Connective Tissue Oncology Society 11 th Annual Meeting

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

Connective Tissue Oncology Society 11 th 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

Functional telomeres are essential for maintaining the stability and integrity of chromosomes by preventing

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.

Telomerase Alternative lengthening of telomere (ALT) mechanisms

Telomerase Alternative lengthening of telomere (ALT) mechanisms

HUMAN TELOMERASE Telomerase is a ribonucleoprotein complex that maintains and elongates telomeres by the

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

Telomerase activity is generally absent in normal somatic cells. Telomerase is reactivated in 80

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

ALT MECHANISMS Telomere dynamics in ALT cells are consistent with a recombination-based mechanism Characteristics

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 TRF 1 and TRF 2, and proteins involved in DNA recombination and replication.

Background Based on limited information available thus far, it appears that ALT is more

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).

Study Aims ü To determine the prevalence of TMM in human liposarcoma ü To

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

Methods The presence of telomere maintenance mechanisms (TMMs) in human liposarcoma specimens was assessed

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

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

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

Measurement of telomere length by PFGE Telomerase activity: 48, 5 Kb 38, 5 23,

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

139 liposarcoma lesions 19 primary lesions 103 recurrent lesions 21 patients with different recurrent

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

Case Series Recruitment: 93 patients Age, years median (range): 53 (23 -91) Gender: 39

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

Frequency distribution of TMMs in human liposarcomas ___________________________ N. of lesions ALT+/TA- ALT-/TA+ ALT+/TA+

Frequency distribution of TMMs in human liposarcomas ___________________________ N. of lesions ALT+/TA- ALT-/TA+ ALT+/TA+ Absent ___________________________ 143* 34 (23. 8%) 3 (2%) 72 (50. 4%) ___________________________ * obtained from 97 patients

Frequency distribution of TMMs as a function of the type of tumor lesion __________________________

Frequency distribution of TMMs as a function of the type of tumor lesion __________________________ N. of lesions ALT+/TA- ALT-/TA+ ALT+/TA+ Absent ___________________________ Primary 21 Recurrence 105 Metastasis 17 4 (19%) 1 (4. 8%) 12 (57. 2%) 27 (25. 7%) 20 (19%) 2 (1. 9%) 56 (53. 3%) 10 (58. 8%) _____ 3 (17. 6%) 4 (23. 5%) ___________________________

Frequency distribution of TMMs as a function of tumor histology _____________________________________ N. of cases

Frequency distribution of TMMs as a function of tumor histology _____________________________________ N. of cases ALT+/TA- ALT-/TA+ ALT+/TA+ Absent _____________________________ 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%) _____________________________ Fisher exact test: P < 0. 0001

Telomere Maintenance Mechanisms : Disease-Specific Survival, % Clinical Outcome in Patients with Liposarcoma TMMTMM+

Telomere Maintenance Mechanisms : Disease-Specific Survival, % Clinical Outcome in Patients with Liposarcoma TMMTMM+ RR=2. 1 (95%CL: 1. 1 -4. 1) p = 0. 02 Months

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

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

Summary Results from our study indicate that ALT and TA: ü are present in

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

 • ALT mechanism more strongly correlates with worse prognosis than TA even by

• 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.

ACKNOWLEDGEMENTS ISTITUTO NAZIONALE TUMORI - Milan Dept. Experimental Oncology Aurora Costa Maria Grazia Daidone

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