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Cancer Research 67, 9221, October 1, 2007. doi: 10.1158/0008-5472.CAN-07-1133
© 2007 American Association for Cancer Research
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Cell, Tumor, and Stem Cell Biology

Whole-Genome Profiling in Liposarcomas Reveals Genetic Alterations Common to Specific Telomere Maintenance Mechanisms

Jay E. Johnson1, Edward J. Gettings2, Jaclyn Schwalm2, Jianming Pei2, Joseph R. Testa2, Samuel Litwin2, Margaret von Mehren2 and Dominique Broccoli1

1 Department of Laboratory Oncology Research, Curtis and Elizabeth Anderson Cancer Institute, Memorial Health University Medical Center, Savannah, Georgia and 2 Department of Medical Oncology, Population Science Division and Human Genetics Program, Fox Chase Cancer Center, Philadelphia, Pennsylvania

Requests for reprints: Dominique Broccoli, Department of Laboratory Oncology Research, Curtis and Elizabeth Anderson Cancer Institute, Memorial Health University Medical Center, 4700 Waters Avenue, Savannah, GA 31404. Phone: 912-350-0957; Fax: 912-350-1269; E-mail: BroccDo1{at}memorialhealth.com.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Telomere attrition ultimately leads to the activation of protective cellular responses, such as apoptosis or senescence. Impairment of such mechanisms can allow continued proliferation despite the presence of dysfunctional telomeres. Under such conditions, high levels of genome instability are often engendered. Data from both mouse and human model systems indicate that a period of genome instability might facilitate tumorigenesis. Here, we use a liposarcoma model system to assay telomere maintenance mechanism (TMM)–specific genetic alterations. A multiassay approach was used to assess the TMMs active in tumors. Genomic DNA from these samples was then analyzed by high-resolution DNA mapping array to identify genetic alterations. Our data reveal a higher level of genome instability in alternative lengthening of telomere (ALT)–positive tumors compared with telomerase-positive tumors, whereas tumors lacking both mechanisms have relatively low levels of genome instability. The bulk of the genetic changes are amplifications, regardless of the mode of telomere maintenance used. We also identified genetic changes specific to the ALT mechanism (e.g., deletion of chromosome 1q32.2-q44) as well as changes that are underrepresented among ALT-positive tumors, such as amplification of chromosome 12q14.3-q21.2. Taken together, these studies provide insight into the molecular pathways involved in the regulation of ALT and reveal several loci that might be exploited either as prognostic markers or targets of chemotherapeutic intervention. [Cancer Res 2007;67(19):9221–8]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Telomeres are specialized structures found at the ends of linear DNA molecules and are required for the maintenance of eukaryotic chromosomes. These nucleoprotein structures guard against chromosome end-to-end fusions and prevent the natural ends of chromosomes from being recognized as dsDNA breaks (1). Telomeres suffer sequence attrition each time a cell replicates its genome due, in part, to the incomplete nature of semiconservative DNA replication. Processing events necessary for the maintenance of telomere structure might also contribute to sequence attrition. Eventually, unchecked loss of telomeric DNA produces a dysfunctional telomere that activates DNA damage checkpoints (2, 3), initiating protective cellular responses, such as apoptosis (4) or senescence (5). Although such responses typically limit the likelihood that an aberrantly proliferating cell will cause a tumor, impairment of pathways responsible for the maintenance of genome integrity can allow continued proliferation despite the presence of dysfunctional telomeres. Under these conditions, high levels of genome instability are often engendered (6). Interestingly, data from both mouse and human model systems indicate that a transient period of genome instability might actually promote cancer formation (6, 7).

Activation of a telomere maintenance mechanism (TMM) has been found to stabilize telomeres, facilitate evasion of cell cycle checkpoints, and allow the unlimited cellular proliferation that is a hallmark of cancer (8, 9). Maintenance of telomeric DNA is usually accomplished through the action of telomerase, a large multisubunit complex that adds polynucleotide repeats to preexisting telomeres (10). In humans, the core telomerase holoenzyme consists of a catalytic protein subunit (hTERT) and a template RNA (hTER). Whereas most tumors use telomerase to maintain telomeric arrays, sarcomas often use a telomerase-independent mechanism called alternative lengthening of telomeres (ALT; refs. 11, 12).

There are several differences between cells that use ALT for telomere maintenance and those that use telomerase. Whereas telomerase-positive cells contain relatively short telomeres, telomeric tracts in ALT-positive cells are heterogeneously sized and have a greater mean length than their telomerase-positive counterparts (11). Another characteristic of ALT-positive cells is the presence of extrachromosomal DNA circles made up of telomeric sequence. ALT-positive cells also contain cell cycle–regulated colocalizations of telomeric DNA, the telomere binding proteins TRF1 and TRF2, and the promyelocytic leukemia (PML) nuclear body in structures called ALT-associated PML bodies (APBs; refs. 13–15). Interestingly, several lines of evidence implicate telomeric recombination as playing a role in ALT. For example, telomere dynamics in ALT-positive cells are consistent with a recombination-based mechanism of elongation (16). Furthermore, a tag from a single marked telomere is readily transferred to other telomeres in ALT cells (17). Moreover, several groups have shown that there is increased telomeric sister chromatid exchange in ALT cells (17–19). Finally, several studies have shown that survivors of telomerase deficiency in yeast maintain their telomeres by a mechanism that is likely functionally similar to ALT in humans and that this mechanism proceeds by DNA recombination (20–22).

Dysfunctional telomeres cause genome instability, whereas both ALT and telomerase rescue telomere dysfunction. Several studies have explored the relationship between genome instability and telomerase activation, including recent work using a human breast cancer model system (23). The effect of ALT activation on the levels of genome instability in human cancer has not yet been determined. Here, we use DNA mapping array technology to assay genomic imbalances in a set of liposarcomas, allowing for direct comparison of the levels of genome instability associated with the various TMMs. This approach has the added benefit of facilitating the identification of TMM-specific genetic alterations, analysis of which should contribute to our understanding of the ALT and telomerase mechanisms.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tumor specimens. Our tumor sample set consisted of 37 liposarcomas collected between 1992 and 2006 and made available for analysis from the Fox Chase Cancer Center (FCCC) Tumor Bank. Of these samples, 34 were described previously with respect to both morphologic characteristics and TMMs (24). In the intervening time, three new samples were obtained and tested for the presence of TMMs. Tumors were obtained from males and females with equal frequency, and the median age of the patients was 65 years. Our collection contained mostly grade 1 tumors (n = 27), several grade 2 tumors (n = 7), and the remainder were grade 3 (n = 3). Eighteen samples were primary tumors, with the remainder (n = 19) representing recurrences. Two tumors thought previously to be related were shown by genotyping to actually be two genetically distinct occurrences. The tumor characteristics (Table 1 ) have been updated given this finding.


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  		Table 1. Telomere maintenance and genome instability in liposarcomas

 
Assays of telomere maintenance. A multiassay approach was used to determine the TMM active, if any, in each tumor. These assays include the telomeric repeat amplification protocol assay (24, 25), reverse transcription PCR–based detection of telomerase mRNA (24, 26), telomere restriction fragment length analysis (24, 25, 27, 28), and indirect immunofluorescent staining of APBs (24) and have been described previously.

DNA copy number and genotyping assay. Genomic DNA was prepared from tumor samples by SDS lysis and phenol extraction and checked for purity using a NanoDrop spectrophotometer. DNA was also checked for excessive shearing by agarose gel electrophoresis. Tumor DNA was then analyzed by the Affymetrix GeneChip Single Nucleotide Polymorphism (SNP) 100K mapping assay to identify genomic alterations. This assay has been described previously (29). The intensity of probe signals following hybridization to the GeneChip was determined using Affymetrix GeneChip Operating software version 1.2. The Affymetrix Copy Number Analysis Tool version 2.1 was used to generate copy number (CN) calls for each of 57,290 discreet SNP markers, using the default genomic smoothing window setting of 0.5 Mb. Both CN and loss of heterozygosity (LOH) calls were generated using the Affymetrix Dynamic Model. A locus was determined to be amplified if the reported sample CN was greater than the wild-type (WT) reference (two copies; 2N) by >0.5N. Similarly, a locus was considered to be deleted if the CN was less than the value of the WT by at least 0.5N. Genotyping was done using the Affymetrix GeneChip DNA Analysis software version 3.0 with default settings of 0.25 for both homozygous and heterozygous call thresholds. The resultant genotype calls were used to determine the likelihood of LOH for each SNP marker. A locus was considered to show LOH if the probability of its homozygosity correlating with diploidy was 0.01% or less. LOH measurements on unpaired samples, as done here, were achieved by comparing the observed genotypes with the heterozygosity rates of 110 normal reference samples. The X and Y chromosomes were omitted from all analyses.

Data analysis. We used nonparametric statistical tests to avoid distributional assumptions. Amplifications were compared with deletions by a two-sided Wilcoxon one-sample test where, for each tumor, the fraction of deleted SNPs was subtracted from the fraction of amplified SNPs. In cases where such differences were all positive, we made use of the simpler two-sided sign test.

To compare the extent of amplification or deletion of commonly altered regions (i.e., chromosome 12q and chromosome 1q) between ALT-positive and ALT-negative tumors, we used Fisher's exact two-sided test. This test was applied at each SNP locus where the fraction of ALT-positive tumors with a CN change was compared with the fraction of ALT-negative tumors also showing a change. This method was used to analyze amplifications of SNPs within chromosome 12q and deletion of SNPs within chromosome 1q. For chromosome 12q, 360 adjacent SNPs were compared. Eight hundred seventy-seven SNPs were compared for chromosome 1q.

We used the Jonckheere-Terpstra test of the null hypothesis that there is no relation of the TMM with the levels of either genome or telomere instability versus the alternative that instability levels vary with the mode of telomere maintenance; specifically, that the instability observed in unknown tumors is less than that of telomerase-positive tumors, which in turn is less than that of ALT-positive tumors.

PCR sequencing of TP53. At least 150 ng of genomic DNA were subjected to PCR amplification using primer pairs to amplify the regions containing exons 2 to 4 (5'-CCAGGTGACCCAGGGTTGGAAG-3' and 5'-GAAGCCAAAGGGTGAAGAGGAAT-3') or 5 to 9 (5'-TTCACTTGTGCCCTGACTT-3' and 5'-CTGGAAACTTTCCACTTGAT-3'). For amplification of exons 2 to 4, reactions were done in a buffer containing 16.6 mmol/L ammonium acetate, 67 mmol/L Tris-OH (pH 8.8), 6.7 µmol/L EDTA (pH 8.0), 1% DMSO, 10 mmol/L ß-mercaptoethanol, and 3.5 mmol/L magnesium chloride. Reactions were incubated at 94°C for 3 min and subjected to two rounds each of the following cycles: 94°C for 1 min, 62°C for 1 min, and 72°C for 1.5 min; 94°C for 1 min, 61°C for 1 min, and 72°C for 1.5 min. Next, reactions were subjected to an additional 33 cycles of amplification using the following conditions: 94°C for 1 min, 60°C for 1 min, and 72°C for 1.5 min. For amplification of exons 5 to 9, the buffer was formulated as described above with the exception of containing ammonium sulfate in place of ammonium acetate and containing 6.7 mmol/L magnesium chloride. Reactions were incubated at 94°C for 3 min and subjected to two rounds each of the following cycles: 94°C for 1 min, 60°C for 1 min, and 72°C for 1.5 min; 94°C for 1 min, 59°C for 1 min, and 72°C for 1.5 min. Next, reactions were subjected to an additional 33 cycles of amplification using the following conditions: 94°C for 1 min, 58°C for 1 min, and 72°C for 1.5 min. Finally, reaction products were extended for an additional 10 min at 72°C. Sequencing of the resulting PCR products was done at either the FCCC Sequencing Facility or at SeqWright (Houston, TX), using sequencing primers specific to exons 2 to 4 (5'-CCAGGTGACCCAGGGTTGGAAG-3' and 5'-GAAGCCAAAGGGTGAAGAGGAAT-3'), exon 5 (5'-TGAGGAATCAGAGGCCTGG-3'), exon 6 (5'-AGAGACGACAGGGCTGGT-3'), exon 7 (5'-GAGGCAAGCAGAGGCTGG-3'), exon 8 (5'-CCTTACTGCCTCTTGCTTC-3'), or exon 9 (5'-TTATGCCTCAGATTCACTTTT-3').


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Analysis of CN changes and LOH. Characterization of telomere maintenance in our liposarcoma sample set revealed that 24% of tumors are ALT-positive (n = 9), 24% are telomerase-positive (n = 9), and the remainder do not have characteristics consistent with either mechanism (n = 19). To identify correlations between the active TMM and genetic alterations, we did DNA CN and genotyping analyses on DNA obtained from our tumor specimens. A total of 24 tumors were analyzed by DNA mapping array, 4 of which represent recurrences of other tumors in the sample set. Genome instability was measured as the percentage of total SNPs assayed (n = 57,290) that deviate from a WT CN. Basal levels of genome instability were determined by mapping array analysis of normal peripheral blood mononuclear cells obtained from the same patient as tumor 32. DNA from these control cells showed, as expected, a relatively low proportion (2%) of the total SNPs deviating from a WT CN. Studies have shown that CN variable regions (CNVR) exist (30), wherein certain SNP markers might show CN variation between individuals without being indicative of genome instability, per se. Therefore, optimal analysis of these samples would entail comparison of SNP CNs observed in tumor samples with those observed in normal DNA obtained from the same individual. Because matched normal DNA was not available for the majority of the tumors interrogated here, we instead compared tumor CN changes against the CNVRs published in the Database of Genomic Variants3 and determined that the genetic alterations observed in the liposarcoma tumors are likely not due to CN variation. Our results indicate that ALT-positive liposarcomas have, on average, more CN changes than telomerase-positive tumors. Furthermore, telomerase-positive tumors have more CN changes than tumors with no evidence of telomere maintenance (ALT>telomerase>unknown; P = 0.031; Fig. 1A ). The majority of ALT tumors (five of seven) exhibited >20% of SNPs deviating from diploid with an average of 21.9% of SNPs having altered CN. Telomerase-positive tumors had on average 11.2% of SNPs deviating from a WT CN, with three of the five tumors exhibiting 15% to 20% of SNPs with CN alterations. None of the telomerase-positive tumors had >20% of SNPs exhibiting CN changes. Although relatively few (6%) of the SNPs deviated from a WT CN in tumor 9, this sample also showed both telomerase activity and an intermediate APB phenotype, possibly indicating the activation of both telomere maintenance pathways within this tumor. Finally, the majority of tumors without evidence of telomere maintenance (six of nine) had <10% of SNPs deviating from a WT CN with an average of only 7.3% of SNPs exhibiting CN changes. One of two tumors in this category (tumor 8) that exhibited a high frequency of CN changes (25%) also had an intermediate APB phenotype (data not shown), possibly indicating that this tumor had a weak ALT phenotype or was composed of a heterogeneous population of cells, some of which were ALT positive. Cultured cells from the other lesion (tumor 32, 19% CN changes) showed transient telomerase activity (data not shown), suggesting that this mechanism might have been active sporadically during the development of this lesion, despite not being detected in the tumor sample, itself. If these tumors were to be excluded from analysis, the remaining tumors without evidence of telomere maintenance would exhibit CN changes in only 4.3% of SNPs, on average.


Figure 1
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  		Figure 1. Levels of genome instability and LOH in liposarcomas. Stacked bar graphs showing the relative levels of overall genome instability (A), peritelomeric genome instability (B), and LOH (C) in liposarcoma tumors.

 
Telomere dysfunction drives genome instability. Accordingly, it is possible that alterations in CN at telomere-proximal loci might precede changes at other genetic loci, producing a higher frequency of CN changes at peritelomeric regions. Thus, instability at telomere-proximal regions was also measured, only considering SNPs positioned within the terminal 10 Mb of each chromosome. Similar to the results for overall instability, the levels of peritelomeric CN change were higher in ALT-positive tumors than in their telomerase-positive counterparts, which in turn showed more CN changes than those tumors lacking evidence of both mechanisms (P = 0.0128). Interestingly, instability near the telomere was typically higher than that for the genome as a whole (Fig. 1B).

Analysis of tumor genomic DNA by mapping array also allows the identification of SNP markers that show a high likelihood of LOH. Our results indicate that LOH is more prevalent in ALT-positive tumors than in those lesions that use telomerase or have no evidence of telomere maintenance (Fig. 1C). On average, 38% of SNPs in ALT-positive tumors showed evidence of LOH, with the majority of tumors (five of seven) showing levels much greater than what was observed for the normal peripheral blood control (14%). SNPs (16.4%) exhibit LOH in the average telomerase-positive liposarcoma (n = 5), a value similar to that of the WT control. The same is true of those tumors without evidence of telomere maintenance, which show LOH at an average of only 18.8% of SNPs. Only two of these samples exhibit LOH at a substantially greater frequency than the WT control, tumors 8 and 32 (29% of SNPs, each). As discussed above, it is possible that these two lesions exhibited either low-level ALT (tumor 8) or transient telomerase activity (tumor 32). Excluding these samples from analysis reveals LOH at an average of 15.3% of SNPs in the remaining tumors. The data indicate that ALT utilization is associated with relatively high levels of LOH. In addition, LOH does not seem to simply be a function of the tendency toward genetic rearrangements, as there is little difference between the extent of LOH in telomerase-positive tumors (intermediate levels of genome instability) and those tumors without detectable telomere maintenance (low levels of genome instability).

The majority of CN changes are amplifications. Regardless of the category of tumor analyzed (ALT, telomerase, and unknown), CN changes tend to represent amplifications rather than deletions (Fig. 1; Tables 1 and 2 ). This tendency toward amplifications is seen both at interstitial regions of the genome and at telomeres and is most striking for ALT-positive tumors (overall and peritelomeric; P = 0.0156). In addition, a similar but less significant tendency is observed in telomerase-positive lesions (P = 0.0625 and 0.0431, respectively) and those tumors lacking both mechanisms (P = 0.0313 and 0.236, respectively). In the most extreme case (analysis of overall genome instability in tumors with no evidence of ALT or telomerase), ~80% of all markers that deviate from a WT CN are amplified. The least relative amplification is seen by analysis of overall genome instability in telomerase-positive tumors, wherein ~60% of all non-2N markers represent amplifications. Although these numbers represent averages obtained from analysis of all tumors in each category, the propensity toward amplifications is also observed in individual tumor samples (Table 1). In only 2 (9%) of the 24 tumors analyzed (tumors 8 and 26) do the frequency of deletions exceed that of amplifications. For tumor 26, this is true only if the CN changes at telomere-proximal loci. In tumor 8, however, the number of deletions exceeds that of amplifications in both interstitial and telomeric regions. Interestingly, tumor 8 also represents the only tumor with no strong evidence of ALT or telomerase that also exhibited relatively high levels of genome instability (25% overall and 29% peritelomeric). As mentioned previously, this tumor showed an APB phenotype intermediate to those tumors that use ALT and those that do not, which, in connection with its high level of genome instability, makes it atypical in the context of the sample set.


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  		Table 2. Frequency of amplifications and deletions in liposarcomas

 
Analysis of telomere maintenance, genome instability, and tumor progression. In a recent study, positive correlations were identified between activation of telomere maintenance and tumor grade in liposarcomas. The authors found that the majority (~70%) of grade 1 lesions showed no evidence of ALT or telomerase (31). In grade 2 and 3 lesions, however, the converse was true, with ~70% of tumors showing evidence of a TMM. We sought to extend these studies by considering possible associations between telomere maintenance and tumor progression in our sample set. For grade 1 lesions, tumors that were TMM positive (n = 12, 46%) and tumors that lacked both ALT and telomerase (n = 14, 54%) were present in comparable numbers. In grade 2 and 3 lesions, similar frequencies were observed, with exactly equal numbers of tumors showing telomere maintenance as those that did not (n = 5, each). Therefore, in contrast with previous studies, our data show no particular correlation between tumor grade and TMM and are consistent with the activation of a telomere maintenance being an early event in the development of liposarcomas.

In consideration of recurrent tumors, telomere attrition correlates with increased instability of both the telomere and the whole genome. For tumor 26, each successive recurrence shows progressively shorter average telomere lengths compared with the primary tumor (Fig. 2 ). In terms of the associated levels of genome instability, an inverse proportionality is indicated, with telomere and overall instability increasing as average telomere length decreases. Of these three samples, telomerase activity is detected only in the last occurrence. With respect to tumor 27 and its recurrences, a reduction in average telomere length was observed from the primary tumor to the first recurrence, whereas there is relatively little increase in either telomere or overall genome instability. Furthermore, there is relatively little telomere shortening from the first recurrence to the second, presumably due to the action of telomerase, and the levels of peritelomeric and overall instability are similarly maintained.


Figure 2
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  		Figure 2. Telomere maintenance and genome instability in recurrent tumors. Recurrent tumor sets derived from tumor 26 (A) and tumor 27 (B) were analyzed. Average telomere lengths (black). Levels of instability (gray). Tumors in which telomerase activity was detectable (italics). The remaining two tumors showed no evidence of telomerase or ALT.

 
Identification of TMM-specific genetic changes by whole-genome profiling. Previous studies assessing genetic changes in soft tissue sarcomas, and specifically in liposarcomas, using genome-wide approaches have relied largely on comparative genomic hybridization using bacterial artificial chromosome arrays and also failed to stratify tumors, or their associated changes, with respect to TMM. The experiments done here allow both a higher resolution analysis of CN changes and identification of any changes correlated with a specific subset of tumors. The most common alteration observed in liposarcomas, amplification of an interstitial region of chromosome 12q, is also the most common alteration in our sample set. Based on our results, chromosome 12q also represents the portion of the genome featuring the highest average CNs in all liposarcomas, ranging from four to eight copies. Our analyses have identified the minimal common amplicon as being chromosome 12q14.3-q21.2 (Fig. 3A ) and showed that this amplification is underrepresented among ALT-positive tumors. Interestingly, the CDK4 gene, which has been shown previously to be amplified in soft tissue sarcomas (32), is not part of the contiguous chromosome 12q14.3-q21.2 amplicon identified in this study. Nevertheless, it is similarly amplified in at least half of tumors lacking ALT and in only one ALT-positive sample (14%). Additionally, we identified deletion of chromosome 1q32.2-q44 as being a genetic change frequent in tumors that use the ALT mechanism (Fig. 3B). This is a large deletion spanning over 40 Mb and encompassing several genes whose functions make them candidate regulators of ALT. For example, the deletion spans the gene encoding the p53-binding protein 2 (TP53BP2/ASPP). Binding of 53BP2 to p53 competes with the association of the latter factor with promoter regions involved in the activation of cellular senescence, thereby sensitizing cells to p53-dependent apoptotic death. As discussed below, it is possible that impairment of p53 might facilitate ALT activation. Additional candidate genes that might play a role in the regulation of ALT, telomerase, or tumorigenesis, in general, are summarized in Table 3 .


Figure 3
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  		Figure 3. High-resolution analysis of chromosome 12q amplifications and 1q deletions as a function of TMM in liposarcomas. Stacked area graphs showing the proportion of tumors that show amplification or deletion of the indicated regions of chromosome 12q (A) and chromosome 1q (B). Regions wherein the CNs in ALT-positive and ALT-negative lesions vary significantly from one another (P < 0.05; tall red columns). Regions wherein such differences approach significance (0.10 > P > 0.05; narrow red columns).

 

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  		Table 3. TMM-specific genetic alterations of chromosome 1q and chromosome 12q identified by whole-genome profiling

 
The TP53 DNA surveillance gene is WT in liposarcomas. Considered the "guardian of the genome," the TP53 gene is mutated in nearly half of all cancers. In a study assaying p53 status in soft tissue sarcomas, it was found that there was a high incidence of TP53 mutations in tumors of histologic grade 3 (33). In addition, our laboratory and others have suggested previously that impairment of the activity of p53 in suppressing DNA recombination might cause a more permissive environment for the activation of the ALT mechanism (34). Apparently, such inhibition might also result in the generation of a potentially tumor-promoting unstable genetic milieu.

To assess TP53 status, we did PCR sequencing of exons 2 to 9, which would contain the majority of all reported mutations should any be present in these tumors. The sequence of TP53 was interrogated in a total of 24 tumors (ALT, n = 7; telomerase, n = 7; unknown, n = 10). The WT status of TP53 was confirmed in all of the lesions interrogated, with the exception of tumor 2, which showed a missense mutation at codon 60 (CCA>GCA). This mutation has not been reported previously. Sequence analyses have also identified several commonly observed polymorphisms, including the well-characterized codon 72 (proline/arginine) polymorphism.4 The proline and arginine variants are present in 45% and 55%, respectively, of nonrecurrent liposarcoma tumor samples (data not shown); these frequencies are not appreciably different from what is observed in the U.S. population as a whole. Detection of such changes shows the efficacy of this approach in identifying sequence alterations when present. Our data indicate that mutation of the TP53 DNA surveillance gene does not play a significant role in the activation of ALT or the accrual of genome instability in liposarcomas. As mentioned above, previous work has shown that mutation of TP53 seems to correlate with high histologic grade, whereas our sample set contains mostly low and intermediate grade lesions. Thus, although our data suggest that mutation of TP53 is not required for the initial development of liposarcomas, it remains possible that TP53 inactivation might play a role in the evolution of more advanced malignancies.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
DNA mapping analyses have revealed that ALT-positive tumors have, on average, higher levels of genome instability than do their telomerase-positive counterparts. This finding suggests that, compared with telomerase, ALT might be less efficient at stabilizing telomere-driven genome instability or activated following a longer period of telomere dysfunction. Another possibility is that the spectrum of mutations that causes ALT also results in high levels of genome instability independent of telomere status. This possibility is intriguing given the likelihood that the ALT mechanism functions by DNA recombination.

Our sample set contained tumors that showed no evidence of telomere maintenance, analysis of which showed that the majority of these lesions had relatively few CN changes. These findings are consistent with previous reports that neither activation of a TMM nor telomere-driven genome instability is obligatory steps in the formation of a clinically detectable lesion. The fact that tumors lacking both known TMMs typically have relatively low levels of genome instability suggests that these lesions have not yet undergone a telomere crisis event. Theoretically, this is possible if the progenitor cells that cause such tumors possess sufficient telomeric reserves to overcome the proliferation-induced telomeric shortening that is usually associated with tumorigenesis. Indeed, it has been suggested that under conditions of low cell turnover, transformed cells can produce a clinically significant and aggressive tumor within relatively few population doublings (35).

Analysis of genotyping data indicates that LOH is prevalent in liposarcomas that use ALT. Furthermore, the observed LOH does not correlate with the level of CN changes, as the extent of LOH in telomerase-positive tumors (11.2% SNP CN changes) was similar to what was observed in those tumors without detectable telomere maintenance (4.3% SNP CN changes). Therefore, it is probable that the LOH observed in ALT-positive liposarcomas is either due to activities associated with activation of the ALT mechanism or a consequence of the ALT mechanism, itself. Currently, the data are insufficient to distinguish between these possibilities.

The observation that instability at telomere-proximal sites is consistently higher than at interstitial sites of the genome raises the possibility that the observed instability might be driven by the breakage-fusion bridge cycle associated with dysfunctional telomeres. This observation might also be due, however, to preferential breakage at telomere-proximal fragile sites compared with those located interstitially (36, 37). Alternatively, abrogation of p53 function might result in the genome instability observed in our panel of liposarcomas. Although sequence analyses have shown that the TP53 gene is WT in nearly all tumors assayed, the possibility remains that TP53 function is impaired indirectly. Indeed, amplification of the MDM2 gene is observed in more than half of liposarcomas and its overexpression has been shown to impair p53 function by targeting the protein for ubiquitin-dependent degradation (38). However, amplification of MDM2 is observed infrequently in ALT-positive tumors, which also have the highest average levels of genome instability. Therefore, in these lesions, impairment of p53 might be achieved by mutation of one of its downstream effectors. It is also probable that the observed instability is the combined result of multiple mechanisms affecting the stability of the genome. Additional experiments will be necessary to determine the actual causes of genome instability in liposarcomas, as well as their respective contributions to this phenomenon.

With the exception of tumor 8, all liposarcoma tumors show a greater proportion of amplifications than deletions. Our data suggest that tumor 8 might have either been heterogeneous with respect to ALT activation or simply displayed a weak ALT phenotype. However, the molecular mechanisms underlying the unique pattern of genome instability observed in this tumor (deletions exceeding amplifications) remain unclear. Future identification and analysis of other samples that display similar characteristics should help to explain this observation.

Taken together, our data indicate that ALT-positive and ALT-negative tumors are fundamentally different biological entities, with ALT-positive tumors showing significantly higher levels of genome instability and LOH than their ALT-negative counterparts. Moreover, whole-genome profiling has revealed two sizable genetic alterations further distinguishing ALT-positive and ALT-negative lesions. Whereas amplification of chromosome 12q has been identified previously as the most common genetic change in liposarcomas, here we show that amplification of the minimum region, chromosome 12q14.3-q21.2, is common only to telomerase-positive tumors and those tumors that lack evidence of telomere maintenance. In addition, deletion of chromosome 1q32.2-q44 is a frequent event only in ALT-positive samples. A recent study reported that both ALT and telomerase positivity are associated with unfavorable disease outcome in liposarcomas, although prognosis is poorest for patients whose tumors use ALT (31). Apparently, differences in TMM-specific genetic alterations, such as those shown in the current study, might underlie the observed differences in patient prognosis. Analysis of candidate factors encoded within the altered regions might aid development of novel anticancer therapeutics that specifically target the ALT or telomerase mechanisms. Additionally, these regions might also encode genes that, while not playing a role in telomere maintenance per se, are tightly linked to either of the specific TMMs. Given the positive correlations of these mechanisms with tumor aggressiveness, such factors might represent excellent biomarkers for the rapid identification of the active TMM and, consequently, could serve as efficient indicators of prognosis. Finally, identification of ALT- or telomerase-associated factors might also serve as predictors of tumor drug response. For example, ALT-positive tumors show a higher level of genome instability than tumors lacking ALT and, consequently, are likely to display differential sensitivity to DNA-damaging agents. Conversely, ALT-positive tumors should prove refractory to treatment with intervention strategies that specifically inhibit telomerase. Rapid identification of the active TMM in liposarcomas would allow treatment to be tailored to the individual, achieving the highest possible tumor cell killing with the lowest possible patient toxicity.


   Acknowledgments
 
Grant support: NIH grants CA117675-01 (J.E. Johnson) and CA098087-03 (D. Broccoli).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank the members of the Broccoli Lab for helpful discussion; P. Cairns of FCCC for assistance with TP53 sequencing; FCCC Sequencing Facility; C. Renner, K. Kaputa, and R. Page of the FCCC Histopathology Center and Tumor Bank; and S. Jablonski of the FCCC Cell Imaging Facility.


   Footnotes
 
3 http://projects.tcag.ca/variation/ Back

4 http://p53.bii.a-star.edu.sg/index.php Back

Received 3/26/07. Revised 7/23/07. Accepted 7/26/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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