Friday 16th November
Osteosarcoma
Poster discussion
L
1425144
M
1426725
N
1427271
O
1433633
P
1425094
Bass Hassan
University of Oxford
Sir William Dunn School of Pathology
Oxford Sarcoma Service at the Oxford University Hospitals Trust
Oxford Cancer and Haematology Centre
Nuffield Orthopaedic Centre
Osteosarcoma Genomics- Highly Complex
The HALLMARKS
Paper Presentation Summary
Adjuvant chemotherapy plus early >90% necrosis :
OS=57% at 25 years
miR-133a suppression improves outcome in mice
Caprin 1 promotes SaOS-2 cell metastasis in mice
Intercalary allografts not recommended
Need for a chemotherapy trial age >40 year
RNA Interference
miRNA
Families and targets
L 1425144 Micro-RNA expression and functional profiles of osteosarcoma
Kobayashi et al National Cancer Research Centre, Tokyo
24 fresh frozen OS
8 cell lines
miRNA microarrays (933)
anti-miR library
Targets proteins identified
(liquid chromatography and
mass spec- 2DICAL)
• Blocking let-7 suppressed proliferation
• Main targets Serpin H1 and vimentin
L 1425144
24 OS samples
microRNA microarray analysis
Global expression of miRNAs in OS samples
Frequency of
miRNA expression
Flow diagram
Anti-miR inhibitors library, MTT assay
In 8 OS cells
Identification of miRNAs regulating OS cell proliferation
Proteomic approach (2DICAL)
In U2 cells
Sample
108 commonly expressed miRNAs in OS samples
515 miRNAs
108 miRNAs
100
75
50
25
0
OS03
OS13
OS17
OS18
OS20
OS25
OS26
OS27
OS28
OS32
OS35
OS36
OS38
OS44
OS45
OS46
OS48
OS49
OS50
OS51
OS52
OS64
OS65
OS67
200
400
600
800
Number of miRNAs (n=939)
Identification of miRNA targeted protein
Index
L 1425144
Survey of miRNAs regulating OS cell proliferation
Change of ATP production (%)
U2
NOS1
MNNGHOS
SaOS2
9N2
HsOS1
80
60
40
20
0
-20
-40
Inhibited miRNA
3N1
MG63
Identification of proteins regulated
by let-7a family miRNAs
1,093.924m/z (1,093.824~1,094.024)
Vimentin
let-7a
let-7a
let-7f
let-7f
let-7g
let-7g
let-7i
let-7i
NC1
NC1
NC2
NC2
Serpin H1
NC2
NC1
-Actin-
let-7i
-Actin-
let-7g
Serpin H1-
let-7f
let-7a
NC2
NC1
let-7i
let-7g
let-7f
let-7a
Vimentin-
M 1426725 Evaluation of the role of metadherin in osteosarcoma metastasis
Zhu et al University of Texas MD Anderson Cancer Centre
Metadherin (MTDH) Expression is Up-regulated in Osteosarcoma
Cells
A
B
C
M 1426725
MTDH Knockdown Decreases Cell Motility and Invasiveness
Invasion
Migration
Blockade of Cell Surface MTDH Impedes Migration and Invasion
Invasion
Migration
M 1426725
Knockdown of MTDH Inhibits Pulmonary Metastasis of Osteosarcoma Cells
A
C
B
D
IHC-MTDH
N 1427271 Targeting liposomal nanoparticles to a mouse model of osteosarcoma
Vaghasia et al UCLA, Los Angeles
Federman N. Targeting liposomes toward novel pediatric
anticancer therapeutics. Pediatr Res. 67(5), 514-9
ALCAM (CD 166) has been previously
shown to be highly expressed in
osteosarcoma cell lines
Previous in vitro work demonstrated
targeting and improved cytotoxicity
in ALCAM targeted nanoparticles
when compared to a conventional
liposomal Doxorubin.
Federman N. Enhanced growth inhibition of osteosarcoma by cytotoxic polymerized liposomal
nanoparticles targeting the alcam cell surface receptor. Sarcoma. 2012; 2012:126906.
N 1427271
Osteosarcoma cell lines KHOS and MNNG were transduced
with a CMV-luciferase-puromycin lentiviral plasmid
N 1427271
O 1433633 Prediction of a patient survival by MMP1 and P16 immunoreactivity
In osteosarcoma tumour samples before and after neoadjuvant chemotherapy
Robl et al Zurich Switzerland
Methods:
Tissue micro array (TMA): samples of 86 patients, taken before (biopsy, Bx) and after
neoadjuvant chemotherapy (resection, Rx) to analyze 36 known tumor marker proteins
Only non-necrotic tissue sections were graded (controlled by a pathologist)
 semi-quantitative grading performed in MATLAB1
Mean values of grades (Bx and Rx) were used for further analysis:
 downregulation / no change / upregulation
 “proportion of change” (PoC) =
(downregulation + upregulation) / total # of samples
1Ruifrok,
AC, Johnston, DA. (2001). Anal Quant Cytol Histol, 23:291-299.
O 1433633
Results
30
P16
Neuropilin2
Irx4
CXCR7_11G8
CCN1
MMP1
CD44
CXCR4
Caprin
Pten
Vimentin
•
•
•
•
All markers showed a change in expression before and after neoadjuvant
chemotherapy
The PoCs ranged from 3 to 87 % based on samples fulfilling all criteria:
-Bx and Rx available
-no metastasis at diagnosis
-neoadjuvant chemotherapy
Number of Patients
•
Downregulation
No change
Upregulation
25
20
15
10
5
0
PoC
(87%)
(84%)
(79%)
(77%) (73%)
(73%)
(69%)
(67%)
(67%)
(67%)
Fig. 1: Markers ranked according to the largest PoC in immunoreactivity (Bx vs Rx).
(59%)
O 1433633
Results
• If correlated with 5-YS (overall five-year survival) only expression changes
of P16 (P=0.044) and MMP1 (P=0.012) showed a significant correlation
P16-immunoreactivity
(Ntotal = 37 patients)
MMP1-immunoreactivity
(Ntotal = 33 patients)
20
15
10
5
0
25
deceased
alive
Number of patients
Number of patients
25
20
deceased
alive
15
10
5
0
Fig. 2: Change in immunoreactivity (Bx vs. Rx) of P16 and MMP1 in non-metastatic OS patients
related to patient status after five years.
P 1425094 Micro RNA expression profiling
indentifies mir-15B as a regulator of multi-drug
resistance in human osteosarcoma cell lines
Duann et al MGH, Boston
Values of expression
P<0.01
KHOS
KHOSR2
U-2OS
U-2OSMR
SKOV-3
SKOV-3TR
P 1425094
U-2OSMR
Absorbance
U-2OSMR+ miR-15b 1nM
U-2OSMR+ miR-15b 5nM
U-2OSMR+ miR-15b 10nM
U-2OSMR+ miR-15b 20nM
Doxorubicin (μM)
U-2OSMR
Absorbance
U-2OSMR+ nonspecific miR precursor 5nM
U-2OSMR+ nonspecific miR precursor 10nM
U-2OSMR+ nonspecific miR precursor 20nM
Doxorubicin (μM)
P 1425094
Wee1 target gene
miR-15b precursor (nM)
KHOSR2
U-2OSMR
0
1
5
10
20
miR- precursor nonspecific control
40
10
20
40
Wee1 98kD
Actin 42kD
Wee1 98kD
Actin 42kD
microRNA
Thank you to all the speakers, poster presenters
and organisers