CANCER-ASSOCIATED FIBROBLASTS and MICROVASCULAR PROLIFERATION  in   NEUROBLASTOMA TUMORS RANA ZEINE, M.D., Ph.D., Research...
 
ACTIVATED   FIBROBLASTS   Kalluri & Zeisberg. Nat. Rev. Cancer 6, 2006 <ul><li>MYOFIBROBLASTIC Differentiation  </li></ul>...
Induction of GALLBLADDER CARCINOMA Cell Invasion in a Co-Culture System with Stromal FIBROBLASTS  Blocked by NK4 a specifi...
BREAST CARCINOMA  Co-MIXED with Cancer-Associated Fibroblasts resulted in ENHANCED  Xenograft Volume, Microvascular Densit...
BASAL CELL CARCINOMA GREMLIN 1 EXPRESSION in STROMAL FIBROBLASTS Sneddon et al.  PNAS 103, 2006 CANCER-ASSOCIATED FIBROBLA...
EXPRESSION of EPSTI1 at EPITHELIAL – STROMAL INTERFACE in  BREAST CARCINOMA De Neergaard et al.  Am. J. Pathol., 2010  EPS...
aSMA Desmin Desmin smooth muscle  Myosin   heavy chain  SMA CANCER-ASSOCIATED FIBROBLASTS EXPRESS α SMA,  sm-MYOSIN, some...
CANCER-ASSOCIATED FIBROBLASTS are NEGATIVE for hMW-CALDESMON  SMA Nakayama et al.  J. Clin. Pathol  55, 2002 Vessel PERIC...
FIBROBLAST ACTIVATION PROTEIN EXPRESSION in CANCER ASSOCIATED FIBROBLASTS  CORRELATION with POOR PROGNOSIS in  COLON CANCE...
3D-Reconstruction of Vasculature from  Serial Sections Stained for  Endothelium (CD34) Gijtenbeek et al.  Angiogenesis 8, ...
<ul><li>Hashizume </li></ul><ul><li>et  </li></ul><ul><li>al </li></ul><ul><li>Am J Path </li></ul><ul><li>156 </li></ul><...
MICROVASCULAR PROLIFERATION  with Partitioning of the Lumen Glioblastoma Multiforme   Montefiore Medical Center - AECOM   ...
Kim et al. J. Neurosurg. 74, 1991   GLOMERULOID  MICROVASCULAR PROLIFERATION  ASSOCIATED with  SHORTER SURVIVAL  in ASTROC...
MELANOMA BREAST CARCINOMA ENDOMETRIAL CARCINOMA PROSTATE CARCINOMA MICROVASCULAR  PROLIFERATION SHORTER SURVIVAL Endotheli...
Wesseling et al.  J Neuropathology and Experimental Neurology 54, 1995 EN-4 ENDOTHELIAL CELLS  and    -SM Actin PERICYTES...
HISTOGENESIS of NEUROBLASTOMA <ul><li>Definition:  Childhood embryonal tumors of migrating neuroectodermal cells derived f...
INCIDENCE of NEUROBLASTOMA <ul><li>Constitutes 8-10% of all childhood cancers </li></ul><ul><li>Affects ~8.0 children per ...
SCHWANNIAN STROMA − POOR  NEUROBLASTOMA 1.   UNDIFFERENTIATED 2.   POORLY DIFFERENTIATED  3.   DIFFERENTIATING  SCHWANNIAN...
EVENT-FREE SURVIVAL by DIAGNOSIS  (patients  > 18 months; n=908)  (1986-2001:CCG; POG; COG) London WB, Children’s Oncology...
PROGNOSTIC CATEGORIES  DIAGNOSIS MITOSIS-KARYORRHEXIS INDEX (MKI)   Qualman et al. Archives Pathology Laboratory Medicine ...
RISK GROUPS  <ul><ul><li>MYCN Amplification • CHROMOSOMAL Aberrations PLOIDY • CLINICAL Parameters  </li></ul></ul>Qualman...
1. To Determine the Presence and Prognostic Significance of Microvascular Proliferation in Neuroblastoma and in Relation t...
<ul><li>Children’s Memorial Hospital (CMH),  Northwestern University </li></ul><ul><ul><li>Case Series: 60 Tumors, full se...
CASE SERIES ANALYZED  for MICROVASCULAR PROLIFERATION Clinical Parameter CMH (1) CHOP GENDER M,F  23, 28 70, 73 AGE at dia...
CASE SERIES ANALYZED  for CANCER-ASSOCIATED FIBROBLASTS Clinical Parameter CMH (2) AGE at diagnosis 34>1yr, 19<1yr Dx  GN,...
Ganglioneuroma  Schwannian Stroma-Dominant   THIN-WALLED, DILATED & CAPILLARY-LIKE VESSELS H&E
Ganglioneuroma  Schwannian Stroma-Dominant   PERICYTE COVERAGE in THIN-WALLED VESSELS  SMA Pericytes CD31 Endothelial Cells
Ganglioneuroblastoma  Intermixed  Schwannian Stroma-Rich   THIN-WALLED, CLASSIC ANGIOGENIC VESSELS with SPROUTING H&E
Ganglioneuroblastoma Nodular  MICROVASCULAR PROLIFERATION  SS-POOR REGION  NEUROPIL H&E
Microvascular Proliferation  Thin-Walled vessels SS-RICH SS-POOR GNB Nodular   H&E
ENDOTHELIAL CELLS within MICROVASCULAR PROLIFERATION  in SS-POOR REGION  NEUROPIL CD31
Neuroblastoma  Differentiating  Schwannian-Stroma Poor GLOMERULOID MICROVASCULAR PROLIFERATION
Neuroblastoma Differentiating  Schwannian Stroma-Poor    SMA ENDOTHELIAL CELLS and PERICYTES WITHIN STROMA  CD31
MICROVASCULAR PROLIFERATION is PRESENT in SCHWANNIAN STROMA-POOR REGIONS and TUMORS Percent of Cases 8/8 4/4 53/82 11/16 1...
MICROVASCULAR PROLIFERATION is ASSOCIATED with SHORTER SURVIVAL in NEUROBLASTOMA  (patients all ages; n=46 CMH) Peddinti ,...
MICROVASCULAR PROLIFERATION is ASSOCIATED with SHORTER SURVIVAL in NEUROBLASTOMA   (patients all ages; n=143 CHOP) Peddint...
MICROVASCULAR PROLIFERATION is a  POOR PROGNOSTIC FACTOR in NEUROBLASTOMA Single-Predictor Cox Regression Analysis (TMA-CH...
HIGH RISK NEUROBLASTOMA TUMORS are  ASSOCIATED with MICROVASCULAR PROLIFERATION  (143 tumors, CHOP) Peddinti, Zeine et al....
<ul><li>There were  no deaths  in the subset of  </li></ul><ul><li>patients with MYCN amplified tumors that lacked Microva...
Neuroblastoma  Undifferentiated  Schwannian Stroma-Poor  FIBROVASCULAR STROMA
CANCER-ASSOCIATED FIBROBLASTS in Neuroblastoma Undifferentiated   SMA+ve
NO CANCER-ASSOCIATED FIBROBLASTS in Ganglioneuroma  SMA positive pericytes
 SMA hCD Pericytes FEW CANCER-ASSOCIATED FIBROBLASTS in Ganglioneuroblastoma Intermixed  Cancer-Associated Fibroblasts
MORE CANCER-ASSOCIATED FIBROBLASTS in Ganglioneuroblastoma Nodular  SMA hCD Pericytes Cancer- Associated Fibroblasts
ABUNDANT CANCER-ASSOCIATED FIBROBLASTS  in Neuroblastoma Differentiating
Quantitative IHC  (Clarient-Chromavision) Automated Cellular Imaging System II Scanned the   SMA-stained Slides Set Color...
0.0% 1.0% 2.0% 3.0% 4.0% 5.0% 6.0% 7.0% 8.0% PERCENT   SMA+ve REGIONS in NEUROBLASTOMA TUMORS  SMA +ve AREA NBU n=5 NBPD...
CANCER-ASSOCIATED FIBROBLASTS ASSOCIATED with  SS-POOR STROMA  and  MICROVASCULAR PROLIFERATION  Zeine et al. Mod. Pathol....
0.0% 1.0% 2.0% 3.0% 4.0% 5.0% GN n=7 GNBI n=7 GNBN n=4 NB (D+PD+U) n=42 INCREASED   SMA +ve AREAS in SS-POOR STROMA  Mean...
INCREASED   SMA +ve AREAS in  NEUROBLASTOMA TUMORS with  MICROVASCULAR PROLIFERATION  Mean ±SD Percent    -SMA  POSITIVE...
SS-Rich,  No MVP, No CAFs  Tumor-INHIBITORY Role SS-Poor, with MVP &  CAFs Tumor-PROMOTING Role Ganglioneuroma Ganglioneur...
SCHWANNIAN-ENRICHED MICROENVIRONMENT  in NEUROBLASTOMA XENOGRAFT MODEL <ul><li>Human Cell Line </li></ul><ul><li>SMS-KCNR ...
Control, SS-Poor Outside Sciatic Nerve Cancer-Associated Fibroblasts Schwann-Enriched Xenograft Inside Sciatic Nerve Peric...
Control n=9 Inside ScN. n=10  Mean ±  SD  SMA +ve  Cells / mm 2 LESS Cancer-Associated Fibroblasts  in Neuroblastoma Xeno...
ANTI-ANGIOGENIC TREATMENT in NEUROBLASTOMA XENOGRAFT MODELS <ul><li>Two NMYC-Amplified Neuroblastoma Cell Lines:  NMB </li...
BLOCKING ANGIOGENESIS Valproic Acid <ul><li>Branched Chain Fatty Acid </li></ul><ul><li>Inhibits Histone Deacetylase (HDAC...
VALPROIC ACID INHIBITS TUMOR GROWTH  IN VIVO DAYS Tumor Volume mm 3 Yang,… Zeine et al.  Cancer Res. 26(4), 2007
Control SMS-KCNR Xenografts Micro-Vascular Proliferation ValproicAcid  Treatment Thin Walled Vessels ANTI-ANGIOGENIC TREAT...
Control NMB MICROVASCULAR PROLIFERATION Valproic Acid VASCULAR NORMALIZATION FibroCollagenous Stroma H&E Masson Trichrome ...
‘ Normalization’ of Vessels Tong et al. Cancer Research 64 (11), 2004 Murine Mammary Carcinoma  Two-Photon Microscopy Cont...
Abrogation of Microvascular Proliferation by Anti-Angiogenic Treatment with Valproic Acid   Percent of Total Vessels  With...
FUNCTIONAL IMPROVEMENT  LESS LEAKY Reduced Intratumoral Vessel Permeability by Anti-Angiogenic Treatment with Valproic Aci...
Control SMS-KCNR Xenografts ValproicAcid  Treatment LESS CANCER-ASSOCIATED FIBROBLASTS Following ANTI-ANGIOGENIC TREATMENT...
1- Microvacular Proliferation is a Poor Prognostic    Indicator present in SS-Poor Neuroblastoma Regions. 2- Cancer-Associ...
SIGNIFICANCE <ul><li>Histopathologic Evaluation of Neuroblastoma Tumors for Microvascular Proliferation and Cancer-Associa...
ACKNOWLEDGEMENTS University of Pennsylvania (CHOP) John M. Maris, MD  Kristina Cole, MD  Bruce Pawel, MD Statistics (CMH a...
FUTURE DIRECTIONS <ul><li>Elucidate the Molecular Mechanisms for Tumor-Stromal Interactions and the Antagonism Between Fib...
New ‘ANTI-STROMAL’ Agents <ul><li>Aptenins NBRI23477 A and B  (antibiotics) </li></ul><ul><li>(Prostate cancer co-cultures...
STAGING  SYSTEM in Neuroblastoma Brodeur et al. J Clin Oncol 11,1993 STAGE DEFINITION 1 Localized Tumor with Complete Gros...
FIBROVASCULAR STROMA in SS-Poor NB Tumors Masson Trichrome special stain
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Zeine Seminar 2010, Cancer Associated Fibroblasts and Microvascular Proliferation in Neuroblastoma Tumors

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World Cancer Congress 2010
Presence of Cancer-Associated Fibroblasts correlates with Microvascular Proliferation which is a Poor Prognostic Factor in Neuroblastoma Tumors

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  • Zeine Thank you Dr. Zhao, Ladies and Gentlemen. 2010
  • Zeine Tumor Stroma provides the Microenvironment which nurtures the Growth and Progression of tumors. Stromal Cells include fibroblasts, endothelial cells, pericytes and inflammatory cells. Today we will focus on two features of Tumor Stroma, Blood Vessels and Activated Fibroblasts. 2010
  • Zeine Cancer-Associated Fibroblasts are believed to Originate from either Local Tissue Fibroblasts, or Bone Marrow Progenitors, or Neoplastic cells undergoing Epithelial-Mesenchymal Transition, or from Pericytes. They EXHIBIT Myofibroblastic Cytodifferentiation to become Activated Fibroblasts and ACQUIRE motility through formation of alpha-Smooth Muscle Actin Stress Fibers. They have Enhanced Proliferation, and Secrete Extracellular Matrix Proteins and Chemokines. Kalluri and Zeisberg ‘06 2010
  • Cancer-Associated Fibroblasts promote tumor cell invasion via the Hepatocyte Growth Factor – Met Receptor System. In this two-chamber co-culture system, presence of the fibroblasts induced Gallbladder carcinoma cells to invade matrigel; and this was blocked by NK4, a specific competitive antagonist against HGF. Matsumoto and Nakamura ’06 Zeine 2010
  • Cancer-Associated Fibroblasts Promote Tumor Growth and Angiogenesis via the Stromal Derived Factor 1 – CXCR4 Chemokine Receptor System. In this experiment, Breast Carcinoma Co-Mixed with Cancer-Associated Fibroblasts resulted in Enhanced Xenograft Volume, Microvascular Density and Precursor Cell Recruitment, over co-mixing with ‘Counterpart Fibroblasts’. These effects could be blocked by a neutralizing Antibody to SDF-1. Orimo et al. ’05 Zeine 2010
  • Cancer-Associated Fibroblasts may promote Cancer Stem Cell Renewal via Gremlin Antagonism of BMP, Bone Morphogenetic Protein. Gremlin 1 Expression is seen here in stromal fibroblasts in Basal Cell Carcinoma. Sneddon et al. ’06 Zeine 2010
  • More Recently De Neergaard et al. have localized Epithelial-Stromal Interface 1, a Nucleo-Cytoplasmic protein, at the junction between tumor cell nests and surrounding stroma in Breast Carcinoma; and demonstrated that it substitutes for tumor fibroblasts (morphogenesis) and up-regulates markers of epithelial-mesenchymal transition (fibronectin, integrin-alpha2-beta1) . Zeine 2010
  • Zeine In addition to expressing alpha  SMA, Cancer-Associated Fibroblasts may express smooth muscle Myosin heavy chain, and a minority express Desmin, as seen here in breast carcinoma. They do not express Smoothelin which is the marker for mature smooth muscle cells. Gabbiani ’97 2010
  • Cancer-Associated Fibroblasts are negative for high molecular weight Caldesmon which distinguishes them from true Pericytes, seen here in gastric carcinoma. Nakayama et al. ’02 Zeine 2010
  • Zeine FIBROBLAST ACTIVATION PROTEIN, is another marker for CANCER ASSOCIATED FIBROBLASTS. FAP is a seprase, dipeptidyl peptidase, and its expression in this study on COLON Cancer reveals clinical Correlation with POOR PROGNOSIS. Henry et al. ’07 2010
  • Zeine Vascular Architecture Appears More Tortuous, Distorted and Disorganized in aggressive cancers, as seen here in this 3D-Reconstruction of CD34 staining in Gliobastoma Multiforme as compared to normal brain tissue. Gijtenbeek et al. ’05 2010
  • Zeine Scanning Electron Microscopy through blood vessels of Mouse Mammary Carcinoma reveals that ENDOTHELIAL Cells Begin to Overlap, Become Branched, Extend Multiple Projections Forming Bridges and Tunnels that Eventually Partition the Lumen. Hashizume et al. 2000. 2010
  • Zeine In this section from a Glioblastoma Multiforme, Partitioning of the Vascular Lumen can be Discerned on H&amp;E. 2010
  • Zeine In 1991 it was reported that presence of Glomeruloid Microvascular Proliferation was Associated with Shorter Survival in Astrocytoma Tumors. Original curve was kindly provided to me by Professor Tessa Hedley-White, at Harvard, from Kim et al. ’91 2010
  • Zeine Subsequently, Microvascular Proliferation has been found to be a poor prognostic indicator in Melanoma, Breast, Endometrial and Prostate Carcinoma. Straume et al. ’02 2010
  • Zeine Immuno-Electron-microscopy revealed that both ENDOTHELIAL Cells as well as PERICYTES were present within Microvascular Proliferation, as seen here in Glioblastoma Multiforme. Wessling et al. ’95 2010
  • Zeine Neuroblastomas are Embryonal tumors of Migrating Neuroectodermal cells, derived from the neural crest and destined for the adrenal medulla and sympathetic nervous system. Primary Sites can be Adrenal, Abdominal, or anywhere along the Chain of Sympathetic Ganglia. 2010
  • Zeine Neuroblastoma constitutes 8-10% of all childhood cancers. Affecting about 8.0 children, per million per year. Median age of diagnosis is 22 months. The Male to female ratio is 1.2 to 1. 2010
  • Zeine There are 7 Diagnostic types of Neuroblastoma Tumors. 3 are Schwannian Stroma-Poor NEUROBASTOMAs: either Undifferentiated, Poorly Differentiated or Differentiating. 2 are Schwannian Stroma-Rich GANGLIONEUROBLASTOMAs: either Nodular or Intermixed. And 2 are Benign, Schwannian Stroma-Dominant, GANGLIONEUROMAs with either Maturing or fully Mature Nerve Fascicles. 2010
  • Zeine Statistical Data from the Children’s Oncology Group shows a dramatic split between Nodular and Intermixed Ganglioneuroblastoma with regards to event-free survival, whereby the Nodular Ganglioneuroblastomas have a much poorer prognosis, indeed, no better than that for the Schwannian Stroma-Poor Neuroblastoma Tumors. Original Data provided by Wendy London from COG in ’06 2010
  • Zeine Neuroblastoma tumors are Classified as having either Favorable or Unfavorable Prognosis based on Age, Diagnosis and Mitosis-Karyorrhexis Index. The algorithm summarized here is from Qualman et al. ‘05 2010
  • Zeine Furthermore, Neuroblastoma Cases are classified into 3 RISK GROUPS, Low, Intermediate or High Risk, based on MYCN Amplification Status, Ploidy, Chromosomal aberrations and Clinical parameters. 2010
  • Zeine Our Objectives were 1. To Determine the Presence and Prognostic Significance of Microvascular Proliferation in Neuroblastoma and in Relation to Schwannian Stroma and Risk Factors. 2. To Assess Cancer-Associated Fibroblasts in Neuroblastoma and in Relation to Schwannian Stroma and Microvascular Proliferation. 3. To Evaluate the Effects of Anti-Angiogenic Treatment on Tumor Microenvironment in Neuroblastoma. 2010
  • Zeine We examined archival Formalin-Fixed Paraffin-Embedded tissues from 60 tumors diagnosed at Children’s Memorial Hospital (CMH), full sections from one block per tumor, and 155 tumors on a tissue microarray constructed at Children’s Hospital of Philadelphia (CHOP), 1-4 cores per tumor. 2010
  • Zeine Clinical Parameters were collected for Gender, Age, Diagnosis, Stage, MYCN status, Prognostic Category, Risk Group and Survival. The Overall Survival was higher than the National Average in both series, perhaps due to selection bias for cases requiring surgical tissue for diagnosis. 2010
  • Zeine Cancer Associated Fibroblasts were analyzed only in the CMH series where full sections were available. 2010
  • Zeine In Ganglioneuroma Blood Vessels were Thin-Walled, Dilated and Capillary-Like. 2010
  • Zeine Immunohistochemistry revealed single layers of CD31 positive endothelial cells, and orderly coverage by alpha-SMA positive pericytes. 2010
  • Zeine In Ganglioneuroblastoma Intermixed, blood vessels were also of the Thin-Walled, Classic-Angiogenic type, often showing sprouting 2010
  • Zeine By CONTRAST, Microvascular Proliferation was seen in Ganglioneuroblastoma Nodular; note the multiple layers of plump endothelial cells and multiple microvessel lumena. 2010
  • Zeine We found that Microvascular Proliferation was Confined to the Schwannian Stroma-Poor Regions, and examination of the Schwannian Stroma-Rich areas revealed only Thin-Walled vessels. 2010
  • Zeine CD31 immunostain highlighted disorderly Endothelial cells within the microvascular proliferations. 2010
  • Zeine In the Schwannian-Stroma-POOR Neuroblastomas, Florid Glomeruloid Microvascular Proliferation could be seen, as shown here in Neuroblastoma Differentiating. 2010
  • Zeine 11/28/07 Multiple layers of pericytes were present surrounding the microvessels.
  • Zeine In both the CMH series and the CHOP series, Microvascular Proliferation was present only in Schwannian Stroma-Poor Regions and Tumors. Not all Neuroblastomas had Microvascular Proliferation, about 35% did not. 2010
  • Zeine Kaplan-Meier curves Demonstrated Shorter Survival for cases WITH Microvascular Proliferation versus those without; the difference reaching statistical significance at p-value 0.017 in the CMH series, 2010
  • Zeine and p-value 0.014 in the CHOP series. 2010
  • Zeine Univariate Regression Analysis revealed that Presence of Microvascular Proliferation was a Poor Prognostic Factor similar to the other known Risk Factors in Neuroblastoma. 2010
  • Zeine Furthermore, the High Risk Factors in neuroblastoma were Associated with Microvascular Proliferation at a statistically significant level in the CHOP series. 2010
  • Zeine There were no deaths in the subset of patients with MYCN amplified tumors that lacked Microvascular Proliferation (5 cases). The outcome was significantly better for patients with stage 4 disease who lacked Microvascular Proliferation. 2010
  • Zeine Fibrovascular Stroma was present In Schwannian Stroma-POOR Neuroblastoma Tumors. 2010
  • Zeine We then looked for Cancer-Associated Fibroblasts. 2010
  • Zeine No Cancer-Associated Fibroblasts were detected in Schwannian Stroma Dominant Ganglioneuroma. 2010
  • Zeine Few Cancer-Associated Fibroblasts were Present in Ganglioneuroblastoma Intermixed, focally in the vicinity of Neuroblasts where the Schwannian Stroma is somewhat Looser. Hi Molecular Weight Caldesmon staining reveals the Pericytes. 2010
  • Zeine More Cancer-Associated Fibroblasts were seen in Ganglioneuroblastoma Nodular, mainly infiltrating the Schwannian Stroma Poor Regions. Again, Pericytes are distinguished by staining for h-Caldesmon on an Adjacent Section. 2010
  • Zeine Abundant Cancer-Associated Fibroblasts were Present in Neuroblastoma Differentiating, and everywhere within the Fibrovascular Stroma of many of the Schwannian Stroma-Poor Neuroblastoma tumors. 2010
  • Zeine In order to quantitate the results, Automated Cellular Imaging System II was used. We Scanned the alpha  SMA-stained Slides Set Color Detection Thresholds Delineated Tumor Regions Calculated Ratios of Areas Stained to Total Areas Analyzed Of Course, Stain included Pericytes and Cancer Associated Fibroblasts 2010
  • Zeine The PERCENT alpha-SMA positive Regions varied from low to high in the Neuroblastomas, but remained uniformly low in the Ganglioneuromas and Intermixed tumors. Thresholds could be defined at 1.1 and 3 Percent to designate low, medium and high levels. It is reasonable to consider that the Low levels represented mostly Pericytes, while the Medium and High Levels represented accumulation of Cancer Associated Fibroblasts. 2010
  • Zeine Analysis of Both the Categorical Data and the Continuous Data revealed Statistically Significant Associations of CANCER-ASSOCIATED FIBROBLASTs with SS-POOR Histology and with Microvascular Proliferation; Chi square and t-test p values less than 0.001 2010
  • Zeine There was an Inverse Correlation between the Levels of Alpha-SMA and the Amounts of Schwannian Stroma. 2010
  • Zeine And a Direct Correlation between the Levels of Alpha-SMA and the Presence of Microvascular Proliferation. 2010
  • Zeine Our findings are Consistent with a Pro-angiogenic / Tumor Promoting Role for Cancer-Associated Fibroblasts; and an Anti-Angiogenic / Tumor Inhibitory role for Schwannian Stroma Derived Factors. 2010
  • Zeine In order to Experimentally Enrich the Tumor Microenvironment for Schwannian Stroma, Neuroblastoma Xenografts were Engrafted Inside the Sciatic Nerve of Nude Mice previously in Liu et al. 2005. A MYCN Amplified cell line SMS-KCNR, Derived from Human Neuroblastoma, was used. Xenografts grown inside the Nerve were shown to have S-100 positive Schwann Cells as well as Reduced Vascular Density. Liu et al. from Sue Cohn’s lab ’05 2010
  • Zeine We Examined 10 of those tumors and found that as compared to controls, Cancer-Associated Fibroblasts were absent or reduced in the Schwann-Infiltrated Xenografts. 2010
  • Zeine The inverse relation between Schwann Cell Infiltration and Cancer-Associated Fibroblast Accumulation suggests that the Schwannian Microenvironment might interfere with either cancer-associated fibroblast migration or activation, thus Slowing down angiogenesis. 2010
  • Zeine For testing the effects of Anti-Angiogenic Treatment: Two, MYCN-amplified neuroblastoma Cell lines, NMB, and SMS-KCNR, were inoculated subcutaneously into female 4-6 week old Athymic Nude Mice. Treatment with 400 mg/Kg Valproic Acid was given once a day for 20 days for mice with small tumors, and for 10 days in mice with large tumors. 2010
  • Zeine Valproic Acid is a Branched Chain Fatty Acid; It Inhibits Histone Deacetylase (HDAC) It Inhibits Growth of Neuroblastoma and Other Cancers; and Blocks VEGF- and hypoxia-induced Angiogenesis. HDAC inhibitors are believed to exert their anti-neoplastic effects through Histone Acetylation. 2010
  • Zeine Treatment in vivo with VALPROIC ACID inhibited TUMOR GROWTH significantly. 2010
  • Zeine Valproic Acid Treatment Abrogated Microvascular Proliferation and resulted in Thin-Walled Vessels, Often Embedded in Fibrocollagenous Stroma. 2010
  • Zeine Masson Trichrome special stain highlighted the Fibro-Collagenous Stroma surrounding the Thin-Walled vessels found in Valproic Acid Treated Xenografts. In untreated Controls, the Majority of Vessels showed Microvascular Proliferation. 2010
  • Zeine Our Observations Represent the Histopathologic Counterpart of the Phenomenon of ‘Normalization’ of Vessels Demonstrated here by Tong et al. by comparing Two-Photon Microscopy on Murine Mammary Carcinoma with and without Anti-angiogenic Treatment. Tong et al. ’04 2010
  • Zeine Semiquantitative Analysis illustrates Abrogation of Microvascular Proliferation Following in vivo treatment with Valproic Acid in the two neuroblastoma models. 2010
  • Zeine Functional improvement, evidenced by Less leaky blood vessels, could be demonstrated by Spectrophotometric Quantitation of Evan’s Blue Extravasation. Mice bearing xenografts received a 0.2% solution of Evan’s Blue by tail vein injection. After 20 minutes in circulation, excess dye was removed by saline perfusion. Tumors were cut into pieces and placed in formamide for 72 hours at room temperature to facilitate dye extraction. We demonstrated a significant reduction in vessel permeability following treatment. 2010
  • Zeine We Also Noted Less Cancer-Associated Fibroblasts in the Valproic Acid Treated Xenografts. 2010
  • Zeine Conclusions: 1- Microvacular Proliferation is a Poor Prognostic Indicator present in SS-Poor Neuroblastoma Regions. 2- Cancer-Associated Fibroblasts Colocalize with Microvacular Proliferation and are Precluded from SS-Rich Regions of Neuroblastoma Tumors. 3- It is Possible to Reduce Cancer-Associated Fibroblast Infiltration by Increasing the Schwannian Component within the Tumor Microenvironment. 4- Anti-Angiogenic Treatment Reduced Microvascular Proliferation and Cancer-Associated Fibroblasts in Neuroblastoma Xenografts. 2010
  • Zeine Significance: Histopathologic Evaluation of Neuroblastoma Tumors for Microvascular Proliferation and Cancer-Associated Fibroblasts May Help Refine Risk Group and Prognostic Classification. May Have Impact on Eligibility Criteria for Stratification of Patients for Inclusion in Clinical Trials. May Guide Development of Novel Anti-Angiogenic and Anti-Stromal Treatment Strategies for Children with Aggressive Neuroblastoma. Results are Consistent with Tumor-Inhibitory Roles for Schwannian Derived Factors, and Tumor-Promoting Roles for Cancer-Associated Fibroblasts. 2010
  • Zeine I thank Sue Cohn and her lab members Radhika Peddinti, Alex Chlenski, Qiwei Yang, Helen Salwen, Yufeng Tian, Lisa Guerrero as well as the Pathcore staff at Northwestern University and University of Chicago I thank Maria Tretiakova for her help with ACIS II. We thank the statisticians Roopa Seshadri at Children’s Memorial Hospital and Wendy London of COG; and our collaborators at Children’s Hospital of Pennsylvania, John Maris, Kristina Cole and Bruce Pawel. 2010
  • Zeine Future Directions would be to Elucidate the Molecular Mechanisms for Tumor-Stromal Interactions and the Antagonism Between Fibrovascular Stroma and Schwannian Stroma. Discover and Identify Stromal Elements of Biologic and Clinical Significance in Cancer. Identify Cancer Stem Cells within tumors. Pursue Preclinical Testing of Therapeutic Strategies Targeting Tumor Stroma or Altering Tumor Microenvironment. 2010
  • Zeine Some of the Anti-Stromal Agents that are currently being Explored Experimentally include Aptenins, Phthoxazolin, Inthomycin B, Pioglitazone and Ciglitazone. Bundscherer et al. ’09, Kawada et al. ’08 and ’09 2010
  • Zeine The Prognosis is also impacted by Stage of Tumor; Stage 4 carrying a worse prognosis than stage 4S which was defined as dissemination limited to skin, liver and/or bone marrow in infants younger than 12 months. 2010
  • Zeine Masson Trichrome special stain Highlights Fibrovascular Stroma in the Schwannian-Stroma-POOR Neuroblastoma Tumors 2010
  • Zeine Seminar 2010, Cancer Associated Fibroblasts and Microvascular Proliferation in Neuroblastoma Tumors

    1. 1. CANCER-ASSOCIATED FIBROBLASTS and MICROVASCULAR PROLIFERATION in NEUROBLASTOMA TUMORS RANA ZEINE, M.D., Ph.D., Research Assistant Professor & Associate Director (former) Northwestern University, Chicago, IL, USA
    2. 3. ACTIVATED FIBROBLASTS Kalluri & Zeisberg. Nat. Rev. Cancer 6, 2006 <ul><li>MYOFIBROBLASTIC Differentiation </li></ul><ul><li> -Smooth Muscle ACTIN Expression </li></ul><ul><li>Enhanced PROLIFERATION </li></ul><ul><li>Matrix Proteins♦Chemokine Secretion </li></ul>FIBROBLASTS CANCER-ASSOCIATED FIBROBLASTS ? ORIGINS Local Tissues Marrow Progenitors Epithelial-Mesenchymal Transition Pericytes  SMA Stress Fibers
    3. 4. Induction of GALLBLADDER CARCINOMA Cell Invasion in a Co-Culture System with Stromal FIBROBLASTS Blocked by NK4 a specific competitive antagonist against the Hepatocyte Growth Factor – Met Receptor system Matsumoto & Nakamura, Int. J. Cancer 119, 2006 CANCER-ASSOCIATED FIBROBLASTS PROMOTE TUMOR INVASION via HGF-MET Ohnishi & Daikuhara 2003 FIBROBLASTS MOTILITY CARCINOMA
    4. 5. BREAST CARCINOMA Co-MIXED with Cancer-Associated Fibroblasts resulted in ENHANCED Xenograft Volume, Microvascular Density and Precursor Cell Recruitment over ‘Counterpart Fibroblasts’ Orimo et al. Cell 121, 2005 CANCER-ASSOCIATED FIBROBLASTS PROMOTE TUMOR GROWTH & ANGIOGENESIS via SDF1 – CXCR4 Kucia et al. 2004 Blocked by Antibody to Stromal-Derived Factor-1
    5. 6. BASAL CELL CARCINOMA GREMLIN 1 EXPRESSION in STROMAL FIBROBLASTS Sneddon et al. PNAS 103, 2006 CANCER-ASSOCIATED FIBROBLASTS MAY PROMOTE CANCER STEM CELL RENEWAL via GREMLIN ANTAGONISM of BONE MORPHOGENETIC PROTEIN (BMP)
    6. 7. EXPRESSION of EPSTI1 at EPITHELIAL – STROMAL INTERFACE in BREAST CARCINOMA De Neergaard et al. Am. J. Pathol., 2010 EPSTI1 Substitutes for fibroblasts in tumor micro-environment assays and upregulates Epithelial-Mesenchymal Transition
    7. 8. aSMA Desmin Desmin smooth muscle Myosin heavy chain  SMA CANCER-ASSOCIATED FIBROBLASTS EXPRESS α SMA, sm-MYOSIN, some DESMIN in BREAST CARCINOMA Schürch, Seemayer & Gabbiani 1997 ch.7 in Sternberg (Ed.) Histology for Pathologists smooth muscle Myosin heavy chain
    8. 9. CANCER-ASSOCIATED FIBROBLASTS are NEGATIVE for hMW-CALDESMON  SMA Nakayama et al. J. Clin. Pathol 55, 2002 Vessel PERICYTES hCD +ve hCD GASTRIC CARCINOMA Cancer-Associated FIBROBLASTS + PERICYTES
    9. 10. FIBROBLAST ACTIVATION PROTEIN EXPRESSION in CANCER ASSOCIATED FIBROBLASTS CORRELATION with POOR PROGNOSIS in COLON CANCER Henry et al. Clin. Cancer Res. 13, 2007 FAP / Seprase, 97-kDa Glycoprotein, Gelatinase, Dipeptidyl Peptidase
    10. 11. 3D-Reconstruction of Vasculature from Serial Sections Stained for Endothelium (CD34) Gijtenbeek et al. Angiogenesis 8, 2005 Glioblastoma Multiforme VASCULAR ARCHITECTURE APPEARS MORE TORTUOUS, DISTORTED and DISORGANIZED in AGGRESSIVE CANCER Normal Grey Matter
    11. 12. <ul><li>Hashizume </li></ul><ul><li>et </li></ul><ul><li>al </li></ul><ul><li>Am J Path </li></ul><ul><li>156 </li></ul><ul><li>2000 </li></ul>Mous e Mammary Carc I noma Normal Overlapping Branched Not Connected Multiple Projections Bridges Tunnels Partitioned Lumen
    12. 13. MICROVASCULAR PROLIFERATION with Partitioning of the Lumen Glioblastoma Multiforme Montefiore Medical Center - AECOM H&E
    13. 14. Kim et al. J. Neurosurg. 74, 1991 GLOMERULOID MICROVASCULAR PROLIFERATION ASSOCIATED with SHORTER SURVIVAL in ASTROCYTOMA ‒ GLIOBLASTOMA Tumors
    14. 15. MELANOMA BREAST CARCINOMA ENDOMETRIAL CARCINOMA PROSTATE CARCINOMA MICROVASCULAR PROLIFERATION SHORTER SURVIVAL Endothelial stain FACTOR VIII Straume et al. Cancer Research 62, 2002
    15. 16. Wesseling et al. J Neuropathology and Experimental Neurology 54, 1995 EN-4 ENDOTHELIAL CELLS and  -SM Actin PERICYTES Vascular Lumena Immuno-Electron Microscopy of Glomeruloid Microvascular Proliferation in GLIOBLASTOMA Multiforme
    16. 17. HISTOGENESIS of NEUROBLASTOMA <ul><li>Definition: Childhood embryonal tumors of migrating neuroectodermal cells derived from the neural crest and destined for the adrenal medulla and sympathetic nervous system. </li></ul><ul><li>Primary Sites: Adrenal 40% </li></ul><ul><li>Abdominal 25% </li></ul><ul><li>Sympathetic Ganglia 25% </li></ul><ul><li>Thoracic 15% </li></ul><ul><li>Cervical 5% </li></ul><ul><li>Pelvic 5% </li></ul>
    17. 18. INCIDENCE of NEUROBLASTOMA <ul><li>Constitutes 8-10% of all childhood cancers </li></ul><ul><li>Affects ~8.0 children per million per year under the age of 15 years </li></ul><ul><li>Median age of diagnosis is 22 months </li></ul><ul><li>Male to Female ratio: 1.2 to 1 </li></ul>
    18. 19. SCHWANNIAN STROMA − POOR NEUROBLASTOMA 1. UNDIFFERENTIATED 2. POORLY DIFFERENTIATED 3. DIFFERENTIATING SCHWANNIAN STROMA − RICH GANGLIONEUROBLASTOMA 4. NODULAR 5. INTERMIXED SCHWANNIAN STROMA − DOMINANT GANGLIONEUROMA 6. MATURING 7. MATURE GANGLIONEUROMA WHO DIAGNOSTIC CLASSIFICATION International Neuroblastoma Pathology Committee
    19. 20. EVENT-FREE SURVIVAL by DIAGNOSIS (patients > 18 months; n=908) (1986-2001:CCG; POG; COG) London WB, Children’s Oncology Group, Statistics and Data Center, 2006 0 20 40 60 80 100 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Years Probability (%) Neuroblastoma (n=785) Ganglioneuroblastoma, Intermixed, (n=44) Ganglioneuroblastoma, Nodular (n=79) Kaplan-Meier Curves
    20. 21. PROGNOSTIC CATEGORIES DIAGNOSIS MITOSIS-KARYORRHEXIS INDEX (MKI) Qualman et al. Archives Pathology Laboratory Medicine 129,2005 UNFAVORABLE FAVORABLE HISTOLOGY AGE
    21. 22. RISK GROUPS <ul><ul><li>MYCN Amplification • CHROMOSOMAL Aberrations PLOIDY • CLINICAL Parameters </li></ul></ul>Qualman et al. Archives Pathology Laboratory Medicine 129,2005 Biologic and Clinical Risk Factors and Groups in Neuroblastoma Parameter Low Risk Intermediate High Risk MYCN status Ploidy 17q gain 11q, 14q LOH 1p LOH TRK A TRK B TRK C Age Stage 3-yr survival Normal Hyperdiploid Near-triploid Rare Rare Rare High Truncated High Usually < 1 yr 1, 2, 4S > 90% Normal Near-diploid Near-tetraploid Common Common Uncommon Low or Absent Low or Absent Low or Absent Usually > 1 yr Usually 3 or 4 30%-50% Amplified (>10 copies) Near-diploid Near-tetraploid Common Rare Common Low or Absent Low or Absent Low or Absent Usually 1-5 yr Usually 3 or 4 < 20%
    22. 23. 1. To Determine the Presence and Prognostic Significance of Microvascular Proliferation in Neuroblastoma and in Relation to Schwannian Stroma and Risk Factors. 2. To Assess Cancer-Associated Fibroblasts in Neuroblastoma and in Relation to Schwannian Stroma and Microvascular Proliferation. 3. To Evaluate the Effects of Anti-Angiogenic Treatment on Tumor Microenvironment in Neuroblastoma. OBJECTIVES
    23. 24. <ul><li>Children’s Memorial Hospital (CMH), Northwestern University </li></ul><ul><ul><li>Case Series: 60 Tumors, full section from 1 block </li></ul></ul><ul><li>Children’s Hospital of Philadelphia (CHOP), University of Pennsylvania </li></ul><ul><ul><li>Tissue Microarray (TMA): 155 Tumors, 1-4 cores </li></ul></ul>TUMOR SPECIMENS
    24. 25. CASE SERIES ANALYZED for MICROVASCULAR PROLIFERATION Clinical Parameter CMH (1) CHOP GENDER M,F 23, 28 70, 73 AGE at diagnosis Age Range 16<1yr, 30>1yr 6d – 14y 90>1yr, 51<1yr 5d – 17y Dx GN, GNB, NB 5, 13, 33 12, 24, 118 STAGE 1, 2, 3, 4, 4s 18, 8, 8, 12 45, 39, 25, 27, 1 MYCN A, S, u 10, 33, 3 11, 85, 47 PROGNOSTIC F,U,u 24, 19, 3 80, 58, 5 RISK High, Low 15, 31 38, 104 Overall SURVIVAL 82 % 84 %
    25. 26. CASE SERIES ANALYZED for CANCER-ASSOCIATED FIBROBLASTS Clinical Parameter CMH (2) AGE at diagnosis 34>1yr, 19<1yr Dx GN, GNB, NB 7, 11, 42 STAGE 1+2+3, 4 34, 15 MYCN A, S, u 9, 40, 4 PROGNOSTIC F,U,u 28, 23, 2 RISK High, Low 15, 25 5-Yr Survival: Hi, Lo Risk 61%, 100% MVP Yes, No 38, 22
    26. 27. Ganglioneuroma Schwannian Stroma-Dominant THIN-WALLED, DILATED & CAPILLARY-LIKE VESSELS H&E
    27. 28. Ganglioneuroma Schwannian Stroma-Dominant PERICYTE COVERAGE in THIN-WALLED VESSELS  SMA Pericytes CD31 Endothelial Cells
    28. 29. Ganglioneuroblastoma Intermixed Schwannian Stroma-Rich THIN-WALLED, CLASSIC ANGIOGENIC VESSELS with SPROUTING H&E
    29. 30. Ganglioneuroblastoma Nodular MICROVASCULAR PROLIFERATION SS-POOR REGION NEUROPIL H&E
    30. 31. Microvascular Proliferation Thin-Walled vessels SS-RICH SS-POOR GNB Nodular H&E
    31. 32. ENDOTHELIAL CELLS within MICROVASCULAR PROLIFERATION in SS-POOR REGION NEUROPIL CD31
    32. 33. Neuroblastoma Differentiating Schwannian-Stroma Poor GLOMERULOID MICROVASCULAR PROLIFERATION
    33. 34. Neuroblastoma Differentiating Schwannian Stroma-Poor  SMA ENDOTHELIAL CELLS and PERICYTES WITHIN STROMA CD31
    34. 35. MICROVASCULAR PROLIFERATION is PRESENT in SCHWANNIAN STROMA-POOR REGIONS and TUMORS Percent of Cases 8/8 4/4 53/82 11/16 17/28 6/11 4/4 5/5 0/6 0/5 0/15 0/9 0/12 0/7 █ TMA-CHOP n=155 █ CMH n=53
    35. 36. MICROVASCULAR PROLIFERATION is ASSOCIATED with SHORTER SURVIVAL in NEUROBLASTOMA (patients all ages; n=46 CMH) Peddinti , Zeine et al. Clin. Cancer Res., 13, 2007 Kaplan-Meier Curves Log-Rank test p=0.017 Percent Survival No Microvascular Proliferation ( n=20: 13 NB, 7 GNB ) Survival in Years Microvascular Proliferation ( n=26: 20 NB, 6 GNB ) 0 2 4 6 8 10 12 14 0 20 40 60 80 100 65% ± 3 100%
    36. 37. MICROVASCULAR PROLIFERATION is ASSOCIATED with SHORTER SURVIVAL in NEUROBLASTOMA (patients all ages; n=143 CHOP) Peddinti , Zeine et al. Clin. Cancer Res., 13, 2007 No Microvascular Proliferation (n=60: 41 NB, 19 GNB) Survival in Years Percent Survival Microvascular Proliferation (n=83: 77 NB, 5 GNB) Kaplan-Meier Curves Log-rank test p=0.014 0 5 10 15 0 20 40 60 80 100 78% 95%
    37. 38. MICROVASCULAR PROLIFERATION is a POOR PROGNOSTIC FACTOR in NEUROBLASTOMA Single-Predictor Cox Regression Analysis (TMA-CHOP) RISK FACTOR ASSOCIATION with SHORTER SURVIVAL p-value Age ( ≥ 1 year vs < 1 year) 0.014 Stage (4 vs <4) <0.001 MYCN (Amplified vs Single Copy) 0.003 Prognostic Category (Unfavorable vs Favorable) <0.001 MVP (Present vs. Absent) 0.02
    38. 39. HIGH RISK NEUROBLASTOMA TUMORS are ASSOCIATED with MICROVASCULAR PROLIFERATION (143 tumors, CHOP) Peddinti, Zeine et al. Clinical Cancer Research, vol.13, June 2007 RISK FACTORS ASSOCIATION with MVP Chi-Square test p-value High-Risk Group <0.001 Unfavorable Prognosis <0.001 Advanced Stage 0.008 MYCN Amplification 0.006 Stroma-poor <0.001
    39. 40. <ul><li>There were no deaths in the subset of </li></ul><ul><li>patients with MYCN amplified tumors that lacked Microvascular Proliferation (n=5). </li></ul><ul><li>The outcome was significantly better for patients with stage 4 disease who </li></ul><ul><li>lacked Microvascular Proliferation. </li></ul>SURVIVORS LACKED MICROVASCULAR PROLIFERATION
    40. 41. Neuroblastoma Undifferentiated Schwannian Stroma-Poor FIBROVASCULAR STROMA
    41. 42. CANCER-ASSOCIATED FIBROBLASTS in Neuroblastoma Undifferentiated  SMA+ve
    42. 43. NO CANCER-ASSOCIATED FIBROBLASTS in Ganglioneuroma  SMA positive pericytes
    43. 44.  SMA hCD Pericytes FEW CANCER-ASSOCIATED FIBROBLASTS in Ganglioneuroblastoma Intermixed Cancer-Associated Fibroblasts
    44. 45. MORE CANCER-ASSOCIATED FIBROBLASTS in Ganglioneuroblastoma Nodular  SMA hCD Pericytes Cancer- Associated Fibroblasts
    45. 46. ABUNDANT CANCER-ASSOCIATED FIBROBLASTS in Neuroblastoma Differentiating
    46. 47. Quantitative IHC (Clarient-Chromavision) Automated Cellular Imaging System II Scanned the  SMA-stained Slides Set Color Detection Thresholds Delineated Tumor Regions Calculated Ratios of Areas Stained to Total Areas Analyzed Stain included Pericytes and Cancer Associated Fibroblasts Neuroblastoma Ganglioneuroma
    47. 48. 0.0% 1.0% 2.0% 3.0% 4.0% 5.0% 6.0% 7.0% 8.0% PERCENT  SMA+ve REGIONS in NEUROBLASTOMA TUMORS  SMA +ve AREA NBU n=5 NBPD 14 NBD 23 NGNB GNBI 7 GN 7 4 Low Med High
    48. 49. CANCER-ASSOCIATED FIBROBLASTS ASSOCIATED with SS-POOR STROMA and MICROVASCULAR PROLIFERATION Zeine et al. Mod. Pathol. 22(1s), 2009
    49. 50. 0.0% 1.0% 2.0% 3.0% 4.0% 5.0% GN n=7 GNBI n=7 GNBN n=4 NB (D+PD+U) n=42 INCREASED  SMA +ve AREAS in SS-POOR STROMA Mean±SD  - SMA POSITIVE AREA student’s t-test p<0.001   SS-Rich Tumors SS-Poor Tumors or Regions Zeine et al. Mod. Pathol. 26(4), 2009
    50. 51. INCREASED  SMA +ve AREAS in NEUROBLASTOMA TUMORS with MICROVASCULAR PROLIFERATION Mean ±SD Percent  -SMA POSITIVE AREA 0.0% 1.0% 2.0% 3.0% 4.0% 5.0% 6.0% GN n=7 NB+GNBI 15 NB+GNBN 38 p<0.001  student’s t-test No MVP With MVP Zeine et al. Mod. Pathol. 26(4), 2009
    51. 52. SS-Rich, No MVP, No CAFs Tumor-INHIBITORY Role SS-Poor, with MVP & CAFs Tumor-PROMOTING Role Ganglioneuroma Ganglioneuroblastoma Intermixed NB Differentiated NB Poorly Differentiated Zeine et al. Mod. Pathol. 26(4), 2009
    52. 53. SCHWANNIAN-ENRICHED MICROENVIRONMENT in NEUROBLASTOMA XENOGRAFT MODEL <ul><li>Human Cell Line </li></ul><ul><li>SMS-KCNR </li></ul><ul><li>MYCN amplified </li></ul><ul><li>~10 7 cells </li></ul><ul><li>Athymic Female </li></ul><ul><li>Nude Mice </li></ul><ul><li>SCIATIC NERVE </li></ul>Liu et al. Am. J. Pathol. , vol.166 (3), ’05 Control OUTSIDE Sciatic Nerve INSIDE Sciatic Nerve S-100 Schwann Cells
    53. 54. Control, SS-Poor Outside Sciatic Nerve Cancer-Associated Fibroblasts Schwann-Enriched Xenograft Inside Sciatic Nerve Pericytes Absent Fibroblasts  SMA Zeine et al. Mod. Pathol. 26(4), 2009
    54. 55. Control n=9 Inside ScN. n=10 Mean ± SD  SMA +ve Cells / mm 2 LESS Cancer-Associated Fibroblasts in Neuroblastoma Xenografts ENGRAFTED INSIDE the SCIATC NERVE Zeine et al. Mod. Pathol. 26(4), 2009 The Inverse Relation between Schwann Cells and Cancer-Associated Fibroblasts Suggests that the Schwannian Microenvironment might Interfere with either Fibroblast Infiltration or Activation, thus inhibiting Angiogenesis. p<0.001 *
    55. 56. ANTI-ANGIOGENIC TREATMENT in NEUROBLASTOMA XENOGRAFT MODELS <ul><li>Two NMYC-Amplified Neuroblastoma Cell Lines: NMB </li></ul><ul><li>SMS-KCNR </li></ul><ul><li>~10 7 cells s/c </li></ul><ul><li>Female 4-6wk Athymic Nude Mice </li></ul><ul><li>Treatment Protocols: Valproic Acid 400 mg/Kg </li></ul><ul><li>Small Tumors (70 mm 3 ) treated 1x/d for 20d </li></ul><ul><li>Large Tumors (380 mm 3 ) treated 1x/d for 10d </li></ul>
    56. 57. BLOCKING ANGIOGENESIS Valproic Acid <ul><li>Branched Chain Fatty Acid </li></ul><ul><li>Inhibits Histone Deacetylase (HDAC) </li></ul><ul><li>Inhibits Growth of Neuroblastoma and Other Cancers </li></ul><ul><li>Blocks VEGF – and Hypoxia – induced Angiogenesis </li></ul><ul><li>Michaelis et al. Mol. Pharmacol. 65, 2004 </li></ul>HDAC inhibitors Histone acetylation Anti-neoplastic
    57. 58. VALPROIC ACID INHIBITS TUMOR GROWTH IN VIVO DAYS Tumor Volume mm 3 Yang,… Zeine et al. Cancer Res. 26(4), 2007
    58. 59. Control SMS-KCNR Xenografts Micro-Vascular Proliferation ValproicAcid Treatment Thin Walled Vessels ANTI-ANGIOGENIC TREATMENT ABROGATES MICROVASCULAR PROLIFERATION in NB H&E Yang,… Zeine et al. Cancer Res. 26(4), 2007
    59. 60. Control NMB MICROVASCULAR PROLIFERATION Valproic Acid VASCULAR NORMALIZATION FibroCollagenous Stroma H&E Masson Trichrome Yang,… Zeine et al. Cancer Res. 26(4), 2007
    60. 61. ‘ Normalization’ of Vessels Tong et al. Cancer Research 64 (11), 2004 Murine Mammary Carcinoma Two-Photon Microscopy Control Anti-Angiogenic VEGF2R Blocker DC101 Day 0 5 1 2 3 4
    61. 62. Abrogation of Microvascular Proliferation by Anti-Angiogenic Treatment with Valproic Acid Percent of Total Vessels With Microvascular Proliferation Neuroblastoma Xenografts
    62. 63. FUNCTIONAL IMPROVEMENT LESS LEAKY Reduced Intratumoral Vessel Permeability by Anti-Angiogenic Treatment with Valproic Acid Spectrophotometric Quantitation of Evan’s Blue Extraction into formamide for 72 hr, RT 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 VPA Treated n=4 Control n=5 p=0.04 * Relative OD 620 nm Neuroblastoma SMS-KCNR Xenografts (student’s t-test) Yang,… Zeine et al. Cancer Res. 26(4), 2007 Extravasation 100  l, 0.2% Evan’s Blue by Tail-Vein Injections Let Circulate 20 min. 25 ml Saline Perfusion
    63. 64. Control SMS-KCNR Xenografts ValproicAcid Treatment LESS CANCER-ASSOCIATED FIBROBLASTS Following ANTI-ANGIOGENIC TREATMENT x200  SMA x400
    64. 65. 1- Microvacular Proliferation is a Poor Prognostic Indicator present in SS-Poor Neuroblastoma Regions. 2- Cancer-Associated Fibroblasts Colocalize with Microvacular Proliferation and are Precluded from SS-Rich Regions of Neuroblastoma Tumors. 3- It is Possible to Reduce Cancer-Associated Fibroblast Infiltration by Increasing the Schwannian Component within the Tumor Microenvironment. 4- Anti-Angiogenic Treatment Reduced Microvascular Proliferation and Cancer-Associated Fibroblasts in Neuroblastoma Xenografts. CONCLUSIONS
    65. 66. SIGNIFICANCE <ul><li>Histopathologic Evaluation of Neuroblastoma Tumors for Microvascular Proliferation and Cancer-Associated Fibroblasts May Help Refine Risk Group and Prognostic Classification. </li></ul><ul><li>May Have Impact on Eligibility Criteria for Stratification of Patients for Inclusion in Clinical Trials. </li></ul><ul><li>May Guide Development of Novel Anti-Angiogenic and Anti-Stromal Treatment Strategies for Children with Aggressive Neuroblastoma. </li></ul><ul><li>Results are Consistent with Tumor-Inhibitory Roles for Schwannian Derived Factors, and Tumor-Promoting Roles for Cancer-Associated Fibroblasts. </li></ul>
    66. 67. ACKNOWLEDGEMENTS University of Pennsylvania (CHOP) John M. Maris, MD Kristina Cole, MD Bruce Pawel, MD Statistics (CMH and COG) Roopa Seshadri, PhD Wendy B. London, PhD Susan L. Cohn, MD Radhika Peddinti, MD Alex Chlenski, PhD Qiwei Yang, PhD Helen Salwen Yufeng Tian Lisa Guerrero Maria Tretiakova, MD, PhD Northwestern University Robert H. Lurie Comprehensive Cancer Center University of Chicago
    67. 68. FUTURE DIRECTIONS <ul><li>Elucidate the Molecular Mechanisms for Tumor-Stromal Interactions and the Antagonism Between Fibrovascular Stroma and Schwannian Stroma. </li></ul><ul><li>Discover and Identify Stromal Elements of Biologic and Clinical Significance in Cancer. </li></ul><ul><li>Identify Cancer Stem Cells within tumors. </li></ul><ul><li>Pursue Preclinical Testing of Therapeutic Strategies Targeting Tumor Stroma or Altering Tumor Microenvironment. </li></ul>
    68. 69. New ‘ANTI-STROMAL’ Agents <ul><li>Aptenins NBRI23477 A and B (antibiotics) </li></ul><ul><li>(Prostate cancer co-cultures with stromal cells), Kawada et al. J. Antibiot (Tokyo) 62(5), 2009 </li></ul><ul><li>Phthoxazolin, Inthomycin B </li></ul><ul><li>Inhibit Expression of (IGF)-1 and binding proteins </li></ul><ul><li>Inhibit TGF  1 activity </li></ul><ul><li>(Prostate cancer co-cultures with stromal cells), Kawada et al. Cancer Sci. 100(1), 2008 </li></ul><ul><li>Pioglitazone, Ciglitazone </li></ul><ul><li>Peroxisome Proliferator Activated Receptor Agonists </li></ul><ul><li>(PPAR: members of Ligand Activated Nuclear Receptor Superfamily, transcriptional activators) </li></ul><ul><li> (Liver cancer), Bundscherer et al. Anticancer Agents Med Chem, 2009 </li></ul>
    69. 70. STAGING SYSTEM in Neuroblastoma Brodeur et al. J Clin Oncol 11,1993 STAGE DEFINITION 1 Localized Tumor with Complete Gross Excision; -ve Nodes 2A Localized Tumor with Incomplete Gross Excision; -ve Nodes 2B Localized Tumor with +ve Ipsilateral Nodes 3 Localized Tumor with +ve Contralateral Nodes, or Unresectable Tumor; Infiltrating Across Midline 4S Localized Tumor, in Infant < 1 yr, with Dissemination Limited to Skin, Liver and/or Bone Marrow (Minimal) 4 Any Primary Tumor with Distant Metastasis (except 4S)
    70. 71. FIBROVASCULAR STROMA in SS-Poor NB Tumors Masson Trichrome special stain

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