Liposarcoma ClassificationWHO 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 alBill et alCrago et alZhang et al

Myxoid Round cell

translocation t(12;16)(q13p11) DDIT3 & FUS (EWSR1)

Hoffman et al, LazarDi Giadomenico et al

Pleomorphic Complex karyotype Torres et al

??

SURGICAL CONSIDERATION FROM THE ANALYSIS OF TWO LARGE SERIES OF ATYPICAL LIPOMATOUS TUMORS (ALT) OF EXTREMITIES AND SUPERFICIAL TRUNK Mussi et al.

Marginal surgery, n=95IRCCS Istituto Clinico Humanitas

Simple resection/shelling out, n=76 Istituto Ortopedico Gaetano Pinii

1998 – 2010

LR in 5/95 (5%) LR in 11/76 (14%)

No difference in postoperative complication rate (9%)

Tumor and patients characteristics

 Total Intitution 1 Institution 2

**

*

Results: prognostic factors for LDFS

Univariate Multivariable

• 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”?

Conclusions and Implications: ALT

Localized and Metastatic Myxoid/Round Cell Liposarcoma: Clinical and Molecular Observations

Hoffman et. al., pr: Lazar

• n = 207 localized , 1990-2010 • 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 % 5 yr: 93 % 10 yr: 87%• poor outcome (MV): age>45, male, LR disease

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

In vivo ML xenografts with postgrafting histology and molecular features

0

500

1000

1500

2000

2500

3000

95 105 115 125 135 145 155 165 175

Tu

mo

r w

eig

ht

(mg

±

se)

Days from inoculum

Trabectedin

Placebo

Treatment

Ctr Treated

0

200

400

600

800

1000

1200

1400

1600

1800

109 119 129 139 149 159 169

Tum

or w

eigh

t (m

g ±se)

Days from inoculum

Trabectedin

Placebo

Treatment

Ctr Treated(ML004) Type III FUS-CHOP translocation

(ML015) Type II FUS-CHOP translocation

Mode of Action of Trabectedin in Myxoid LPSDi Giandomenico et. al., D’Incalci Lab, Mario Negri Institute

Poster Y

• Trabectedin caused detachment of the FUS-CHOP from target promoters @ 24h• FUS-CHOP rebinds to target promoters 15 days post-treatment

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.

Conclusions and ImplicationsTrabectedin 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

ROLE OF AXL AS A POTENTIAL THERAPEUTIC TARGET FOR PLEOMORPHIC LPS AND DDLPS Torres et. al.

Poster AA

Sarcoma Research LaboratoryUniversity of Texas MD Anderson Cancer Center

• 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

• Goal: small molecule inhibitor of AXL• Further dissection of signaling pathways/networks• Validation as a therapeutic target

Conclusions and Implications

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 PatientsMonths (95% CI)

R0/R1 resectionMonths (95% CI)

R2 resection n=13Months (95% CI)

Median PFS 21.1 (15.0-27.2) 22.1 (16.9-27.3) 6.6 (1.6-9.3)

Median LRFS 21.5 (14.5-28.5) 23.9 (19.5-28.3) 5.7 (3.8-7.6)

Median DRFS 45.8 (29.7-61.8) 51.1 (32.3-59.0) 17.4 (8.4-27.9)

Median OS 59.0 (51.8-66.2) 63.2 (55.8-83.3) 17.8 (10.0-41.5)

High Local Recurrence Rate

Retroperitoneal Dedifferentiated Liposarcoma

100/119 patients (84%) had disease recurrence or progression

92/100 (92%) were local recurrences

R0/R1 resection – 82% had disease recurrence

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 6 (6)Skin/subcutaneous 3 (3)

Retroperitoneal Dedifferentiated Liposarcoma

Predictors of LRFS

Multivariable Cox Regression Analysis

HR 95% CI P value

0.61 0.38-0.99 0.045

1.75 1.01-3.03 0.047

Univariate Analysis

VariableMedian

(months) 95% CI P value

Location of Initial Surgery 0.006

OSH, n=82 15.3 8.31-22.29

BWH, n=37 27.9 11.99-43.81

Multifocality 0.003

Single tumor, n=74 24.5 19.29-29.71

Multifocal disease, n=21 11.4 6.96-15.84

Extent of Resection 0.018

R0/R1, n=80 23.9 19.50-28.30

R2, n=11 5.7 3.83-7.57

Retroperitoneal Dedifferentiated Liposarcoma

Predictors of OS

Multivariable Cox Regression Analysis

HR 95% CI P value

1.83 1.04-3.21 0.037

4.00 1.50-10.67 0.006

2.20 1.02-4.74 0.045

Univariate Analysis

Variable Median (months) 95% CI P value

Gender 0.010

Female 77.8 57.7-111.9

Male 49.1 34.8-60.8

FNCLCC grade 0.019

Intermediate 59.3 49.1-83.3

High 38.9 25.6-60.8

Extent of Resection <0.001

R0/R1 63.2 55.8-83.3

R2 17.8 10.0-41.5

Tumor Integrity <0.001Intact tumor specimen(s) 64.2 55.8-91.7 Fragmented tumor specimen(s) 21.0 9.2-46.7

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 1+ Neoadjuvant chemotherapy 4 3+ Neoadjuvant radiation therapy 11 9

Postoperative chemotherapy 10 8Intraoperative radiation therapy 7 6Postoperative radiation therapy 14 12

UNRAVELING MOLECULAR DEREGULATION IN DDLPS CELLS LINES Bill et. al.

Poster Z

Sarcoma Research LaboratoryUniversity of Texas MD Anderson Cancer Center

• 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

• Recapitulation of clinically relevant molecular alterations

• Modeling disease progression and therapeutic response

• Systems biology approach

Conclusions and Implications

CDK4 and MDM2 cooperate to inhibit senescence and promote liposarcomagenesis

Crago et. al.

Well differentiated liposarcoma

growth

Loss chromosome 11qand 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

Pre-

adip

ocyt

eLS

8817

LS00

82LS

8107

LS77

85LS

7302

LS05

57LS

6736

MDM2

CDK4

actin

MDM2amp - + + + + + + + + CDK4amp - + + + + + + - -

LS69

60-1

F

LS8817

LS0557

CDK4amplified

No CDK4 amplification

LS6736

Chromosome 12q and senescence

LS cells with no CDK4 amplification undergo senescence and have wild type levels of MDM2 despite MDM2 amplification

A. B.

CDK4/6 inhibitor PD0332991 (PD) inhibits growth in LS cell lines with CDK4 amplification

Chromosome 12q and senescence

days

A.

untreated CDK4i x48h (PD)

B.LS8817

G1: 57%S: 28%G2: 14%

G1: 86%S: 5%G2: 7%

LS88

17

C.

LS14

1LS

0082

LS81

07

LS77

85-1

LS77

85-1

0

no drug48h CDK4i

* No evidence of apoptosis by annexin staining or differentiation of cells by RT-PCR for markers of adipogenesis

Identical results with CDK4 shRNA

CDK4 inhibitor induces senescence in a subset of LS cell lines

Chromosome 12q and senescence

Responders Non-Responders

A.

B.Responders Non-Responders

MDM2

p53

p16

p21

LS8817 LS0082 LS8107 LS7785-10 - + - + - + - +CDK4i tx

↓0.43x ↓0.47x

• MDM2 knockdown induces senescence in LS cells• even in lines not responsive to PD

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

Potent Inhibition of Human Liposarcoma Growth and Survival

by a Novel Modulator of MDM2-p53 Interaction

CTOS 17th Annual Meeting 26

Yi-Xiang Zhang, PhD

MDM2

p53p53

Induction of p21 Cell Cycle Cell Cycle

ArrestArrest

Induction of BAX, PUMA,

NOXACell DeathCell Death

MDM2Antagonists

Other Biological Functions

MDM2/p53 Feed Back Loop

SAR299155 is a Potent Inducer of p53 Activity

SAR299155 Decreases Cell Viability in Liposarcoma Cells with Wild-type p53

27

A B

DC

LP6 (p53 wt) LP6 (p53 mut)

LP6 (+p53 siRNA)

SAR299155 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.

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

29

Liposarcoma – Revealing Molecular Correlates of Tumor Progression

1 2 3

Myxoid LPS Round Cell LPS

Pleomorphic LPS

De-Diff LPSALT/WD LPS De-Diff LPS

A. Lazar

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!!!