1. Liposarcoma Classification
WHO 2013
Molecular Feature
Author, Presenter
ALT
Mussi et al, Quagliolo
Well Diff
adipocytic
sclerosing
inflammatory
amplification 12q13~15 (MDM2+, CDK4+/-)
Crago et al
Spindle cell
M2M2-
De Diff
amplification 12q13~15 (MDM2, CDK4)
Keung et al
Bill et al
Zhang et al
Myxoid
Round cell
translocation t(12;16)(q13p11)
DDIT3 & FUS (EWSR1)
Hoffman et al, Lazar
Di Giadomenico et al
Pleomorphic
Complex karyotype
Torres et al

2. SURGICAL CONSIDERATION FROM THE ANALYSIS OF TWO LARGE SERIES OF ATYPICAL LIPOMATOUS TUMORS (ALT) OF EXTREMITIES AND SUPERFICIAL TRUNK Mussi et al.
– 2010 ?
Marginal surgery, n=95
IRCCS Istituto Clinico Humanitas
Simple resection/shelling out, n=76
Istituto Ortopedico Gaetano Pinii
LR in 5/95 (5%)
LR in 11/76 (14%)
No difference in postoperative complication rate (9%)

3. Tumor and patients characteristics
Total Intitution Institution 2 * * *

4. Results: prognostic factors for LDFS
Univariate Multivariable

5. Conclusions and Implications: ALT
sclerosing subtype unfavorable for LR
high rate residual disease after UPS (46%)
conservative resection appropriate
piecemeal resection inappropriate
no dedifferentiation, no distant metastases, no death in 171 patients
preop recognition
should ALT be considered a “sarcoma”?

6. Metastasis = 13 %; median time = 34 mos
Localized and Metastatic Myxoid/Round Cell Liposarcoma: Clinical and Molecular Observations
Hoffman et. al., pr: Lazar
n = 207 localized ,
median follow-up = 68 mos
LR in 7.4 %, median time = 31 mos
Risk: prior LR, non-extremity disease
Metastasis = 13 %; median time = 34 mos
Risk: male gender (MV)
DSS 1 yr: 99 % yr: 93 % yr: 87%
poor outcome (MV): age>45, male, LR disease
MLS comprises approximately a 1/3 of LPS and 10% of all adult STS, and is therefore one of the most common STS
It’s a disease of 40 and 50 YO
Appears to be gender predilection
It frequently presents as a slow-growing, deep seated tumor which predominantly arises in the lower extremities and the buttock, although it can appear almost anywhere in the body.
It is not uncommon for patients to have tumors for months and even years before they seek medical attention due to the slow-growing nature of the disease

7. Conclusions and Implications
Round cell phenotype associated with distinct molecular signature vs. myxoid:
CXCR4, PDGFRB, P53, adipophilin, PDGFRA, VEGF: RC > myxoid (TMA n=110 patients)
Metastatic disease associated with molecular signature vs. localized:
CXCR4 & PDGFRB: mets > primary
Alteration in molecular features with progression
Role for rebiopsy upon progression
AXL a predictor of DSS

8. Mode of Action of Trabectedin in Myxoid LPS
Di Giandomenico et. al., D’Incalci Lab, Mario Negri Institute
Poster Y
In vivo ML xenografts with postgrafting histology and molecular features
Ctr
Treated
(ML004) Type III FUS-CHOP translocation
(ML015) Type II FUS-CHOP translocation
Trabectedin caused detachment of the FUS-CHOP from target 24h
FUS-CHOP rebinds to target promoters 15 days post-treatment

9. Different FUS-CHOP Detachment Kinetics Between Type II and III Tumors
Type II (ML015)
Type III (ML004)
Different FUS-CHOP Detachment Kinetics Between Type II and III Tumors
FUS-CHOP Type III was rebound to the promoters already 72 hours after the first dose.

10. Conclusions and Implications Trabectedin in MLS
correlation between molecular response and biological activity
effect on transcriptional targets as a read out predicting clinical response
role for scripted re-biopsy during treatment

11. Sarcoma Research Laboratory
University of Texas MD Anderson Cancer Center
ROLE OF AXL AS A POTENTIAL THERAPEUTIC TARGET FOR PLEOMORPHIC LPS AND DDLPS Torres et. al Poster AA
Objective: evaluate the role of the RTK AXL signaling pathway in PLS (n=56)and DDLPS (n=60)
AXL highly expressed in PLS and DDLPS tumor samples and cell lines (TMA, qRT-PCR, WB, P-blot)
knockdown of AXL via siRNA or shRNA inhibited growth and suppressed migration/invasion in vitro
knock down in the pleomorphic cell line PLS-1 impaired tumorigenicity in vivo

12. Conclusions and Implications
Goal: small molecule inhibitor of AXL
Further dissection of signaling pathways/networks
Validation as a therapeutic target

13. primary RP DDLPS Rx’d at DF 1998 and 2008 (n=119)
Keung et. al.
Clinicopathologic Characteristics and Predictors of Outcomes in Patients with Primary Retroperitoneal Dedifferentiated Liposarcoma Undergoing Surgery
DDLPS (vs. WDLPS): Higher LR, DR, worse survival
primary RP DDLPS Rx’d at DF 1998 and 2008 (n=119)
median fup time 74 mos.
All Patients
Months (95% CI)
R0/R1 resection
R2 resection n=13
Median PFS
21.1 ( )
22.1 ( )
6.6 ( )
Median LRFS
21.5 ( )
23.9 ( )
5.7 ( )
Median DRFS
45.8 ( )
51.1 ( )
17.4 ( )
Median OS
59.0 ( )
63.2 ( )
17.8 ( )
I would to thank Drs Swallow and Chibon and CTOS for the opportunity to present our data on prognostic factors in primary retroperitoneal dedifferentiated liposarcoma

14. High Local Recurrence Rate
Retroperitoneal Dedifferentiated Liposarcoma
High Local Recurrence Rate
Variable
n (%)
Number of recurrences 1
43 (45) 2
24 (25) 3
9 (10) 4
2 (2)
Unknown
17 (18)
Site of recurrence or progression
Retroperitoneal
92 (92)
Intraperitoneal
26 (26)
Lung
14 (14)
Liver
6 (6)
Bone
Skin/subcutaneous
3 (3)
100/119 patients (84%) had disease recurrence or progression
92/100 (92%) were local recurrences
R0/R1 resection – 82% had disease recurrence
Among patients who underwent R0/R1 resection, 82% had disease recurrence. Among those who underwent R2 resection, 77% had disease progression.

15. Multivariable Cox Regression Analysis
Retroperitoneal Dedifferentiated Liposarcoma
Predictors of LRFS
Univariate Analysis
Variable
Median (months)
95% CI
P value
Location of Initial Surgery
0.006
OSH, n=82
15.3
BWH, n=37
27.9
Multifocality
0.003
Single tumor, n=74
24.5
Multifocal disease, n=21
11.4
Extent of Resection
0.018
R0/R1, n=80
23.9
R2, n=11
5.7
Multivariable Cox Regression Analysis HR
95% CI
P value
0.61
0.045
1.75
0.047
On univariate analysis, location of initial surgery, multifocality, and extent of resection were associated with LRFS.
On multivariable analysis, initial surgery at BWH was associated with improved LRFS while multifocal disease was associated with worse LRFS.

16. Multivariable Cox Regression Analysis
Retroperitoneal Dedifferentiated Liposarcoma
Predictors of OS
Univariate Analysis
Variable
Median (months)
95% CI
P value
Gender
0.010
Female
77.8
Male
49.1
FNCLCC grade
0.019
Intermediate
59.3
High
38.9
Extent of Resection
<0.001
R0/R1
63.2 R2
17.8
Tumor Integrity
Intact tumor specimen(s)
64.2
Fragmented tumor specimen(s)
21.0
Multivariable Cox Regression Analysis HR
95% CI
P value
1.83
0.037
4.00
0.006
2.20
0.045
On univariate analysis, male gender, high FNCLCC tumor grade, R2 resection, and tumor fragmentation during resection were associated with worse overall survival.
On multivariable analysis, high FNCLCC grade, R2 resection, and tumor fragmentation during resection remained independent predictors of worse overall survival.

17. Conclusion and Implications
high risk histo- and anatomic- subtype
microscopic margins usually positive (~90%)
1/4 of patients from outside institutions had surgery by urologists or gynecologists
*Neither chemotherapy nor radiation therapy at initial presentation was associated with PFS, LRFS, DRFS, or OS
Treatment at Initial Presentation (n=119) n %
Surgery
119
100
Surgery alone
103 88
+ Neoadjuvant chemotherapy + radiation therapy 1
+ Neoadjuvant chemotherapy 4 3
+ Neoadjuvant radiation therapy 11 9
Postoperative chemotherapy 10 8
Intraoperative radiation therapy 7 6
Postoperative radiation therapy 14 12
In conclusion, we found that primary RP DDLPS are large tumors with median size of 20.5cm and usually require multi-organ resection. Resections were often macroscopically complete but margins are rarely negative.

18. MDM2 amp, copy #=mRNA, =protein CDK4 amp variable P53 wt
Sarcoma Research Laboratory
University of Texas MD Anderson Cancer Center
UNRAVELING MOLECULAR DEREGULATION IN DDLPS CELLS LINES Bill et. al Poster Z
Objective: establish and genetically/epigenetically characterize a panel of human LPS cell lines
10 DD-LPS cell lines in long term passage (FISH, qPCR, SNP array, WB, expression array, RPPA)
Recurrent gains 12q13-15
MDM2 amp, copy #=mRNA, =protein
CDK4 amp variable
P53 wt

20. Conclusions and Implications
Recapitulation of clinically relevant molecular alterations
Modeling disease progression and therapeutic response
Systems biology approach

21. CDK4 and MDM2 cooperate to inhibit senescence and promote liposarcomagenesis Crago et. al.
Well differentiated liposarcoma growth
Loss chromosome 11q
and others
? Chromosome 12 gene amplification
Dedifferentiated liposarcoma
Well-differentiated liposarcoma with
SENESCENCE
CDK4
MDM2 ?
> 90% WDLS and DDLS show amplification of 12q13-15 genes MDM2 and CDK4
WD/DDLS without CDK4 amplification have improved prognosis

22. LS cells with no CDK4 amplification undergo senescence and
Chromosome 12q and senescence
LS cells with no CDK4 amplification undergo senescence and
have wild type levels of MDM2 despite MDM2 amplification A. B.
Pre-adipocyte
LS6960-1F
LS8817
LS0082
LS8107
LS7785
LS7302
LS0557
LS6736
LS0557
MDM2amp
CDK4amp
No CDK4
amplification
MDM2
LS6736
Same phenomenon observed in cell lines without CDK4 amplification
- However, also noted that in cell lines without CDK4 amplification, MDM2 levels were not clearly upregulated even in the context of MDM2 amplification
- Does CDK4 and/or MDM2 modulate senescence in LS
CDK4
CDK4
amplified
actin
LS8817

23. Chromosome 12q and senescence
CDK4/6 inhibitor PD (PD) inhibits growth in LS cell lines with CDK4 amplification C. A.
no drug
48h CDK4i
days
LS8817
LS141
LS0082
LS8107
LS7785-1 B. LS
Instead inhibited CDK4 activity in cell lines with both MDM2 and CDK4 amplification (using a drug currently in clinical trials)
See inhibition of cell proliferation as assayed by:
DNA content
FACS analysis
BrdU incorporation
Observed growth arrest in six cell lines tested; in all observe a decreased in Rb phosphorylation confirming drug activity.
No evidence that this arrest was associated with apoptosis or with cell differentiation.
LS8817
G1: 57%
S: 28%
G2: 14%
G1: 86%
S: %
G2: 7%
* No evidence of apoptosis by annexin staining or differentiation of cells by RT-PCR for markers of adipogenesis
untreated
CDK4i x48h (PD)

24. CDK4 inhibitor induces senescence in a subset of LS cell lines
Chromosome 12q and senescence
CDK4 inhibitor induces senescence in a
subset of LS cell lines B.
Responders
Non-Responders A.
LS LS LS LS
CDK4i tx
p16
p21
p53
MDM2
↓0.43x ↓0.47x
We did observe senescence in 3/6 cell lines as asssayed by beta-galactosidase and senescence-associated heterochromatic foci.
Cells are termed responders (vs. non-responders which undergo growth arrest without senescence after inhibition of CDK4 activity
SAHF is seen here in a representative responder cell line
Examined the effect of CDK4 inhibition on classic mediators of senescence – no increase in p16, p21, or p53
Did see consistent downregulation of MDM2 in responder cell lines after drug treatment, but not in non-responders
Responders
Non-Responders
Identical results with CDK4 shRNA
MDM2 knockdown induces senescence in LS cells
even in lines not responsive to PD

25. Conclusions and Implications
MDM2 as a final common target
Induction of senescence by PD, by knockdown of MDM2:
no induction of p53; doesn’t require p53
pathway as a therapeutic target
SENESCENCE
CDK4
MDM2

26. Other Biological Functions
Potent Inhibition of Human Liposarcoma Growth and Survival by a Novel Modulator of MDM2-p53 Interaction
Yi-Xiang Zhang, PhD
MDM2
p53
Induction of p21
Cell Cycle Arrest
Induction of
BAX, PUMA, NOXA
Cell Death
Antagonists
Other Biological Functions
MDM2/p53 Feed Back Loop
SAR is a Potent Inducer of p53 Activity
CTOS 17th Annual Meeting

27. SAR299155 Restores p53 Activity in vivo
SAR Decreases Cell Viability in Liposarcoma Cells with Wild-type p53 A B
LP6 (p53 wt)
LP6 (p53 mut) C
LP6 (+p53 siRNA) D
SAR Restores p53 Activity in vivo

28. Complete Regression of Primary Liposarcoma Tumor Xenograft LPS3 Treated with SAR299155
*, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001; compared with respective control group treated with vehicle.

29. Conclusions and Implications
SAR299155: 5-fold more potent than Nutlin-3
correlation between biochemical assays and biological activity
reproducibility with other LPS cell lines as xenograft, patient tumor-derived xenografts
identifying new biomarkers of response in DD-LPS

30. Liposarcoma – Revealing Molecular Correlates of Tumor Progression1 2 3
ALT/WD LPS
De-Diff LPS
De-Diff LPS
Myxoid LPS is considered a intermediate grade malignancy, somewhere between WDLPS to DDLPS,pleomorphic and RC. This is evident by its level of differentiation
Round Cell LPS
Myxoid LPS
Pleomorphic LPS
A. Lazar 30

31. Liposarcoma Themes
Requirement for accurate clinical, histological and molecular data
Inter- and intra- subtype heterogeneity
Temporal heterogeneity
Stratification by molecular properties
For prognostication
For targeted therapy
For clinical trials of other modalities
Splitters > Lumpers
Numbers game: Collaborative Imperative!!!