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Neuro Oncol. 2010 Mar 11.
Introduction
• Pilocytic astrocytoma–m/c pediatric brain tumor– Excellent prognosis, indolent nature• 10yr survival : 90~96.8%• 20yr survival : 70% , progression-free survival :
40%
– Long-term sequeale related disease & treatment
• Development of new therapies to de-crease morbidity and improve survival
Introduction
• Hedgehog(Hh) signaling–Medulloblastoma (MBL) : therapeutically
benefit!–Malignant glioma• Included small collection of pilocytic astrocy-
tomas from adult patients• Protein & transcript expression profiles for Hh
receptor Patched (PTCH) : activated to a small extent
Aims of study
• Hh pathway in a larger panel of pilocytic astrocytoma specimens from pediatric patients
1) Hh pathway is operational in sporadic pilocytic astrocytomas
2) Hh pathway is activated to a greater extent in tumors from younger patients
3) Expression of the Hh pathway signal transduction components correlates with expression of the cellular proliferation marker Ki-67
Tissue Procurement
• Brain tumor and epilepsy specimens– Vanderbilt Medical Center, 2003 ~ 2008
20 specimens classified as pilocytic astrocytoma• Ages : 1 ~ 22 yrs
• neurofibromatosis type 1 and pilomyxoid astrocytoma : not included
epilepsy control specimens : 37–57 yrs
– Various location
• RNA extration : quantitative real-time PCR (qRT-PCR)– 18 of tumor speciemen : available for banking in a research tissue
repository
• Microarray construction– 17 of tumor speciemen : paraffin blocks
• Primary cell cultures– 4 of the tumor speciemen : adequate excess tissue was available
RNA Extraction, cDNA Synthesis & qRT-PCR
• Total RNA : brain tissue or primary cell cultures– Genomic DNA removal & purification
– Single-stranded cDNA synthesis
• Negative control : synthesis reaction without reverse transcriptase (-RT)– qRT-PCR : triplicate with negative control
• Primary brain tumor and epilepsy specimens– SYBR Green Supermix (Bio-Rad)
– cDNA template
– TaqMan primers for PTCH (Hs00970979_m1) & GAPDH (Hs99999905_m1)
• Primary cell cultures– TaqMan Fast Universal PCR Master Mix (Applied Biosystems)
– cDNA template
– Primers : TaqMan Gene Expression Assay, Applied Biosystems• hPTCH (Hs00970979_m1), hGLI1 (Hs00171790_m1), and hGAPDH (Hs99999905_m1)
• Standard curve : serial dilutions of a human cDNA mixture
TMA Construction & IHC Analysis
– FFPE : 1mm diameter 4 core• 20 consecutive section
– 1 & 20 : H&E staining
– 2~19 : IHC
– IHC • PTCH-1 (1:100; Santa Cruz Biotechnologies)
• Ki67 (1:50; DAKO)
• Gli-1 (1:50; Cell Signaling)
– Dividing average number of cells that stained positively for PTCH, GLI1, or Ki67 per HPF by the total number of cells per HPF (high power field)• Counted in 9 HPF for each sample of IHC staining
• Total numbers of cells : counted in 4 HPF of H&E staining
Cell culture & Hh signaling assays
• Primary cell cultures– Tumor sample dissorciation, Plating
• Confluent culture – Plated for 7 days in nontreated NeuroCult medium
– Triplicate for 40hours with• Alone
• 50 nM smoothened agonist (SAG)/ 500 nM SAG
• 200 nM smoothened antagonist (SANT1)
• For assays of Hh pathway inhibition– Confluent cultures with 50 nM SAG
– Triplicate for 48 hours with • 50 nM SAG
• 200 nM SANT1
• GLI1 and GAPDH : measured by qRT-PCR
Statistical Analysis
• Pearson product–moment correlation coefficient with a 99% confidence in-terval
• Student’s t-test
• Program : GraphPad Prism TM
RESULT
PTCH mRNA Expression Levels in PAs Correlate Inversely with Age
45% of PA specimen
Significant inverse correlation between PTCH expression levels and patient age(Pearson’s test, r = − 0.59, P = 0.0097)
normalized to endogenous GAPDH levels
: Expressed as fold difference
5.01
1.23Student’s t-test, P=0.013
PTCH mRNA Expression Levels in PAs Correlate Inversely with Age
Hh pathway may be activated in PAs from younger patient
Additional pathologic features : 9 samples
Focal infiltrative growth Oligodendroglioma-like qualities
Pilomyxoid featuresLeptomeningeal spread
Necrosis Focal areas of increased proliferation
More prevalent in patients before the age of 10 yrs
Single-label staining for GLI1
Greater numbers of cells labeled for PTCH than GLI1 in all instances
PAs are heterogeneous with respect to Hh pathway component ex-
pression
CASE # 3 CASE # 20
GLI1 staining
PTCH or GLI1 staining indices from one portion of a tumor
: NOT always correspond with PTCH mRNA expres-sion level
measured in another portion of the same tumor
As for PTCH mRNA measurements,higher staining indices for PTCH and
GLI1 weremeasured in tumors from younger pa-
tients
14.00
6.81
Student’s t-test, P=0.033 Student’s t-test, P=0.034
4.82 0.7
4
Ki-67 indices
Range : 0% ~ 2.77% Higher in younger pa-
tientsSignificant correlation ;• Ki67 and PTCH
Pearson’s test, r=0.68, P=0.0058 • Ki67 and GLI1
Pearson’s test, r=0.82, P=0.0002
Double-labeling experiments: 86% of Ki-67-positive cells expressed PTCH
across all tumors
Primary cell culture in GLI1mRNA expression (P < 0.05, Student’s t-test)
1) Basal expression levels of GLI1 transcript : NOT reduced by the SANT1 treat-ment
2) GLI1 expression levels : NOT induced by the SAG treatment (primary GBM cul-ture)
Hh-responsive astrocytoma
Hh-unresponsive GBM
Hh pathway is operationally intact within a subset of pilocytic astrocy-
tomas
Confirm modulatory status of Hh pathway
dose-dependent induction of GLI1mRNAwith SAG
GLI1 level : In presence of SAG
To basal levels by SANT1 treatment
Hh signaling in PA primary culture : modulated by either activation or inhibi-
tion Confirming the operational status of the
pathway.
After 7 days : re-plated with SANT1
Discussion
• “ligand-independent” activation : spordic medulloblastoma– 3 Hh component mutation
1) PTCH (Patched)2) SMOH (Smoothend)3) SUFU (suppressor-of-fused)
• “ligand-dependent” activation : ma-lignant glioma
Discussion
1) proportion of tumors in which the pathway is activated is less well defined– absence of clear thresholds for PTCH and GLI1
expression levels to define an “on” or “off” state
2) marked cellular heterogeneity in the ex-pression of Hh pathway– Hh pathway is activated in only a portion of tu-
mor cells in malignancies with a ligand-depen-dent mechanism
3) Hh pathway activity has been demon-strated in animal transplantation models
MAPK pathway & BRAF gene
• In 66%–85% of sporadic PAs– BRAF gene rearrangements or mutations
Aberrant activation of the MAPK pathway
• Integration of MAPK and Hh signaling– by ERK-mediated control of the GLI function
– implicated in the regulation of cellular proliferation• Basal cell carcinoma & gastric carcinoma
• Further study : ? MAPK and the Hh pathways function synergisti-
cally regulate the growth of PA
? Animal model of spordic PA
Cancer Res 2009; 69: (4). February 15, 2009
THANK YOU
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