BIOLOGICAL MARKERS OF PROGNOSIS IN EWING'S SARCOMA: THE ROLE OF GAL3BP

CTOS meeting, Seattle 2007

Katia Scotlandi, Diana Zambelli, Monia Zuntini, and Piero Picci

Istituti Ortopedici RizzoliBologna, Italy

From experimental models to clinics

TC-71

MDR transfectants

Drug resistant cells

4 different variants, characterized by:

MDR phenotype

Loss of malignancy

Cells

Incidence Latency (days) Total number Incidence Latency (days)

TC-71 6/10 41 ± 5 10 14/15 13 ± 6TC/MDR-8 0/10 3/5 12 ± 6

TC/MDR-41 0/10 4/5 6 ± 0TC/ET 12nM 0/10 4/5 20 ± 0

Metastasis to bones Tumorigenicity

Gene expression profile of these cells

COMMON

UP-REGULATED

GENES

Gene Name GenBank Description Mean LogR SAM Score(d)

ABCB1 NM_000927 ATP-binding cassette, sub-family B (MDR/TAP), member 1 4.006 12.954HLA-A NM_002116 major histocompatibility complex, class I, A 1.654 9.299PRDX2 NM_005809 peroxiredoxin 2 1.319 7.690IFITM2 NM_006435 interferon induced transmembrane protein 2 (1-8D) 2.487 7.289IFITM1 NM_003641 interferon induced transmembrane protein 1 (9-27) 2.296 6.612IFITM3 NM_021034 interferon induced transmembrane protein 3 (1-8U) 2.674 6.182RPS4Y1 NM_001008 ribosomal protein S4, Y-linked 1.334 6.152MRCL3 NM_006471 myosin, light polypeptide, regulatory, non-sarcomeric (20kD) 1.269 6.146EIF3S6 NM_001568 eukaryotic translation initiation factor 3, subunit 6 (48kD) 2.273 5.984PIP5K2A NM_005028 phosphatidylinositol-4-phosphate 5-kinase, type II, alpha 1.779 5.978YWHAE NM_006761 tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, epsilon 1.520 5.543KPNA2 NM_002266 karyopherin alpha 2 (RAG cohort 1, importin alpha 1) 1.363 5.019B2M NM_004048 beta-2-microglobulin 1.927 4.996YWHAZ NM_003406 tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide 1.987 4.792RAD21 NM_006265 RAD21 (S. pombe) homolog 1.342 4.713TPT1 NM_003295 tumor protein, translationally-controlled 1 2.018 4.452HINT1 NM_005340 histidine triad nucleotide-binding protein 1.269 4.332LGALS3BP NM_005567 lectin, galactoside-binding, soluble, 3 binding protein (galectin 6 binding protein) 1.935 4.162SDHB NM_003000 succinate dehydrogenase complex, subunit B, iron sulfur (Ip) 1.327 3.809CANX NM_001024649 calnexin 1.698 3.809ATP5A1 NM_001001937 ATP synthase, H+ transporting, mitochondrial F1 complex, alpha subunit, isoform 1, cardiac muscle 1.648 3.791MYC NM_002467 v-myc avian myelocytomatosis viral oncogene homolog 1.482 3.726IL15RA NM_002189 interleukin 15 receptor, alpha 1.283 3.719STOML2 NM_013442 stomatin-like protein 2 1.613 3.634UBE2C NM_007019 ubiquitin carrier protein E2-C 1.358 3.392ITM2A NM_004867 integral membrane protein 2A 1.734 3.332SLC25A5 NM_001152 solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), member 5 1.250 3.322IRS1 NM_005544 insulin receptor substrate 1 1.923 3.313IL27RA NM_004843 class I cytokine receptor 1.454 3.294HSP90B1 NM_003299 tumor rejection antigen (gp96) 1 1.258 3.229HLA-B NM_005514 major histocompatibility complex, class I, C 1.576 3.190FVT1 NM_002035 follicular lymphoma variant translocation 1 1.752 3.185CCNI NM_006835 cyclin I 1.920 3.170RPS15A NM_001019 ribosomal protein S15a 1.723 3.129RPS29 NM_001030001 ribosomal protein S29 1.676 3.000TRO NM_001039705 trophinin 1.411 2.966CALM2 NM_001743 calmodulin 2 (phosphorylase kinase, delta) 1.539 2.939FKBP1A NM_000801 FK506-binding protein 1A (12kD) 1.617 2.837CTGF NM_001901 connective tissue growth factor 1.312 2.713RPL9 NM_000661 ribosomal protein L9 1.611 2.666RPS7 NM_001011 ribosomal protein S7 1.812 2.646CDC42EP1 NM_007061 CDC42 effector protein (Rho GTPase binding) 1 1.279 2.605ATP6V1F NM_004231 ATPase, vacuolar, 14 kD 1.237 2.599SPTBN1 NM_003128 Homo sapiens mRNA for short form of beta II spectrin, partial 1.727 2.580RNP24 NM_006815 coated vesicle membrane protein 1.462 2.567GSTM3 NM_000849 glutathione S-transferase M3 (brain) 1.534 2.545

gene functional Networks operating in the regulation of mets capabilities

Primary lesions

(n = 42)

Metastasis(n = 7)

Recurrences(n = 7)

Sex Male Female

2616

52

34

Age ≤ 14 years > 14 years

1527

25

16

Tumor Site Extremity Pelvis Other

2589

511

5-2

Metastasis at Diagnosis

No Yes

366

--

--

Type of Translocation

EWS/Fli1 Type 1 EWS/Fli1 Type 2

EWS/Fli1 Type 3

Other

231018

5101

5200

Validation by TaqMAN Real Time PCR array

Genes:

CDC42EP1,

CTGF,

FVT1,

IRS1

LGALS3BP

HINT

IFITM2,

STOML2

RPS4Y1

CANX

CTGF

KPNA2

RNP24

Comparison of the mean expression values of primary lesions with recurrences or metastases for each gene did not provide significant results. However remarkable reduction in the recurrences and metastasis groups was observed for some genes

0

.2

.4

.6

.8

1

Ove

rall

Sur

viva

l

0 10 20 30 40 50 60 70

Time (Months)

p = 0.03

H

L

0

.2

.4

.6

.8

1

Eve

nt-F

ree

Sur

viva

l

0 10 20 30 40 50 60 70Time (Months)

p = 0.04

H

L

Prognostic Value of 6 out of 12 genes

IFITM2

EFS OVS

RPS4Y1

0

.2

.4

.6

.8

1

Eve

nt-F

ree

Sur

viva

l

0 10 20 30 40 50 60 70Time (Months)

0

.2

.4

.6

.8

1

Ove

rall

Sur

viva

l0 10 20 30 40 50 60 70

Time (Months)

p = 0.04

H

L

0

.2

.4

.6

.8

1

Ove

rall

Sur

viva

l

0 10 20 30 40 50 60 70

Time (Months)

p = 0.03

H

L

Prognostic Value

HINT1

EFS OVS

0

.2

.4

.6

.8

1

Eve

nt-F

ree

Sur

viva

l

0 10 20 30 40 50 60 70Time (Months)

p = 0.06

H

L

0

.2

.4

.6

.8

1

Eve

nt-F

ree

Sur

viva

l

0 10 20 30 40 50 60 70Time (Months)

p = 0.06

H

L

0

.2

.4

.6

.8

1

Ove

rall

Sur

viva

l

0 10 20 30 40 50 60 70Time (Months)

p = 0.03H

L

STOML2

Prognostic Value

None of these 6 prognostic genes displayed correlation with clinical pathological features. However, GALS3BP status showed a statistically significant,

positive correlation with the fusion type translocation (p= 0.0007, Fisher’s exact test), EWS/Fli-1 type 1 fusion occurring more frequently in LGALS3BP high-

expressing cases (17/20, 85% vs 7/22, 32%)

LGALS3BP

EFS OVS

0

.2

.4

.6

.8

1

Eve

nt-F

ree

Sur

viva

l

0 10 20 30 40 50 60 70Time (Months)

p = 0.008

H

L

0

.2

.4

.6

.8

1

Ove

rall

Sur

viva

l

0 10 20 30 40 50 60 70Time (Months)

p = 0.005H

L

Prognostic Value

As expected, EWS/Fli-1 type 1 expression also showed significant correlation with survival

Prognostic Value:Multivariate Analysis

Event-Free Survival Overall Survival

Evaluation of Protein Expression by Tissue Array Analysis

PRIMARY LESIONS

(N = 60)

Sex Male Female

3327

Age ≤ 14 years > 14 years

2832

Tumor Site Extremity Pelvis Other

331116

Metastasis at Diagnosis

No Yes

519

In collaboration with A.Llombart-Bosch, University of Valencia, Spain

Prognostic Value:LGALS3BP Protein Expression

39 LOW 21 HIGH

(Monia Zuntini1*, Diana Zambelli1*, Filippo Nardi1, Maria Cristina Manara1, Massimo Serra1, Stefano Ferrari2, Patrizia Bacchini3, Antonio

Llombart-Bosch4, Stefano Iacobelli5, Piero Picci1, and Katia Scotlandi1 Biological indicators of prognosis in Ewing’s sarcoma: an emerging role for lectin galactoside-binding soluble 3 binding protein (LGALS3BP) (Submitted to JCO))

0

.2

.4

.6

.8

1

Eve

nt-F

ree

Sur

viva

l

0 10 20 30 40 50 60 70Time (Months)

H

L

p = 0.03

0

.2

.4

.6

.8

1

Ove

rall

Sur

viva

l

0 10 20 30 40 50 60 70Time (Months)

H

L

p = 0.03

OVSEFS

GAL3-BP

LGALS3BP (also known as 90K or Mac-2 binding protein) was originally identified as a tumor-associated antigen in breast cancer

It binds galectins and appears to be have a role in cell-cell adhesion

A dualistic role depending on the cellular context In EWS, high expression is associated with better

prognosis Molecular mechanisms?? Studies are in progress

LGALS3BP/90K in EWS cell lines

59.96

5.832.11

14.9

59.7

-1.3

79.87

29.3432

15.92

-10

0

10

20

30

40

50

60

70

80

90

RDES SKES-1 IOR/BRZ IOR/CAR IOR/RCH IOR/NGR 6647 LAP35 SKNMC TC71

ng/m

l

Protein

4.70

0.80 0.80

8.74

36.56

5.236.90

10.00

6.20

1.48

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

40.0

RDES SKES IOR/BRZ IOR/CAR IOR/RCH IOR/NGR 6647 LAP-35 SKNMC TC71

RQ

mRNA

TC/90K CLONES

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

TC

-71 29

100 3

5

51 4 89 5 84

80

46

97

52

94

81

76

44

14

96

62

50

90K

(cl

on

e/T

C-7

1)

Screening of 90 clones by ELISA

20 CLONES

Future plans

Analyze the presence of 90K in the serum of EWS patients

Growth ability, both in monolayer and soft-agar

Adhesion to different substrates

Migration and invasion processes

Chemo sensitivity to drugs commonly applied in the treatment of EWS patients

Evaluate the biological behavior of TC/90K clones

Thanks to:

EU project EUROBONET

AIRC

Regione Emilia Romagna and Italian Ministry of Health