R2 서자희

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

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