recurrent chromosomal copy number alterations in chordoma g. petur nielsen; john iafrate; zhenfeng...
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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 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