Connective Tissue Oncology Society 11th Annual Meeting Boca Raton, November 19-21 2005

1. Connective Tissue Oncology Society 11th Annual Meeting Boca Raton, November 19-21 2005 Expression and clinical relevance of telomere manteinance mechanism (TMM) in liposarcoma Alessandro Gronchi alessandro.gronchi@istitutotumori.mi.it

2. Functional telomeres are essential for maintaining the stability and integrity of chromosomes by preventing degradation and end-to-end fusion. Telomeres of human somatic cells shorten with each round of cell division because of the incomplete replication of linear DNA. The sequential loss of telomeric DNA limits the proliferative lifespan of cells to a definite number of cell divisions before they enter a state of replicative senescence.

3. Telomerase Alternative lengthening of telomere (ALT) mechanisms

4. HUMAN TELOMERASE Telomerase is a ribonucleoprotein complex that maintains and elongates telomeres by the de novo synthesis of telomeric repeats (TTAGGG). Telomerase components: hTERT the catalytic reverse transcriptase subunit hTR the template-containing RNA subunit

5. Telomerase activity is generally absent in normal somatic cells. Telomerase is reactivated in 80 - 90% of human cancers of different histotypes.

6. ALT MECHANISMS Telomere dynamics in ALT cells are consistent with a recombination-based mechanism Characteristics of ALT cells include unusually long and heterogeneous telomeres and subnuclear structures calleded ALT-associated promyelocytic leukemia (PML) bodies (APBs) that contain telomeric DNA, telomere-specific binding proteins TRF1 and TRF2, and proteins involved in DNA recombination and replication.

7. Background Based on limited information available thus far, it appears that ALT is more frequently present in cell lines and tumors of mesenchymal origin than in those of epithelial origin. Although telomerase and ALT are both able to support immortalization, they may confer different properties to tumor cells in vivo, thus making it important to investigate the prognostic implications of telomere maintenance mechanisms in clinical tumors. The only clinical studies available thus far indicate that i) patients with ALT+ high grade glioblastoma have significant longer survival than those that are ALT- (Hakin-Smith et al., 2003: Henson et al., 2005); ii) patients with ALT+ or telomerase+ osteosarcomas have a similar prognosis (Ulaner et al., 2003).

8. Study Aims • To determine the prevalence of TMM in human liposarcoma • To assess whether TMM is associated with recurrent or metastatic phenotype • To correlate TMM with clinical outcome

9. Methods • The presence of telomere maintenance mechanisms (TMMs) in human liposarcoma specimens was assessed by: • TRAP assay • immunofluorescence/FISH for APB detection • PFGE for telomere length measurement Telomere repeat amplification protocol (TRAP) It measures the telomerase activity as the ability of the enzyme to add telomeric repeats to a synthetic telomerase substrate. The products of telomerase activity are amplified by PCR and resolved on a polyacrylamide gel. A combined immunofluoresce (with a anti-PML antibody)/ FISH (fluorescence in situ hybridization, with a telomeric probe) is used for APB detection. APB are present where there is a colocalization of both signals (PML and telomere) Pulse-field gel electrophoresis (PFGE) is used to resolve and detect telomere fragments on agarose gel by the use of a labeled telomeric probe.

10. Detection of ALT-associated PML-bodies (APBs) by combined PML immunofluorescence /telomere FISH Nuclei Telomeres PML Merge

11. Telomerase activity: ALT + ALT - 48,5 Kb 38,5 - - - - + + + + 23,1 19,4 17 15 9,3 6,4 4,3 Measurement of telomere length by PFGE

12. 139 liposarcoma lesions TMM characterization 19 primary lesions 103 recurrent lesions 17 metastases 21 patients with different recurrent lesions 3 patients with different metastatic lesions TMM Stability study 3 patients with primary and recurrent lesions 4 patients with recurrent and metastatic lesions 1 patient with primary and metastatic lesions 19 patients at 1st presentation 67 recurrent patients 7 metastatic patients Follow-up study 93 patients with follow-up data

13. Case Series Recruitment: 93 patients Age, years median (range): 53 (23-91) Gender: 39 Females 54 Males Primary site: Abd/retrop 35 Extremity 58 Treatment: Surgery + CT Median DSS: 120 mos (95%CL:104-172) Unfavorable events: 41

14. Frequency distribution of TMMs in human liposarcomas _____________________________________________________ N. of ALT+/TA- ALT-/TA+ ALT+/TA+ Absent lesions _____________________________________________________ 143*34 (23.8%) 34 (23.8%) 3 (2%) 72 (50.4%) _____________________________________________________ *obtained from 97 patients

15. Frequency distribution of TMMs as a function of the type of tumor lesion ____________________________________________________ N. of ALT+/TA- ALT-/TA+ ALT+/TA+ Absent lesions _____________________________________________________ Primary 21 4 (19%) 4 (19%) 1 (4.8%) 12 (57.2%) Recurrence 105 27 (25.7%) 20 (19%) 2 (1.9%) 56 (53.3%) Metastasis 17 3 (17.6%) 10 (58.8%) _____ 4 (23.5%) _____________________________________________________

16. Frequency distribution of TMMs as a function of tumor histology _________________________________________________________________________ N. of ALT+/TA- ALT-/TA+ ALT+/TA+ Absent cases _________________________________________________________ Well-differentiated 39 6 (15.4%) 1 (2.6%) ____ 32 (82%) Dedifferentiated 36 10 (28%) 7 (19.4%) 1 (2.6%) 18 (50%) Mixoid 23 3 (13%) 9 (39.1%) ____ 11 (47.9%) Mixoid – round cells 27 5 (18.5%) 18 (66.7%) 2 (7.4%) 2 (7.4%) _________________________________________________________ Fisher exact test: P < 0.0001

17. Telomere Maintenance Mechanisms : Clinical Outcome in Patients with Liposarcoma TMM- TMM+ Disease-Specific Survival, % RR=2.1 (95%CL:1.1-4.1) p = 0.02 Months

18. TMM- Probability, % TA+ ALT+ Survival (months)

19. Summary • Results from our study indicate that ALT and TA: • are present in one half of human liposarcomas examined • they can be the sole mechanism of telomere maintenance also in tumor recurrences and metastases • they are related to histological tumor progression • are associated to adverse prognosiswith different pattern

20. ALT mechanism more strongly correlates with worse prognosis than TA even by multivariable analysis. • However, this result should be interpreted by considering TMM distribution within the two liposarcoma subgroups, also characterized by different clinical histories: • ALT mechanism, principally expressed in WD/DD liposarcomas with a prevalence in DD subgroup, contributed to accurately segregate among the aggressive liposarcomas those with a worse prognosis • The prevalence of TA mechanism in mixoid liposarcomas, with a peak of 70% in the RCM subgroup, made it less specific in identifying, among putatively high-risk patients, those who will die. This observation is also coherent with the clinical outcome of RCM liposarcomas, characterized by a homogeneous dismal prognosis with metastasis-related death.

21. ACKNOWLEDGEMENTS ISTITUTO NAZIONALE TUMORI - Milan Dept. Experimental Oncology Aurora Costa Maria Grazia Daidone Raffaella Villa Laura Daprai Rosita Motta Roberta Erdas Dept. Surgical Oncology Alessandro Gronchi Dept. Pathology Silvana Pilotti CHILDREN’S MEDICAL RESEARCH INSTITUTE – Sydney Roger R. Reddel Jeremy H. Henson