Transcript 35055 - Connective Tissue Oncology Society

Microarray gene expression
utilized to identify putative
serum biomarkers of NF1
associated tumors
Connective Tissue Oncology Society
November 14, 2008
Trent Hummel, Yonatan Mahler, Shyra Miller,
Timothy Cripe, Walter Jessen,
Bruce Aronow, Nancy Ratner
Cincinnati, OH, USA
Background
• 30% of NF1 patients develop plexiform neurofibromas (PNF)
– these typically arise in childhood
– Benign tumors involving larger nerves
– Can invade critical structures causing death and significant morbidity
– Irregular borders make growth calculations difficult with CT and MRI
scans
– Surgery is the only reliable treatment
– Plexiform neurofibromas can undergo transformation to malignant
Huson SM, et al. Brain 1988
peripheral nerve sheath tumors (MPNST)
Rasmussen SA, et al Am J Epidemiol 2000
Background
• Malignant peripheral nerve sheath tumors
(MPNST)
• Highly aggressive sarcomas
– 5 year survival approximately 30%
• Lifetime risk in NF1 patients is 8-13%
• No reliable method to detect transformation of
plexiform neurofibromas to MPNSTs
Huson SM, et al. Brain 1988
Rasmussen SA, et al Am J Epidemiol 2000
Hypotheses
• There are unique serum proteins that mark
malignant peripheral nerve sheath tumors
• There are unique proteins in the peripheral
blood of NF1 patients with PNF that
correlate with tumor burden/growth.
NF1 Microarray Consortium
86 microarrays in 9 batches
(77 samples plus 9 references)
45 Cell culture samples
NHSC
10 Normal human Schwann cell
dNFSC +/-
2 Dermal neurofibroma NF1 +/- Schwann cell
dNFSC -/-
9 Dermal neurofibroma NF1 -/- Schwann cell
11 Plexiform neurofibroma Schwann cell
pNFSC
MPNST cell
13 MPNST cell line
• Gene expression from 10
independent cultures of primary
human Schwann cells were
compared to 67 NF1 tumor
samples
• Included cultured neurofibroma
Schwann cells and primary
tumors on an Affymetrix platform
32 Tumor samples
dNF
13 Dermal neurofibroma
pNF
13 Plexiform neurofibroma
MPNST
6 MPNST
NF1 Microarray Consortium - Nancy Ratner, Shyra Miller, Bruce Aronow, Walter Jessen,
Marco Giovannini, Anat Stemmer-Rachamimov, Peggy Wallace, Conxi Lazaro, and Grier Page
• The degree of
differential expression is
expressed by color
Normal
dNFSC +/-
dNFSC -/-
• Each row represents a
single gene expression
profile
pNFSC
MPNST cell
dNF
pNF
• Genes in blue exhibit
down regulation
MPNST
6.0
0.0
• Genes in red signify
upregulated expression
as compared to normal
human Schwann cells.
• For this project, genes
that demonstrated
differential upregulation
between NHSC and
neurofibromas and/or
MPNSTs were chosen
for further analysis.
Methods
Filter these upregulated genes
through a database
of secreted proteins
Test candidates
in human model
Confirm secretion of
proteins in cell media
Validate candidates with
quantitative RT-PCR
Methods
• Using microarray data from NF1
Microarray Consortium (Miller S, et al. submitted)
• Compared differentially expressed
probesets with the Secreted Protein
Database
– List of probesets that encode secreted
proteins that may be released into the
extracellular milieu
Chen Y, et al. Nucleic Acids Res. 2005
Methods
9473 genes differentially expressed
between cell culture classes
Normal Human Schwann cell
dermal NFSC, plexiform NFSC,
MPNST cell line (FDR=0.05)
3721 genes from the
Secreted Protein Database
with a confidence rank of 0-3
• Analysis yielded 666 genes that
encode putative secreted proteins
•Differentially upregulated
between NHSC and
neurofibromas and/or MPNST
• Subsequent cluster analysis across
both tumors and cell lines reduced
the number of candidates to 132
•Alternatively spliced forms of
genes were removed
•92 genes that encode putative
secreted proteins and that are
upregulated in MPNST and/or
neurofibromas as compared to
normal human Schwann cells
1664 of 3721 genes upregulated from
Normal Human Schwann cell to Neurofibroma
or Normal Human Schwann cell to MPNST
Top Candidates
• Initial Candidates Selected base on:
– Robust expression in microarray data in all
cell lines when compared to NHSC
– Commercially available reagents
– Adrenomedullin (ADM)
Adrenomedullin
• Adrenomedullin (ADM) is a secreted peptide (52 AA)
• Stimulates angiogenesis
– ADM knockout mice die at E14 because of severe vascular
defects
– ADM expression correlates with vascularity in RCC and breast
carcinomas
• Hypoxia up-regulates ADM in human glioma cell lines
• GBM Xenograft tumor model - anti-ADM antibody greatly
reduced both tumor growth and vascularity
Zhao et al 1998
Jougasaki and Burnett, 2000
Kitamuro et al, 2000 Garayoa et al, 2000
Shindo et al., 2001 Oehler et al., 2002
Ouafik et al., 2002
Validation of Array Data
with RT-PCR
ADM fold change by RT-PCR in
MPNST cell lines compared to NHSC
ADM fold change by RT-PCR
in NFSC compared to NHSC
80
193
200
70
100
85
48
50
31
21
6
37
40
20
13
19
1
-/N
FS
C
-/N
FS
C
+/
N
FS
C
+/
FS
C
N
26
T
YS *
T1
*
88
14
S4
62
S5
20
T2
65
90
-8
88
-3
* sporadic MPNST cell lines
H
SC
0
0
H
SC
55
60
N
1
Fold Change
150
N
Fold change
156
•Quantitative RT-PCR confirms microarray data in MPNST cell lines and NFSC
(NF1 +/- and -/-) cell lines as compared to Normal Human Schwann cells
•Greater than 3 fold change considered significant
ADM in Conditioned Media
ADM level in serum free MPNST
conditioned media
1.5
* = p<0.0013
** = p<0.04
*
*
ng/ml
1.0
*
*
0.5
**
**
S4 M
62
S4
C
M
62
-T
Y
C
M
T2
65
C
M
88
-3
C
M
26
T
C
M
C
88
14
N
H
SC
C
M
0.0
Results using a competitive ELISA adrenomedullin detection kit
(Phoenix Pharmaceuticals) following the manufacturer’s protocol
Methods
Filter these upregulated genes
through a database
of secreted proteins
Test candidates
in human model
Confirm secretion of
proteins in cell media
Validate candidates with
quantitative RT-PCR
Characteristics
NF1 w/o
PNF
NF1 w/
PNF
NF1
MPNST*
Controls
10
17
5
25
Female
7
6
Unknown
15
Male
3
11
Range
13-42
5-42
Mean
21
19
13
Median
17
16
15
Total
Sex
10
Age
Unknown
*4/5 MPNST serum samples courtesy of Victor Mautner, 1/5 was a 16 y/o
who had serum collected for presumed PNF and found to have MPNST.
4-19
Serum Concentration of Adrenomedullin
in NF1 patients with and without tumor burden
p=0.0006
p<0.0001
p<0.0001
0.4
p=0.03
p<0.0001
•ADM elevated in patients
with NF1 regardless of tumor
status
•No difference in ADM
concentration when
comparing plexiform
neurofibroma tumor burden,
tumor location, age, or
gender
0.3
ng/ml 0.2
•Adrenomedullin is a
potential serum biomarker of
NF1
0.1
ST
N
F1
M
PN
PN
F
w
/o
F1
N
N
F1
C
w
ith
on
tr
PN
F
ol
s
0.0
•Increased serum levels of
ADM may correlate with
presence of MPNST, though
small sample size limits the
power of this analysis.
4/5 MPNST samples courtesy Victor Mautner
Conclusions
• Feasible to utilize microarray gene expression data to
generate a list of putative secreted biomarkers
– Confirming expression with secondary technique such as RTPCR
– Testing protein expression/secretion in cell lines and
subsequently in human serum
• Adrenomedullin
– Potential biomarker for NF1
– Also potential serum biomarker for NF1 related MPNSTs
Future Directions
• Continue investigating list of 92 genes
• Obtain more human MPNST serum samples
• Further investigate the biological effects of
adrenomedullin on MPNST cell lines
• Envision a panel of proteins used to delineate between
growth of plexiform neurofibromas and transformation to
malignant peripheral nerve sheath tumors
– Add to clinical decision making
– Add to treatment decisions regarding therapy
Thanks to…
• Nancy Ratner
• Shyra Miller
• Members of NF1 Microarray consortium -
Bruce Aronow, Walter Jessen,
Marco Giovannini, Anat Stemmer-Rachamimov, Peggy Wallace, Conxi Lazaro, and Grier Page
•
•
•
•
•
Jon Williams
John Perentesis
Elizabeth Schorry
Tim Cripe
Yoni Mahler
SPD
• Entries
ranked
according to
prediction
confidence
(rank 0 -3)
Chen Y, et al. Nucleic Acids Res. 2005