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R2 서자희

Neuro Oncol . 2010 Mar 11. . R2 서자희. 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

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R2 서자희

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  1. NeuroOncol. 2010 Mar 11. R2 서자희

  2. Introduction • Pilocyticastrocytoma • 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 decrease morbidity and improve survival

  3. Introduction • Hedgehog(Hh) signaling • Medulloblastoma (MBL) : therapeutically benefit! • Malignant glioma • Included small collection of pilocyticastrocytomas from adult patients • Protein & transcript expression profiles for Hh receptor Patched (PTCH) : activated to a small extent

  4. Aims of study Hh pathway in a larger panel of pilocyticastrocytoma specimens from pediatric patients 1) Hhpathway is operational in sporadic pilocyticastrocytomas 2) Hhpathway 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

  5. Tissue Procurement • Brain tumor and epilepsy specimens • Vanderbilt Medical Center, 2003 ~ 2008  20 specimens classified as pilocyticastrocytoma • Ages : 1 ~ 22 yrs • neurofibromatosis type 1 and pilomyxoidastrocytoma : 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

  6. RNA Extraction, cDNASynthesis & 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 tumorand epilepsy specimens • SYBR Green Supermix(Bio-Rad) • cDNA template • TaqManprimers for PTCH (Hs00970979_m1) & GAPDH (Hs99999905_m1) • Primary cell cultures • TaqMan Fast Universal PCR Master Mix (Applied Biosystems) • cDNAtemplate • 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

  7. 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

  8. Cell culture & Hh signaling assays • Primary cell cultures • Tumor sample dissorciation, Plating • Confluent culture • Plated for 7 days in nontreatedNeuroCult medium • Triplicate for 40hours with • Alone • 50 nM smoothened agonist (SAG)/ 500 nMSAG • 200 nMsmoothened antagonist (SANT1) • For assays of Hh pathway inhibition • Confluent cultures with 50 nM SAG • Triplicate for 48 hours with • 50 nM SAG • 200 nMSANT1 • GLI1 and GAPDH : measured by qRT-PCR

  9. Statistical Analysis Pearson product–moment correlation coefficient with a 99% confidence interval Student’s t-test Program : GraphPad Prism TM

  10. Result

  11. PTCH mRNA Expression Levels in PAs Correlate Inversely with Age normalized to endogenous GAPDH levels : Expressed as fold difference 45% of PA specimen Significant inverse correlation between PTCH expression levels and patient age (Pearson’s test, r = − 0.59, P = 0.0097)

  12. PTCH mRNA Expression Levels in PAs Correlate Inversely with Age Hh pathway may be activated in PAs from younger patient 5.01 1.23 Student’s t-test, P=0.013

  13. Additional pathologic features : 9 samples Focal infiltrative growth Oligodendroglioma-like qualities Pilomyxoid features Leptomeningeal spread Necrosis Focal areas of increased proliferation More prevalent in patients before the age of 10 yrs

  14. Single-label staining for GLI1 Greater numbers of cells labeled for PTCH than GLI1 in all instances

  15. PAs are heterogeneous with respect to Hh pathway component expression CASE # 3 CASE # 20 GLI1 staining

  16. PTCH or GLI1 staining indices from one portion of a tumor : NOT always correspond with PTCH mRNA expression level measured in another portion of the same tumor

  17. As for PTCH mRNA measurements,higher staining indices for PTCH and GLI1 weremeasured in tumors from younger patients 14.00 6.81 4.82 0.74 Student’s t-test, P=0.033 Student’s t-test, P=0.034

  18. Ki-67 indices • Range : 0% ~ 2.77% • Higher in younger patients • Significant 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

  19. Double-labeling experiments : 86% of Ki-67-positive cells expressed PTCH across all tumors

  20. Primary cell culture • in GLI1mRNA expression • (P < 0.05, Student’s t-test) Hh-responsive astrocytoma Hh pathway is operationally intact within a subset of pilocyticastrocytomas Hh-unresponsive GBM 1) Basal expression levels of GLI1 transcript : NOT reduced by the SANT1 treatment 2) GLI1 expression levels : NOT induced by the SAG treatment (primary GBM culture)

  21. Confirm modulatorystatus of Hh pathway dose-dependent induction of GLI1mRNAwith SAG After 7 days : re-plated with SANT1 Hh signaling in PA primary culture : modulated by either activation or inhibition  Confirming the operational status of the pathway. • GLI1 level : • In presence of SAG •  To basal levels by SANT1 treatment

  22. Discussion • “ligand-independent” activation : spordicmedulloblastoma • 3 Hh component mutation 1) PTCH (Patched) 2) SMOH (Smoothend) 3) SUFU (suppressor-of-fused) • “ligand-dependent” activation : malignant glioma

  23. 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 expression of Hh pathway • Hh pathway is activated in only a portion of tumor cells in malignancies with a ligand-dependent mechanism 3) Hh pathway activity has been demonstrated in animal transplantation models

  24. 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 Hhsignaling • 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 synergistically regulate the growth of PA ? Animal model of spordic PA

  25. Cancer Res 2009; 69: (4). February 15, 2009

  26. 왜 나만 갖고 그래 … THANK YOU

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