Telomere Maintenance Mechanisms in Liposarcomas

Study Report
Experimental Plan
Funding

By Dominique Broccoli, PhD

Introduction

A fundamental feature of cancer cells is their ability to divide indefinitely. This is achieved through activation of mechanisms that maintain telomeric DNA repeats. In stem cells and most cancers telomeric DNA is replicated and maintained by telomerase, a specialized reverse transcriptase that adds telomeric repeats onto the 3’ end of an existing DNA strand.¹ In contrast, sarcomas activate the Alternative Lengthening of Telomeres (ALT) mechanism² as frequently as telomerase.^3-5 The two mechanisms differ in the efficiency with which they replicate telomeres and ALT-positive cells and tumors generally have higher levels of genome instability.⁶ Intriguingly, ALT-positive liposarcomas are associated with a worse prognosis than their non-ALT counterparts.⁵ The molecular basis for the differential outcome in this disease is unknown.

Telomere Dynamics in Aging and Cancer

Telomeres are structures at the ends of chromosomes that act as caps to protect these ends from degradation and fusion. Each time a cell divides, a bit of DNA is lost from the end. Thus, chromosomes become shorter as cells age. Eventually enough DNA is lost for the capping function of telomeres to be compromised. This elicits a signal that prevents further cell division, limiting the number of times any given cell can replicate. The limitation on cell division contributes to aging and acts as one of the barriers to tumor formation. In order to evade this barrier, tumor cells must activate a mechanism to add DNA to chromosome ends, in order to balance that which is lost as a consequence of replication. The most common mechanism activated in cancers relies upon the protein used by stem cells and the germline to maintain telomeric DNA, telomerase. Sarcomas are unique in that an alternative mechanism is used as frequently as telomerase.

Global Gene Expression Profiles

With the sequencing of the human genome completed, it became possible to query every gene in a single snapshot to determine whether it is turned on in a given tumor. In this approach, total RNA (the molecule that is expressed from a gene) is extracted from a sample, converted into DNA, labeled and hybridized to a chip containing targets representing every known and predicted coding gene. If a gene is expressed, the RNA from it will bind to the target and generate a signal that can be measured. By comparing the genes that are changed, either turned on or turned off, in a tumor relative to normal tissue we can identify changes that accompany tumor formation. These changes may drive the process of tumor formation or contribute to other tumor characteristics such as drug resistance.

Hypothesis

Using whole genome profiling to identify copy number changes, we identified genetic alterations that are more common ALT-positive liposarcomas compared to telomerase-positive liposarcomas.⁶ We hypothesized that genetic differences such as these underlie the differences in patient outcomes that have been reported. Accordingly, we expanded upon these studies to identify gene expression patterns in tumors which have been characterized for telomere maintenance by performing microarray based expression studies on 35 liposarcomas of varying histologic type. The strongest expression signatures, regardless of histologic type or telomere maintenance mechanism, were proliferative versus adipocytic genes. Given the high degree of intertumor heterogeneity, this strong expression pattern would potentially mask more subtle patterns. The goal of the project supported by The Liddy Shriver Sarcoma Initiative was to identify genes that are differentially expressed in liposarcomas of a single histological subtype that differ in the telomere maintenance mechanism (TMM) that is activated.

Results

We characterized liposarcomas with respect to histological subtype and telomere maintenance mechanism. We focused first on pleomorphic and dedifferentiated liposarcomas because our preliminary data indicated that the majority of these high grade tumors had activation of one of these two telomere maintenance mechanisms. A group of dedifferentiated liposarcomas that had activated either ALT or telomerase were identified and used for subsequent analysis of global gene expression profiles. Surprisingly, we were unable to identify an expression pattern that differentiated the tumors based on telomere maintenance mechanism.⁷

While our study was underway, a similar study was published by an independent group. In this study a 297 gene expression profile distinguishing ALT-positive and telomerase-positive tumors was described.⁸ Analysis of our data set of 38 tumors of mixed histological subtype suing the 297 gene signature resulted in the tumors clustering based upon histological type rather than telomere maintenance mechanism.⁷ Even when analysis was restricted only to dedifferentiated liposarcomas, the 297 gene signature did not distinguish between ALT and telomerase positive tumors. It is possible that the discrepancies in these data reflect differences in the platforms used. However, the tumors used to generate the 297 gene signature are composed primarily of 2 histological subtypes, myxoid/round cell liposarcomas and dedifferentiated liposarcomas. The telomerase-positive tumors group is composed of predominantly myxoid/round cell liposarcomas while the ALT-positive group is composed predominantly of dedifferentiated liposarcomas.⁹ Thus, it is possible that the 297 gene signature actually identifies differences in histological type rather than those associated with activation of specific telomere maintenance mechanisms.

During the course of characterizing liposarcomas for telomere maintenance mechanism we noted that myxoid liposarcomas which are characterized by the TLS-CHOP fusion do not utilize ALT. This is consistent with earlier reports that translocation associated sarcomas tend to utilize telomerase for telomere maintenance.^10,11 In contrast, we find that pleomorphic sarcomas have the opposite pattern, predominantly utilizing ALT for telomere maintenance. This distinction may reflect fundamental differences in how these cancers evolve.

Future Directions

The analysis described above was unable to identify a distinguishing gene signature. However, the evidence to date indicates fundamental differences in tumors that use ALT versus telomerase for telomere maintenance. There has recently been an explosion in our understanding of the role of non-coding RNAs in regulating myriad biological processes. Accordingly, our next step will be to explore the profiles of these molecules in our well characterized panel of liposarcomas that differ in activation of telomere maintenance mechanism.

By Dominique Broccoli, PhD
Member of the Department of Laboratory Oncology Research
Curtis and Elizabeth Anderson Cancer Institute
Memorial University Medical Center
and Professor at the Department of Biomedical Sciences
Mercer University School of Medicine – Savannah Campus
Savannah, GA

References

1. Shay, JW, Bacchetti, S. A survey of telomerase activity in human cancer. Eur. J. Cancer 1997; 33: 787-791.

2. Bryan, TM, Englezou, A, Gupta, J, Bacchetti, S,Reddel, RR. Telomere elongation in immortal human cells without detectable telomerase activity. Embo J 1995; 14: 4240-4248.

3. Johnson, JE, Varkonyi, RJ, Schwalm, J, et al. Multiple mechanisms of telomere maintenance exist in liposarcomas. Clin Cancer Res 2005; 11: 5347-5355.

4. Ulaner, GA, Huang, HY, Otero, J, et al. Absence of a telomere maintenance mechanism as a favorable prognostic factor in patients with osteosarcoma. Cancer Res 2003; 63: 1759-1763.

5. Costa, A, Daidone, MG, Daprai, L, et al. Telomere maintenance mechanisms in liposarcomas: association with histologic subtypes and disease progression. Cancer Res 2006; 66: 8918-8924.

6. Johnson, JE, Gettings, EJ, Schwalm, J, et al. Whole-genome profiling in liposarcomas reveals genetic alterations common to specific telomere maintenance mechanisms. Cancer Res 2007; 67: 9221-9228.

7. Mitchell, M, Doyle, K, Roberts, C, et al. Gene expression signature reflects differentiation differences rather than telomere maintenance mechanism. Oncogene 2010. in press.

8. Lafferty-Whyte, K, Cairney, CJ, Will, MB, et al. A gene expression signature classifying telomerase and ALT immortalization reveals an hTERT regulatory network and suggests a mesenchymal stem cell origin for ALT. Oncogene 2009; 28: 3765-3774.

9. Cairney, CJ, Hoare, SF, Daidone, MG, Zaffaroni, N,Keith, WN. High level of telomerase RNA gene expression is associated with chromatin modification, the ALT phenotype and poor prognosis in liposarcoma. Br J Cancer 2008; 98: 1467-1474.

10. Montgomery, E, Argani, P, Hicks, JL, DeMarzo, AM,Meeker, AK. Telomere lengths of translocation-associated and nontranslocation-associated sarcomas differ dramatically. Am J Pathol 2004; 164: 1523-1529.

11. Ulaner, GA, Hoffman, AR, Otero, J, et al. Divergent patterns of telomere maintenance mechanisms among human sarcomas: sharply contrasting prevalence of the alternative lengthening of telomeres mechanism in Ewing's sarcomas and osteosarcomas. Genes Chromosomes Cancer 2004; 41: 155-162.

By Dominique Broccoli, PhD

Introduction

There are a number of characteristics that distinguish cancer cells from normal cells. One such characteristic of tumor cells is their infinite ability to grow and divide. These cells are in essence immortal. Indeed, many cell lines used in research laboratories today were originally derived from human tumors decades ago.

Liposarcoma is a tumor derived from primitive cells that undergo adipose differentiation. It is largely a disease of adults, its incidence peaking between the ages of 40 and 60 years, and it shows a slight predominance toward men.

How do cells acquire the ability to divide and multiply indefinitely? The answer lies in the behavior of telomeres, specialized structures at the ends of eukaryotic chromosomes. The genetic material is organized as a group of discrete linear DNA molecules, each containing many genes, which are packaged into structures called chromosomes. Humans have 46 chromosomes. To correctly pass on all the genes each time a cell divides the DNA on every chromosome must first be copied and then separated. Structures on the chromosome help to achieve this. The centromere (see Figure 1) is the region of the chromosome that governs separation. Telomeres are structures at the end of the chromosome that act to cap each end of the linear DNA molecule, much as the plastic tips on shoelaces keep them from unraveling.

An introduction to DNA, RNA and proteins can be found on the Nobel website. After clicking on the above hyperlink, make sure to read the section "Learn how to navigate in the document" to take full advantage of this tutorial.

Telomeres were originally defined based on observations in flies and corn that chromosome ends behave differently from double-stranded DNA breaks induced by radiation or chemicals. Double-stranded DNA breaks are highly unstable genetic structures that activate the cellular DNA damage response and may result in chromosome rearrangements. In contrast, naturally occurring chromosome ends, or telomeres, do not activate these cellular pathways or induce chromosome rearrangements despite being the end of a linear DNA molecule. This is achieved by packaging the chromosome end with specific proteins to form a cap-like structure, thereby sequestering the end of the DNA molecule.

The term cellular pathway refers to the sequence of events that result in a final cellular event. The cellular "DNA damage response pathway" is one such cascade of discrete events that is triggered by alterations to the structure of DNA. In this pathway, proteins in the cell first sense the damage. These sensing molecules then transmit damage information to other proteins, which prevent the cell from dividing, and lead to recruitment of proteins that will repair the DNA damage.

The protective function of telomeres is due to the formation of a nucleoprotein complex, comprised of 6 protein subunits (TRF1, TRF2, RAP1, POT1, TIN2 and TPP1) called shelterin,¹ in complex with the telomeric 5’(TTAGGG)3’ hexanucleotide repeats. The shelterin complex sequesters the end of the DNA molecule and prevents it from activating DNA damage pathways.^1,2 This is thought to be achieved by organizing telomeres into lariat structures, called telomere loops or t-loops, formed by invasion of the 3’ single-stranded overhang into the duplex telomeric repeats, thereby sequestering the chromosome end and providing a chromosome "cap."³

What do telomeres have to do with the infinite cellular division potential of cancer cells? It has become clear over the past two decades that telomeres limit the total number of times many cells can divide. In many cells of the body, telomeres become shorter with each cell division because DNA sequences are lost as a result of DNA replication. This sequence loss is a consequence of the way DNA is replicated. Eventually, insufficient telomeric DNA remains at the chromosome ends to form functional telomeric complexes. The uncapped chromosome end resembles a double-stranded DNA break which activates the cellular DNA damage response and halts further cell division. Thus, proliferation-associated telomere sequence loss acts as a tumor suppressor mechanism by limiting the total number of divisions any given cell can undergo. In other words, normal changes in telomeres that occur as a result of cellular aging protect against cancer.

Hampering Chromosomal Replication

Complete replication of chromosomal termini is hampered by the unidirectional nature and primer requirements of conventional DNA polymerases. Due to these features, a region of unreplicated DNA will remain on the parental DNA strand acting as the template for lagging strand synthesis following removal of the most terminal primer. This has become known as the end replication problem.⁴

Enzymes are proteins in cells that perform biochemical reactions. Most of the DNA on the chromosome is copied by enzymes called DNA polymerases. DNA polymerases make a new linear piece of DNA (a daughter strand) using the original DNA (the parental strand) as a template and can only move in one direction. These enzymes require a primer, a small piece of RNA, in order to start adding new bases (the building blocks of DNA). The RNA is removed leaving a piece of parental DNA at the end of the chromosome that has not been copied. This is why sequences are lost each time the DNA is replicated. This inability of DNA polymerases to copy the end of the DNA molecule is known as the end replication problem.

As discussed above, telomeres also act to confer stability on chromosome ends. Mutations in DNA damage response genes, such as the tumor suppressor p53, may prevent a cell from responding appropriately to shortened telomeres that are seen by the cell as DNA damage. In this scenario the cell is unable to sense or repair the damage. As a consequence, continued cellular divisions occur without chromosome caps. This, in turn, leads to elevated chromosome instability resulting in chromosome rearrangements (mutations). This high level of instability, driven by critically short telomeres, has been observed in human tumors and is thought to actually contribute to, and even promote, tumor formation by increasing the rate at which tumor promoting mutations are acquired.^5-7

Articles Discussing Telomere Loss of Function as a Tumor Promoting Event

L. Chin, S.E. Artandi, Q. Shen, A. Tam, S.L. Lee, G.J. Gottlieb, C.W. Greider and R.A. DePinho. p53 deficiency rescues the adverse effects of telomere loss and cooperates with telomere dysfunction to accelerate carcinogenesis, Cell 97 (1999) 527-538.

R.A. DePinho and K. Polyak Cancer chromosomes in crisis, Nat Genet 36 (2004) 932-934.

Chin, K., de Solorzano, C. O., Knowles, D., Jones, A., Chou, W., Rodriguez, E. G., Kuo, W. L., Ljung, B. M., Chew, K., Myambo, K., Miranda, M., Krig, S., Garbe, J., Stampfer, M., Yaswen, P., Gray, J. W., and Lockett, S. J. In situ analyses of genome instability in breast cancer, Nat Genet (2004), 36(9), 984-988.

From the discussion above it is clear that telomeres play a central role in cancer formation. Ultimately, for a tumor to continue to grow the cell(s) must circumvent the telomere length-dependent limitation on continual cellular division and the ensuing rampant chromosome instability. In most carcinomas, this is achieved through the action of an enzyme called telomerase.⁸ Telomerase is composed of a catalytic reverse transcriptase subunit and an RNA template molecule.⁹ Telomerase is able to add sequences directly on to the end of the DNA molecule causing the telomeres to become longer. Telomerase is normally active in the germline (egg and sperm) thereby ensuring that each generation starts life with telomeres of the appropriate length. But during development and early life telomerase is turned off in many cells. This leads to the replication associated loss of DNA from chromosome ends and contributes to aging. Experiments have demonstrated that forced expression of telomerase is sufficient to confer replicative immortality on human cells. Importantly, these cells are not tumorigenic, but they are capable of unlimited cell division. Because telomerase is activated in many carcinomas, significant effort has been expended to determine the molecular mechanisms that regulate telomerase activity and to validate telomerase inhibition as an anticancer strategy.¹⁰

Telomerase is the enzyme that helps to copy the telomeres at the very ends of the chromosomes. It does this by adding DNA on the end of the parental strand, in the opposite direction from that of DNA polymerases. Because there is no parental molecule to copy telomerase uses an RNA molecule that is part of the enzyme as the template. The end result is that the parental molecule is extended thereby balancing sequence lost due to the end-replication problem.

The DNA sequences that comprise genes code for specific proteins. This DNA is first copied into RNA in a process called transcription. The RNA is then copied into a protein in a process called translation. Because DNA is being made from RNA, rather than the more usual DNA to RNA to protein pathway, the polymerase that makes the DNA is called a reverse trascriptase.

Telomerase activity in germline ensures that every generation starts life with the right size chromosomes. Tumor formation requires mutation in a number of cellular pathways. Some of these mutations lead to activation of growth promoting pathways while others result in the loss of growth inhibiting pathways. By turning off telomerase and limiting the number of divisions most cells can undergo a strong barrier to tumor formation is put in place because this is another pathway that must be activated.

A telomerase-independent mechanism to maintain telomeres, called Alternative Lengthening of Telomeres (ALT) has also been described.¹¹ Various lines of evidence suggest that ALT occurs by recombination-based mechanism(s).^12,13 For example, experimental evidence supports a mechanism wherein a short telomere may invade a longer telomere and uses it as the template to extend the shorter chromosome end.¹³ Telomerase-independent mechanisms for telomere maintenance, such as ALT, provide an alternative route whereby tumor cells may overcome the growth limitation imposed by critically short telomeres. Additionally, tumors using ALT for telomere maintenance will be refractory to treatment with telomerase inhibitors. It is therefore crucial to determine the pathways that regulate these mechanisms if we are to develop strategies that can inhibit telomere maintenance in all tumors. Until recently, we have not been able to study these pathways in human tumors due to the rare activation of telomerase-independent mechanisms in carcinomas.

Alternative Lengthening of Telomeres (ALT)

Formally, ALT describes all telomerase-independent telomere maintenance mechanisms. However, to date all but one of the identified human cell lines that use ALT for telomere maintenance have several distinctive characteristics in common. Telomere length in these cell lines is highly heterogeneous, with repeats ranging in size from less than 4 kb to greater than 25 kb.¹³ ALT-positive cells also have elevated levels of extrachromosomal circular DNA molecules that are composed, at least in part, of telomeric repeats.¹⁴ In addition, the frequency of telomeric recombination, measured as telomere-specific sister chromatid exchange, is elevated in cells that use ALT relative to primary or telomerase-positive cells.¹⁵ Finally, a subset of cells in ALT-positive cell lines contain large multiprotein complexes, termed ALT-associated PML nuclear bodies (APBs), in which telomere binding proteins, such as TRF1 and TRF2, and telomeric DNA co-localize with the promyelocytic leukemia (PML) nuclear body.¹⁶ APBs are tightly correlated with ALT, appearing concomitantly with activation of this telomere maintenance pathway. We, and others, have demonstrated that the formation of APBs is coordinately regulated with the cell cycle.^{17, 18}

Within the last 5 years several groups, including ours, have demonstrated that in certain subsets of tumors, most notably sarcomas, ALT and telomerase are used in an equivalent frequency of tumors.^14-18 Surprisingly, a significant proportion of sarcomas, up to 50% in liposarcomas, do not have characteristics of either telomere maintenance mechanism. Because a high frequency of sarcomas utilize non-telomerase based mechanism(s) of telomere maintenance, these tumors are the only system that can reveal differences in the behavior (aggressiveness, drug sensitivity, etc) of tumors using the different pathways. Activation of ALT as the only means of telomere maintenance within the tumor is correlated with a better prognosis in glioblastoma multiforme.¹⁴ In contrast, in liposarcomas, activation of ALT is associated with slightly worse prognosis than activation of telomerase¹⁹ and, the best prognosis is correlated with the absence of characteristics indicative of activation of either telomere maintenance mechanism, which is also the case in osteosarcomas.¹⁸

We hypothesized that the differences in survival for patients whose tumors differed in the telomere maintenance pathway that had been activated resulted as a consequence of fundamental differences in the biology of these cancers. In recently published work,²⁰ we used a technique called whole genome profiling to compare the genetic composition of a panel of liposarcomas that we had previously characterized for which telomere maintenance mechanism was active. This allowed us to identify regions that were commonly lost or amplified in these tumors. It also allowed us to compare the total number of changes across the tumor subtypes. The results indicate that:

ALT-positive liposarcomas have more changes than telomerase-positive tumors; and
tumors with an active telomere maintenance mechanism have more changes than tumors with no evidence of an active telomere maintenance mechanism.

In addition, this whole genome profiling revealed two sizable genetic alterations distinguishing ALT-positive and ALT-negative lesions. Specifically, ALT-positive liposarcomas frequently lose part of chromosome 1, a genetic change not previously reported for liposarcomas. In contrast, the amplification on chromosome 12 that is characteristic of liposarcomas is rarely observed in ALT-positive tumors. Together these data suggest that ALT-positive and ALT-negative tumors are fundamentally different biological entities. These genetic differences are likely responsible for differences in human tumors using different telomere pathways.

Treatment of soft tissue sarcomas has been hampered by chemotherapy with minimal activity as well as significant toxicity. Selecting patients that would derive benefit from therapy in the form of tumor response would save patients from enduring toxicity and identify a group of patients that would not only benefit from response to therapy, but potentially have prolonged survival. The results discussed above have implications for treatment of liposarocmas. First, evidence suggests that sensitivity to DNA damaging chemotherapy drugs is correlated with genomic instability.^21,22 Our observation that ALT-positive tumors have increased instability relative to non-ALT tumors^17,23 raise the possibility that these tumors might be, in general, more sensitive to chemotherapy than tumors with lower levels of instability. Secondly, the genes contained in the regions exhibiting telomere maintenance mechanism associated changes in DNA content will be good candidates for new drugs targeting specific tumors based upon the telomere maintenance pathway that is active. These studies will provide additional depth to our understanding of factors that predict tumor response to chemotherapy and will provide new insight into the development and evolution of soft tissue sarcomas.

In our new study, entitled "Expression profiles of liposarcomas that have activated different telomere maintenance mechanisms," which is supported by the Liddy Shriver Sarcoma Initiative, we plan to continue our evaluation of telomere maintenance mechanism associated differences in liposarcomas. We hypothesize that the fundamental genetic differences we have already identified in liposarcomas will be paralleled by differences in the constellation of genes that are expressed. Gene expression profiles for a number of soft tissue sarcomas, including liposarcomas, have been reported previously.^24,25 There is a clear difference between liposarcomas in the expression profile based upon histologic subtype. For example, well-differentiated liposarcomas express genes also found to be expressed in normal adipose tissue.²⁶ However, these studies do not stratify tumors based upon telomere maintenance mechanism. Given our recent data suggesting that liposarcomas utilizing different telomere maintenance mechanisms have specific genetic alterations and different prognosis, it is important to carry out these experiments not only controlling for histologic subtype but also for telomere maintenance mechanism. In our current project, we will complete this expression analysis utilizing a panel of liposarcomas that have been characterized with respect to which telomere maintenance mechanism is active. The genes identified through such studies have the potential to be useful diagnostic and/or prognostic markers, may predict response to treatment, and will be tested as potential candidates for new chemotherapy drugs.

By Dominique Broccoli, PhD
Department of Laboratory Oncology Research
Curtis and Elizabeth Anderson Cancer Institute
Memorial University Medical Center
Savannah, Georgia

References

1. de Lange, T. Shelterin: the protein complex that shapes and safeguards human telomeres. Genes Dev, 19: 2100-2110, 2005.

2. Harrington, L. Those dam-aged telomeres! Curr Opin Genet Dev, 14: 22-28, 2004.

3. Griffith, J. D., Comeau, L., Rosenfield, S., Stansel, R. M., Bianchi, A., Moss, H., and de Lange, T. Mammalian telomeres end in a large duplex loop. Cell, 97: 503-514, 1999.

4. Olovnikov, A. M. A theory of marginotomy. J. Theor. Biol., 41: 181-190, 1973.

5. DePinho, R. A. and Polyak, K. Cancer chromosomes in crisis. Nat Genet, 36: 932-934, 2004.

6. Chin, K., de Solorzano, C. O., Knowles, D., Jones, A., Chou, W., Rodriguez, E. G., Kuo, W. L., Ljung, B. M., Chew, K., Myambo, K., Miranda, M., Krig, S., Garbe, J., Stampfer, M., Yaswen, P., Gray, J. W., and Lockett, S. J. In situ analyses of genome instability in breast cancer. Nat Genet, 36: 984-988, 2004.

7. Chin, L., Artandi, S. E., Shen, Q., Tam, A., Lee, S. L., Gottlieb, G. J., Greider, C. W., and DePinho, R. A. p53 deficiency rescues the adverse effects of telomere loss and cooperates with telomere dysfunction to accelerate carcinogenesis. Cell, 97: 527-538, 1999.

8. Shay, J. W. and Bacchetti, S. A survey of telomerase activity in human cancer. Eur. J. Cancer, 33: 787-791, 1997.

9. Blackburn, E. H. Telomeres and telomerase. Keio J Med, 49: 59-65, 2000.

10. Incles, C. M., Schultes, C. M., and Neidle, S. Telomerase inhibitors in cancer therapy: current status and future directions. Curr Opin Investig Drugs, 4: 675-685, 2003.

11. Bryan, T. M., Englezou, A., Gupta, J., Bacchetti, S., and Reddel, R. R. Telomere elongation in immortal human cells without detectable telomerase activity. EMBO J., 14: 4240-4248, 1995.

12. Londono-Vallejo, J. A., Der-Sarkissian, H., Cazes, L., Bacchetti, S., and Reddel, R. R. Alternative lengthening of telomeres is characterized by high rates of telomeric exchange. Cancer Res, 64: 2324-2327, 2004.

13. Dunham, M. A., Neymann, A. A., Fasching, C. L., and Reddel, R. R. Telomere maintenance by recombination in human cells. Nature Genetics, 26: 447-450, 2000.

14. Hakin-Smith, V., Jellinek, D. A., Levy, D., Carroll, T., Teo, M., Timperley, W. R., McKay, M. J., Reddel, R. R., and Royds, J. A. Alternative lengthening of telomeres and survival in patients with glioblastoma multiforme. Lancet, 361: 836-838, 2003.

15. Henson, J. D., Hannay, J. A., McCarthy, S. W., Royds, J. A., Yeager, T. R., Robinson, R. A., Wharton, S. B., Jellinek, D. A., Arbuckle, S. M., Yoo, J., Robinson, B. G., Learoyd, D. L., Stalley, P. D., Bonar, S. F., Yu, D., Pollock, R. E., and Reddel, R. R. A robust assay for alternative lengthening of telomeres in tumors shows the significance of alternative lengthening of telomeres in sarcomas and astrocytomas. Clin Cancer Res, 11: 217-225, 2005.

16. Johnson, J. E., Varkonyi, R. J., Schwalm, J., Cragle, R., Klein-Szanto, A., Patchefsky, A., Cukierman, E., von Mehren, M., and Broccoli, D. Multiple mechanisms of telomere maintenance exist in liposarcomas. Clin Cancer Res, 11: 5347-5355, 2005.

17. Montgomery, E., Argani, P., Hicks, J. L., DeMarzo, A. M., and Meeker, A. K. Telomere lengths of translocation-associated and nontranslocation-associated sarcomas differ dramatically. Am J Pathol, 164: 1523-1529, 2004.

18. Ulaner, G. A., Huang, H. Y., Otero, J., Zhao, Z., Ben-Porat, L., Satagopan, J. M., Gorlick, R., Meyers, P., Healey, J. H., Huvos, A. G., Hoffman, A. R., and Ladanyi, M. Absence of a telomere maintenance mechanism as a favorable prognostic factor in patients with osteosarcoma. Cancer Res, 63: 1759-1763, 2003.

19. Costa, A., Daidone, M. G., Daprai, L., Villa, R., Cantu, S., Pilotti, S., Mariani, L., Gronchi, A., Henson, J. D., Reddel, R. R., and Zaffaroni, N. Telomere maintenance mechanisms in liposarcomas: association with histologic subtypes and disease progression. Cancer Res, 66: 8918-8924, 2006.

20. Johnson, J. E., Gettings, E. J., Schwalm, J., Pei, J., Testa, J. R., Litwin, S., von Mehren, M., and Broccoli, D. Whole-genome profiling in liposarcomas reveals genetic alterations common to specific telomere maintenance mechanisms. Cancer Res, 67: 9221-9228, 2007.

21. Cerone, M. A., Londono-Vallejo, J. A., and Autexier, C. Telomerase inhibition enhances the response to anticancer drug treatment in human breast cancer cells. Mol Cancer Ther, 5: 1669-1675, 2006.

22. Lee, K. H., Rudolph, K. L., Ju, Y. J., Greenberg, R. A., Cannizzaro, L., Chin, L., Weiler, S. R., and DePinho, R. A. Telomere dysfunction alters the chemotherapeutic profile of transformed cells. Proc Natl Acad Sci U S A, 98: 3381-3386, 2001.

23. Scheel, C., Schaefer, K. L., Jauch, A., Keller, M., Wai, D., Brinkschmidt, C., van Valen, F., Boecker, W., Dockhorn-Dworniczak, B., and Poremba, C. Alternative lengthening of telomeres is associated with chromosomal instability in osteosarcomas. Oncogene, 20: 3835-3844, 2001.

24. Francis, P., Namlos, H. M., Muller, C., Eden, P., Fernebro, J., Berner, J. M., Bjerkehagen, B., Akerman, M., Bendahl, P. O., Isinger, A., Rydholm, A., Myklebost, O., and Nilbert, M. Diagnostic and prognostic gene expression signatures in 177 soft tissue sarcomas: hypoxia-induced transcription profile signifies metastatic potential. BMC Genomics, 8: 73, 2007.

25. Nilbert, M., Meza-Zepeda, L. A., Francis, P., Berner, J. M., Namlos, H. M., Fernebro, J., and Myklebost, O. Lessons from genetic profiling in soft tissue sarcomas. Acta Orthop Scand Suppl, 75: 35-50, 2004.

26. Matushansky, I., Hernando, E., Socci, N. D., Matos, T., Mills, J., Edgar, M. A., Schwartz, G. K., Singer, S., Cordon-Cardo, C., and Maki, R. G. A developmental model of sarcomagenesis defines a differentiation-based classification for liposarcomas. Am J Pathol, 172: 1069-1080, 2008..

Grant Funding

The Liddy Shriver Sarcoma Initiative funded this $50,000 grant in October 2008. The study was made possible, in part, by a generous gift from Dr. Laura Somerville.

This grant was dedicated to Rose Burt, a courageous, inspirational, and tireless advocate for sarcoma. She was a wonderfully warm and compassionate person who impacted the lives of hundreds of sarcoma patients, caregivers, and survivors. In the midst of her own 21-year battle with liposarcoma, Rose spent countless hours providing information, referrals, resources and a listening ear as the list manager for some of the online sarcoma support lists of the Association of Cancer Online Resources (ACOR). She was often one of the first persons the newly diagnosed patients would meet in their online quest for answers and hope.

Rose was honored with the 2007 Leadership in Courage Award by the Sarcoma Foundation of America for her support of others living with sarcoma. She was a member of the Sarcoma Advocacy Advisory Committee of the Liddy Shriver Sarcoma Initiative and offered many useful comments and creative suggestions about the information we provide and the projects that we undertake.

Rose and her husband Bill participated in the 2007 Team Sarcoma Bike tour in Vermont. Although we had been communicating with Rose for over three years before then, we had not met her face-to-face. It was a blessing for us to have spent this week with her and Bill.

Rose died Monday, Sept. 15, 2008, at age 71 at Trinity Pathway Hospice in Bettendorf, Iowa, after her courageous battle with sarcoma. She was a very special person and is missed by all those who knew her. Rose, we are all in your debt.

Peace,

Bruce and Bev Shriver
Co-founders, Liddy Shriver Sarcoma Initiative