RECURRENT CHROMOSOMAL COPY NUMBER ALTERATIONS IN CHORDOMA

G. Petur Nielsen; John Iafrate; Zhenfeng Duan; Ramnik Xavier; Joseph Schwab; Andrew Rosenberg; Julie Miller; Edwin Choy; David Harmon; Thomas DeLaney; Vajiya Ramesh; Vikram Deshpande; Francis Hornicek

MGH Chordoma Research GroupDF/Harvard Cancer Center

DefinitionMalignant tumor with a phenotype that recapitulates the notochord and usually arises within the bones of the axial skeleton.

• Incidence – 0.5-1 per million– 300 cases/yr in US

• Gender – M:F 1:1 in skull base and 2:1 in spine and sacrum

• Age – mean 5th -6th decade

Primary Malignant Bone Tumor

Osteosarcoma

Chondrosarcoma

Ewing's sarcoma

ChordomaFibrosarcoma

Symptoms – depend on locationSkull – diplopia, headaches,

cranial nerve palsiesSpine – pain, nerve compressionSacrum – pain,

constipation, incontinence, bladder dysfunction

Treatment

• Surgical resection - primary• Radiation therapy - adjuvant• Local recurrence - problem

Prognosis

• Median survival – 6.29 years• 5 yr survival-68% • 10 yr survival-40%

– Alive with local recurrence

Survival

What is known?

• Little to no data concerning the presence of somatic mutations in chordoma

• Handful of studies have looked at karyotype or Comparative Genomic Hybridization (CGH)

• No correlations with anatomic site or outcome

Clinical Characteristics of 21 Patients with Chordoma

Feature Detailed feature Patients

Gender Male, n (%) 15 (71)

Female, n (%) 6 (29)

Age Median age, yr (range) 63 (46-83)

Location of the tumor Skull base, n (%) 2 (10)

Sacrum, n (%) 12 (57)

Mobile spine, n (%) 7 (33)

Results

Average time to death: 8.2 yearsLocal Recurrence: 14 patients

Average patient follow-up: 6.1 years

Red= loss in patient.

Yellow shadingYellow shading= normal.

Green= gain in patient

Reference DNAChordoma DNA

Array-CGH detection of chromosomal abnormalities

Agilent 244,000 spot oligonucleotide DNA arrayFrozen tissue from the 21 chordomas: H&E slides reviewedGenomic DNA extracted using Gentra Puregene isolation kit1 microgram of tumor DNA labeled with CY51 microgram of normal DNA labeled with CY32 day array competitive hybridizationChromosome plots are log2 ratios of normalized CY5:CY3 signals

1 3

Frequent Chromosomal Aberrations by Array CGH

Chromosomal aberration

Overall (n=21)

Skull base

(n=2)

Mobile spine (n=7)

Sacrum

(n=12)

Loss of 1p 21 2 7 12

Loss of 3p 20 2 6 11

Loss of 4 11 1 3 7

Loss of 5q 4 0 1 3

Gain of 7 6 1 2 3

Loss of 9p 18 2 5 11

Loss of 10q 19 2 6 11

Loss of 11 7 0 4 3

Loss of 13q 13 2 4 7

Loss of 14q 15 2 3 10

Loss of 18q 10 2 3 5

Gain of 19 7 1 2 4

Loss of 22q 10 1 4 5

Chromosomal Aberrations of Chordomas in Comparison to Chondrosarcoma

Chromosomal aberration

% Chordomas (n=21)

% Chondrosarcoma(Rozeman et al., 2006)

p-values(Fisher’s exact test;

2-tailed)

Loss of 1p 21/21 5/18 <0.001

Loss of 3p 20/21 2/18 <0.001

Loss of 4 11/21 4/18 0.055

Loss of 5q 4/21 4/18 1.000

Gain of 7 6/21 5/18 1.000

Loss of 9p 18/21 7/18 0.0027

Loss of 10q 19/21 5/18 0.0003

Loss of 11 7/21 2/18 0.135

Loss of 13q 13/21 5/18 0.031

Loss of 14q 15/21 5/18 0.010

Loss of 18q 10/21 2/18 0.016

Gain of 19 7/21 6/18 1.000

Loss of 22q 10/21 2/18 0.016

VHLp16

PTEN

RBSMAD4

NF2

1 2 3 4 5 6 7 8 9 10 11 12

Cen

13 14 15 16 17 18 19 20 21 22 TP73

RUNX3

CDKN2C

1

Runx3

Actin

Oste

obla

stG

B60Ch8

UCH

1 Chordoma tissue

3

Loss 3p(only one orange signal

Ch8

9

Hallor et al. 2008, British Journal of Cancer

CDKN2A deletion commonly observed in Chordoma

p16 immunostaining

1 2 3 5 6 7 8 9 10

Cen

A B 10

PTEN

10

PTEN

mTOR Signaling Pathway

mTORPROTEIN

SYNTHESIS &

CELL GROWTH

S6P

S6KP

4EBP1P

Hamartin

Tuberin

Low Energy

Ras

MEK1/2

ERK1/2

RSK1

LKB1

AMPK

Rheb

PI3K

Akt

Growth FactorStimulation

P

P

PTEN

rapamycin

NF1

PP

P

EGFR (55.0)

Oncogenes on Chromosome 7 (7/22)

BRAF (140.0)

HGF (81.0)

SMO (128.0)

SHH (155.0)

MET(116.0)

Chordomas do not possess common cancer gene mutations

HDAC4

Actin

Osteoblas

tGB60

CH8

UCH1

Met

miRNA-1

•miRNA-1 targets: •FoxP1 •MET •HDAC4

•Decreased expression of miRNA-1 in chordoma

N1N2

T2

T1

miRNA-1 miRNA-RNU48

N1N2T1T2

miRNA-1

Rel

ativ

e E

xpre

ssio

n

28S

18S

miRNA-1

N1

N2

T1 T2 TC1

TC2

A B

C D

Real-time RT-PCR and Northern blot detection of mature miRNAs in chordoma

A

bso

rba

nce

(ce

ll g

row

th)

A

bso

rba

nce

(ce

ll g

row

th)

Concentration of pre-miRNA-1 (nmol)

miRNA-1

miRNA-control

Hours after pre-miRNA transfection

A B

C DMet

Actin

miR

NA

-1

miR

NA

-co

ntro

l

UC

H1

Transfection of miRNA-1 into chordoma cells UCH1suppresses Met expression and inhibits cell growth

Expression of hepatocyte growth factor (HGF) and c-METIn skull based chordoma

Naka, Kuester, Boltze, Scheil-Bertram, Samii,Herold, Ostertag, Krueger, RoessnerCancer 2008; 112: 104-110.

Summary• Chordomas have recurrent and remarkably

stereotypic chromosomal profile• 1p loss is observed in 100% of tumors• 9p (CDKN2A; p16) or chromosome 9 loss was seen

tumors (77%) • Common losses also include 3p, 4, 10, 13, 14, 18,

22• Gains include 7, 19• These alterations point to genes for follow-up

studies – therapies?

Thank youThank you