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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 Group
DF/Harvard Cancer Center
Chordoma
Definition
Malignant tumor with a phenotype
that recapitulates the notochord
and usually arises within the bones
of the axial skeleton.
Chordoma
Primary Malignant Bone Tumor
Osteosarcoma
Chondrosarcoma
Ewing's sarcoma
Chordoma
Fibrosarcoma
• 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
Chordoma
Symptoms – depend on location
Skull – diplopia, headaches,
cranial nerve palsies
Spine – pain, nerve compression
Sacrum – pain,
constipation, incontinence, bladder
dysfunction
Chordoma
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 patient follow-up: 6.1 years
Average time to death: 8.2 years
Local Recurrence: 14 patients
Array-CGH detection of chromosomal abnormalities
Chordoma DNA
Agilent 244,000 spot oligonucleotide DNA
array
Frozen tissue from the 21 chordomas:
H&E slides reviewed
Genomic DNA extracted using Gentra
Puregene isolation kit
1 microgram of tumor DNA labeled with
CY5
1 microgram of normal DNA labeled with
CY3
2 day array competitive hybridization
Chromosome plots are log2 ratios of
normalized CY5:CY3 signals
Reference DNA
Red= loss in patient.
Yellow shading= normal.
Green= gain in patient
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
p-values
(Rozeman et al., 2006)
(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
p16
VHL
PTEN
NF2
RB
SMAD4
1
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22
TP73
RUNX3
CDKN2C
Cen
Chordoma tissue
Runx3
Actin
3
Loss 3p
(only one
orange
signal
Ch8
9
CDKN2A deletion commonly observed in Chordoma
Hallor et al. 2008, British Journal of Cancer
p16 immunostaining
10
A
B
1
2
3
5
6
7
8
9 10
Cen
10
PTEN
PTEN
mTOR Signaling Pathway
Growth Factor
Stimulation
Ras
NF1
PI3K
MEK1/2
PTEN
Akt
Low
Energy
LKB1
ERK1/2
P
P
RSK1
P
P
Tuberin
P
AMPK
Hamartin
P
S6K
Rheb
S6
mTOR
P
4EBP1
rapamycin
P
PROTEIN
SYNTHESIS
&
CELL GROWTH
Chordomas do not possess common cancer gene mutations
Oncogenes on Chromosome 7 (7/22)
EGFR (55.0)
HGF (81.0)
MET(116.0)
SMO (128.0)
BRAF (140.0)
SHH (155.0)
HDAC4
Met
Actin
miRNA-1
•miRNA-1 targets:
•FoxP1
•MET
•HDAC4
•Decreased expression of miRNA-1
in chordoma
Real-time RT-PCR and Northern blot
detection of mature miRNAs in chordoma
A
B
N1
N2
T1
T2
N1
N2
T1
T2
C
Relative Expression
miRNA-1
miRNA-RNU48
D
miRNA-1
28S
18S
miRNA-1
B
Absorbance (cell growth)
A
Absorbance (cell growth)
Transfection of miRNA-1 into chordoma cells
UCH1suppresses Met expression and inhibits cell growth
Hours after pre-miRNA transfection
C
miRNA-control
miRNA-1
Concentration of pre-miRNA-1 (nmol)
D
Met
Actin
Expression of hepatocyte growth factor (HGF) and c-MET
In skull based chordoma
Naka, Kuester, Boltze, Scheil-Bertram, Samii,Herold,
Ostertag, Krueger, Roessner
Cancer 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 you