Connective Tissue Oncology Society

11th Annual MeetingBoca Raton, November 19-21 2005

Expression and clinical relevance of telomere manteinance mechanism (TMM)

in liposarcoma

Alessandro Gronchialessandro.gronchi@istitutotumori.mi.it

Functional telomeres are essential for maintainingthe stability and integrity of chromosomes bypreventing 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

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

Telomerase activity is generally absent in normal somatic cells.

Telomerase is reactivated in 80 - 90% of human cancers of different histotypes.

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.

ALT MECHANISMS

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

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

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.

Nuclei Telomeres PML Merge

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

Measurement of telomere length by PFGE

ALT + ALT -

- - -- + + + +Telomerase activity:

48,5 Kb38,5

23,1 19,4

17 15 9,3

6,4

4,3

TMM characterization

139 liposarcomalesions

103 recurrent lesions 17 metastases19 primary lesions

TMMStability study

67 recurrent patients 7 metastatic patients19 patients at

1st presentation

93 patients with follow-up data

1 patient with primary and metastatic lesions

4 patients with recurrent and

metastatic lesions

3 patients with primary and recurrent lesions

Follow-up study

21 patients with different recurrent lesions

3 patients with different metastatic lesions

Recruitment: 93 patients

Age, years median (range): 53 (23-91)

Gender: 39 Females54 Males

Primary site: Abd/retrop 35Extremity 58

Treatment: Surgery + CT

Median DSS: 120 mos (95%CL:104-172)

Unfavorable events: 41

Case SeriesCase Series

Frequency distribution of TMMs in human liposarcomas

_____________________________________________________

N. of ALT+/TA- ALT-/TA+ ALT+/TA+ Absentlesions

_____________________________________________________

143* 34 (23.8%) 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

____________________________________________________

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%)

_____________________________________________________

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

Telomere Maintenance Mechanisms : Telomere Maintenance Mechanisms :

Clinical Outcome in Patients with LiposarcomaClinical Outcome in Patients with Liposarcoma

0 24 48 72 96 1200

25

50

75

100

RR=2.1 (95%CL:1.1-4.1) p = 0.02

TMM-

TMM+

Dis

ease

-Sp

ecif

ic S

urv

ival

, %

Months

0 24 48 72 96 1200

25

50

75

100

Survival (months)Survival (months)

Pro

bab

ilit

y, %

Pro

bab

ilit

y, % TMM-TMM-

TA+TA+

ALT+ALT+

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 prognosis with different pattern

• 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

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