Connective Tissue Oncology Society 11th Annual Meeting Boca Raton, November 19-21 2005 Expression...
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Transcript of Connective Tissue Oncology Society 11th Annual Meeting Boca Raton, November 19-21 2005 Expression...
Connective Tissue Oncology Society
11th Annual MeetingBoca Raton, November 19-21 2005
Expression and clinical relevance of telomere manteinance mechanism (TMM)
in liposarcoma
Alessandro [email protected]
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