft tissue tumors and in all of the bone tumors. Although chromosome 12 was the only chromosome displaying recurrent amplification in the bone tumors, the soft tissue tumors frequently showed recurrent amplicons mapping to other chromosomes, that is, 1p32, 1q23-24, 3p11-12, 6q24-25 and 20q11-12. Of particular interest, amplicons containing genes involved in the c-jun NH2-terminal kinase/mitogen-activated protein kinase pathway, that is, JUN in 1p32 and MAP3K7IP2 (TAB2) in 6q24-25, were found to be independently amplified in eight of 11 cases with 12q amplification, providing strong support for the notion that aberrant expression of this pathway is an important step in the dedifferentiation of liposarcomas.

Truncation and fusion of HMGA2 in lipomas with rearrangements of 5q32-->q33 and 12q14-->q15.

Chromosome segment 12q13-->q15 recombines with many different chromosome bands in lipomas and at least ten recurrent translocations have been identified. The HMGA2 gene is often rearranged, but little is known about the molecular consequences at other breakpoints. Fusion genes between HMGA2 (12q14-->q15) and LPP (3q27-->q28), LHFP (13q12) and CMKOR1 (2q37) have been reported. In the present study, eight lipomas with rearrangements involving chromosome bands 12q14-->q15 and 5q32-->q33 were analyzed. In chromosome 5, five of the cases had a breakpoint in the 5' part of EBF in 5q33, while three cases had breakpoints located about 200 kb 3' of EBF. In chromosome 12, the breakpoints clustered to the region of HMGA2. Four cases had breaks within the gene and four had breaks 5' to HMGA2 where the gene BC058822 is located. Two versions of an HMGA2/EBF fusion transcript were detected in one case; one transcript was in frame and the other out of frame. Identical EBF/BC058822 fusion transcripts, seen in two cases, one of which also had the HMGA2/EBF transcript, were out of frame and resulted in truncation of EBF. Since EBF and HMGA2 have different orientations, the findings must be explained by complex aberrations including multiple breaks. The combined data indicate that the pathogenetically significant event is fusion, truncation or transcriptional activation of HMGA2, but it can not be excluded that EBF, which has been implicated in adipogenesis, contributes to the tumor development.

Fusion of the HMGA2 and NFIB genes in lipoma.

The major cytogenetic subgroup of lipomas is characterized by aberrations of chromosome segment 12q13-15, which recombines with a large number of other chromosomal regions. The gene HMGA2 is the main target in these aberrations. For some recurrent rearrangements, chimeric transcripts, including the 5' part of HMGA2, have been described. The 3' partners identified are LPP, LHFP, CMKOR1, and EBF. In addition, subsets of other benign solid tumors show aberrations of 12q13-15. Among pleomorphic adenomas of the salivary glands, where the preferred recombination partner with 12q13-15 is 9p22-24, an HMGA2/NFIB fusion gene has been reported. In the present study, two cases of lipoma with rearrangements of 9p22-24 and 12q15 were analyzed by reverse transcription polymerase chain reaction to find out if HMGA2/NFIB was also present in lipoma. An in-frame fusion transcript, combining the four first exons of HMGA2 with exon 8 of NFIB, was detected in one case. It was identical to a transcript that was previously described in salivary gland adenoma and contained a stop codon shortly 3' of the fusion point. The finding of the same fusion gene in different tumors is not unique. For example, HMGA2/LPP has been reported in lipoma, pulmonary chondroid hamartoma, and soft tissue chondroma. Since similar 9;12 translocations have been described also in rare cases of hamartoma and uterine leiomyoma, the occurrence of HMGA2/NFIB could be postulated in these tumors as well.

Retained heterodisomy for chromosome 12 in atypical lipomatous tumors: implications for ring chromosome formation.

Atypical lipomatous tumor (ALT) is an intermediate malignant mesenchymal tumor that is characterized by supernumerary ring chromosomes and/or giant rod-shaped marker chromosomes (RGMC). Fluorescence in situ hybridization (FISH) and molecular genetic analyses have disclosed that the RGMCs always contain amplified sequences from the long arm of chromosome 12. Typically, RGMCs are the sole clonal changes and so far no deletions or other morphologic aberrations of the two normal-appearing chromosomes 12 that invariably are present have been detected. The mechanisms behind the formation of the RGMCs are unknown, but it could be hypothesized that RGMC formation is preceded by trisomy 12 or, alternatively, that ring formation of one chromosome 12 is followed by duplication of the remaining homolog. The latter scenario would always result in isodisomy for the two normal-appearing chromosomes 12, whereas the former would yield isodisomy in one-third of the cases. In order to investigate these possible mechanisms behind ring formation, we studied polymorphic loci on chromosome 12 in 14 cases of ALT showing one or more supernumerary ring chromosomes and few or no other clonal aberrations at cytogenetic analysis. The molecular genetic analyses showed that the tumor cells always retained both parental copies of chromosome 12, thus refuting the trisomy 12 and duplication hypotheses.

Telomere shortening and mitotic dysfunction generate cytogenetic heterogeneity in a subgroup of renal cell carcinomas.

Most renal cell carcinomas (RCC) show only simple chromosomal changes. However, a more complex cytogenetic pattern has been found in a subgroup of aggressive RCC, indicating that further accumulation of chromosome changes could play a role in tumour progression. To explore the possible mechanisms behind cytogenetic evolution in RCC, a parallel assessment of chromosome mutations and mitotic segregation pattern in eight tumours was performed. In the majority of cases, no abnormalities in the cell division machinery were found and the rate of alterations in chromosome copy number, as measured by interphase FISH, was similar to that in non-neoplastic cells. This was reflected by relatively simple karyotypes, with little cytogenetic intratumour heterogeneity. In contrast, another group of tumours exhibited several cytogenetically related clones with additional structural chromosomal changes at two or more ploidy levels and a frequency of copy number alterations that was higher than in normal cells. In these cases, the telomere repeat sequences were abnormally short and chromosomal breakage-fusion-bridge events were observed at cell division, as well as multipolar configurations and supernumerary centrosomes. Abnormalities of the cell division machinery may thus contribute to the evolution of complex karyotypes and genetic intratumour heterogeneity in a subgroup of RCC.

Molecular cytogenetic mapping of recurrent chromosomal breakpoints in tenosynovial giant cell tumors.

Tenosynovial giant cell tumor (TGCT) is the most common benign tumor of synovium and tendon sheath. Cytogenetic data indicate that 1p11-13 is the region most frequently involved in structural rearrangements. With the aim of eventually identifying the genes associated with TGCT development, we have investigated 1p11-13 breakpoints using fluorescence in situ hybridization (FISH) analysis, with a panel of yeast artificial chromosome (YAC) probes covering 1p11-21. Twenty-six tumors were analyzed by G-banding, and 24 of these showed a breakpoint in 1p11-13. The cytogenetic findings add to previous observations that, among a variety of translocations involving 1p11-13, chromosome 2 is the most common translocation partner, with a breakpoint in 2q35-37. This aberration was found in eight cases. Other recurrent translocation partners, found in two or three cases, were 5q22-31, 11q11-12, and 8q21-22. Material from 21 tumors was available for FISH analysis, which revealed that the breakpoints clustered to one region spanned by two YAC probes, 914F6 and 885F12 located in 1p13.2, in 18 cases. Bacterial artificial chromosome probes were used to map the recurrent breakpoint on chromosome 2. In four of seven cases there was a breakpoint within the sequence covered by probe 260J21, where the RDC1 gene is located, a gene reported to fuse with HMGIC in lipomas with a 2;12 translocation.

Centrosomal abnormalities, multipolar mitoses, and chromosomal instability in head and neck tumours with dysfunctional telomeres.

Carcinomas of the head and neck typically exhibit complex chromosome aberrations but the underlying mutational mechanisms remain obscure. Evaluation of cell division dynamics in low-passage cell lines from three benign and five malignant head and neck tumours revealed a strong positive correlation between multipolarity of the mitotic spindle and the formation of bridges at anaphase in both benign and malignant tumours. Cells exhibiting a high rate of mitotic abnormalities also showed several chromosome termini lacking TTAGGG repeats and a high frequency of dicentric chromosomes. Multicolour karyotyping demonstrated a preferential involvement in structural rearrangements of chromosomes with deficient telomeres. The majority of malignant, mitotically unstable tumours expressed the reverse transcriptase subunit of telomerase. These data indicate that some of the genomic instability in head and neck tumours is initiated by telomere dysfunction, leading to the formation of dicentric chromosomes. These form chromosome bridges at mitosis that could prevent the normal anaphase-telophase transition. In turn, this may cause an accumulation of centrosomes and mitotic multipolarity. Telomerase expression does not confer total stability to the tumour genome but could be crucial for moderating the rate of chromosomal evolution.

Analysis of the distribution and frequency of trisomy 7 in vivo in synovia from patients with osteoarthritis and pigmented villonodular synovitis.

Osteoarthritis (OA) and pigmented villonodular synovitis (PVNS) are disorders associated with trisomy 7. The aim of the present study was to determine the frequency and distribution of the cells with +7 in vivo by analyzing sections of paraffin-embedded synovia from patients affected by OA, PVNS, other forms of synovitis [hemorragic synovitis (HS) and chronic synovitis (CS)], and from individuals without joint disease. Fluorescence in situ hybridization (FISH), using a centromeric probe for chromosome 7, showed that the mean frequency of trisomic nuclei in 5-microm sections was highest in PVNS (9.0%), followed by CS (5.9%), OA (5.6%), and HS (4.6%), whereas trisomic nuclei were rare (0.7%) in normal tissue. When 8-microm sections were studied, the frequencies of trisomic cells in OA and control synovia increased to 6.7% and 1.5%, respectively. Trisomic nuclei were found in all cases, including those for which cytogenetic analysis of short-term cultures had not disclosed any trisomic cells. Overall, the trisomic cells were scattered within the tissue. However, small clusters of cells with +7 were found in three cases. By hematoxylin-eosin staining of the slides used for FISH analysis it could be shown that the clustered trisomic cells were proliferating synoviocytes within villous extensions of the synovial membrane.

Comparative genomic hybridization of postirradiation sarcomas.

BACKGROUND: Radiotherapy is a known risk factor for sarcoma development. Postirradiation sarcomas arise within the radiation field after a latency period of several years and usually are highly malignant. Very little is yet known about their genetic changes. METHODS: Twenty-seven postirradiation sarcomas were analyzed by comparative genomic hybridization, which allows genome-wide screening of DNA sequence copy number changes. RESULTS: Copy-number aberrations were detected in 20 (74%) tumors. The mean number of aberrations per tumor was 5.3 with gains outnumbering losses. The most frequent gains affected the minimal common regions of 7q11.2-q21 and 7q22 in 30% and 7p15-pter in 26%. Gain of 8q23-qter was detected in 22%. The most frequent losses affected 11q23-qter and 13q22-q32 in 22%. In osteosarcomas, the most frequent aberration was loss of 1p21-p31, in malignant fibrous histiocytomas (MFH) gain of 7cen-q22, and in fibrosarcomas gain of 7q22. The findings in postirradiation osteosarcomas and MFHs were compared with findings in sporadic osteosarcomas and MFHs, reported previously by the authors. In sporadic osteosarcomas, gains outnumbered losses, but, in postirradiation osteosarcomas, losses were more frequent than gains. Loss at 1p was rare in sporadic osteosarcoma (3%) but frequent (57%) in postirradiation osteosarcomas. Gains at 7q were frequent both in postirradiation and sporadic MFH. CONCLUSIONS: According to previous studies on different types of sporadic sarcomas, gains at 7q or 8q are associated with poor prognosis or large tumor size. Thus, the frequent gains at 7q and 8q might have been responsible in part for the poor prognosis of postirradiation sarcomas. Also, however, some of their clinical features, i.e., high malignancy grade, late diagnosis, and central location, are associated with a poor prognosis.

Identification of cytogenetic subgroups and karyotypic pathways in transitional cell carcinoma.

The clinical course in urinary bladder cancer is difficult or impossible to predict based on conventional disease parameters. It is a reasonable hypothesis that the genetic aberrations acquired by the tumor cells, being instrumental in bringing about the disease in the first place, may also hold the key to more reliable prognostication. However, though 200 transitional cell carcinomas (TCC), the most common bladder cancer in the Western world, with clonal chromosomal abnormalities have been reported, our knowledge about the karyotypic characteristics of these tumors remains insufficient. The aberration pattern is clearly nonrandom, but no completely specific primary or secondary karyotypic abnormality has been identified, and the chronological order in which the aberrations appear during disease progression is not well known. The high degree of karyotypic complexity in epithelial tumors like TCC is one reason why our picture of the sequential order of cytogenetic evolution is unclear. To overcome some of these difficulties we have used several statistical methods that allow analysis and interpretation of the relationship between cytogenetic aberrations in TCC. We show that there exists a temporal order with respect to the appearance of chromosomal imbalances and that this order is highly correlated with tumor stage and grade. Analyzing changes in the distribution of imbalances per tumor in G1, G2, and G3 tumors, we suggest that progression involves the acquisition of cytogenetically detectable and submicroscopic genetic changes at comparable frequencies. By means of computer simulations, we show that the imbalances -9, +7, and 1q+ appear earlier than expected from random events and that -6q, -5q, -18, +5p, -22p, and -15 appear later than expected. Using principal component analysis, we identify two cytogenetic pathways in TCC, one initiated by -9 and followed by -11p and 1q+, the other initiated by +7 and followed by 8p- and +8q. The -9 pathway was correlated with stage Ta-T2 tumors, whereas the +7 pathway was correlated with stage T1-T3 tumors, i.e., +7 tumors appeared to be more aggressive. Although these pathways are well separated at earlier stages, they later converge to contain a common set of imbalances.

Telomere dysfunction triggers extensive DNA fragmentation and evolution of complex chromosome abnormalities in human malignant tumors.

Although mechanisms for chromosomal instability in tumors have been described in animal and in vitro models, little is known about these processes in man. To explore cytogenetic evolution in human tumors, chromosomal breakpoint profiles were constructed for 102 pancreatic carcinomas and 140 osteosarcomas, two tumor types characterized by extensive genomic instability. Cases with few chromosomal alterations showed a preferential clustering of breakpoints to the terminal bands, whereas tumors with many changes showed primarily interstitial and centromeric breakpoints. The terminal breakpoint frequency was negatively correlated to telomeric TTAGGG repeat length, and fluorescence in situ hybridization with telomeric TTAGGG probes consistently indicated shortened telomeres and >10% of chromosome ends lacking telomeric signals. Because telomeric dysfunction may lead to formation of unstable ring and dicentric chromosomes, mitotic figures were also evaluated. Anaphase bridges were found in all cases, and fluorescence in situ hybridization demonstrated extensive structural rearrangements of chromosomes, with terminal transferase detection showing fragmented DNA in 5-20% of interphase cells. Less than 2% of cells showed evidence of necrosis or apoptosis, and telomerase was expressed in the majority of cases. Telomeric dysfunction may thus trigger chromosomal fragmentation through persistent bridge-breakage events in pancreatic carcinomas and osteosarcomas, leading to a continuous reorganization of the tumor genome. Telomerase expression is not sufficient for completely stabilizing the chromosome complement but may be crucial for preventing complete genomic deterioration and maintaining cellular survival.