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    <TD>Journal of Neuro-Oncology</TD></TR>
  <TR>
    <TD>=A9&nbsp;The Author(s)&nbsp;2009</TD></TR>
  <TR>
    <TD>10.1007/s11060-009-9983-4</TD></TR></TBODY></TABLE><!--Begin =
Abstract-->
<H2 class=3Drubric>Laboratory Investigation - Human/Animal Tissue</H2>
<DIV class=3DHeading1><A name=3Dtitle></A>Stem cell associated gene =
expression in=20
glioblastoma multiforme: relationship to survival and the subventricular =
zone=20
</DIV>
<P class=3DAuthorGroup>Melanie&nbsp;Kappadakunnel<SUP>1</SUP>,=20
Ascia&nbsp;Eskin<SUP>1</SUP>, Jun&nbsp;Dong<SUP>1</SUP>,=20
Stanley&nbsp;F.&nbsp;Nelson<SUP>1</SUP>, =
Paul&nbsp;S.&nbsp;Mischel<SUP>1</SUP>,=20
Linda&nbsp;M.&nbsp;Liau<SUP>1</SUP>, Phioanh&nbsp;Ngheimphu<SUP>1</SUP>, =

Albert&nbsp;Lai<SUP>1</SUP>, Timothy&nbsp;F.&nbsp;Cloughesy<SUP>1</SUP>, =

Jonathan&nbsp;Goldin<SUP>1</SUP> and =
Whitney&nbsp;B.&nbsp;Pope<SUP>1&nbsp;<A=20
href=3D"http://www.springerlink.com/content/l26153725wvl0625/fulltext.htm=
l#ContactOfAuthor11"><IMG=20
alt=3D"Contact Information"=20
src=3D"http://www.springerlink.com/content/l26153725wvl0625/contact.gif" =

border=3D0></A></SUP></P>
<TABLE>
  <TBODY>
  <TR vAlign=3Dtop>
    <TD><SPAN class=3DAffiliation><A =
name=3DAff1></A>(1)&nbsp;</SPAN></TD>
    <TD><SPAN class=3DAffiliation>University of California Los Angeles, =
Los=20
      Angeles, CA&nbsp;90095, USA</SPAN></TD></TR></TBODY></TABLE>
<P><A name=3DContactOfAuthor11></A></P>
<TABLE class=3DContact>
  <TBODY>
  <TR>
    <TD vAlign=3Dtop><IMG alt=3D"Contact Information"=20
      =
src=3D"http://www.springerlink.com/content/l26153725wvl0625/contact.gif" =

      border=3D0></TD>
    =
<TD><STRONG>Whitney&nbsp;</STRONG><STRONG>B.&nbsp;</STRONG><STRONG>Pope</=
STRONG><STRONG></STRONG><BR><STRONG>Email:=20
      </STRONG><A=20
    =
href=3D"mailto:wpope@mednet.ucla.edu">wpope@mednet.ucla.edu</A></TD></TR>=
</TBODY></TABLE>
<P class=3DAffiliation><STRONG>Received:=20
</STRONG>1&nbsp;June&nbsp;2009&nbsp;&nbsp;<STRONG>Accepted:=20
</STRONG>23&nbsp;July&nbsp;2009&nbsp;&nbsp;<STRONG>Published online:=20
</STRONG>5&nbsp;August&nbsp;2009 </P>
<DIV class=3DAbstract><A name=3DAbs1></A><SPAN=20
class=3DAbstractHeading>Abstract&nbsp;&nbsp;</SPAN>Current therapies for =

glioblastoma (GBM) target bulk tumor through measures such as resection =
and=20
radiotherapy. However, recent evidence suggests that targeting a subset =
of tumor=20
cells, so-called cancer stem cells, may be critical for inhibiting tumor =
growth=20
and relapse. The subventricular zone (SVZ), which lines the ventricles =
of the=20
brain, is thought to be the origin for the majority of neural stem cells =
and=20
potentially cancer stem cells. Therefore, we assessed the relationship =
between=20
tumor contact with the SVZ as determined by MRI, cancer stem cell gene=20
expression and survival in 47 patients with GBM. Using DNA microarrays, =
we found=20
that genes associated with cancer stem cells were not over-expressed in =
tumors=20
contacting the SVZ. Contact with the SVZ trended with shorter survival =
(median=20
358 versus 644, <I>P</I>&nbsp;=3D&nbsp;0.066). Over-expression of CD133=20
(prominin-1) and maternal embryonic leucine zipper kinase (MELK) was =
associated=20
with shorter survival, whereas mitogen activated protein kinase 8 =
(MAPK8) was=20
associated with longer survival (<I>P</I> values 0.008, 0.005 and 0.002=20
respectively). Thus we found no evidence of a stem-cell derived genetic=20
signature specific for GBM in contact with the SVZ, but there was a =
relationship=20
between stem cell gene expression and survival. More research is =
required to=20
clarify the relationship between the SVZ, cancer stem cells and =
survival. </DIV>
<P class=3DKeyword><SPAN=20
class=3DKeywordHeading>Keywords&nbsp;&nbsp;</SPAN>Glioblastoma&nbsp;-&nbs=
p;Glioma&nbsp;-&nbsp;Subventricular=20
zone&nbsp;-&nbsp;Microarray&nbsp;-&nbsp;Stem=20
cell&nbsp;-&nbsp;Survival&nbsp;-&nbsp;Genomics&nbsp;-&nbsp;MRI </P>
<DIV class=3DArticleNote><SPAN class=3D"">Melanie Kappadakunnel and =
Ascia Eskin=20
contributed equally to this report.</SPAN></DIV>
<DIV class=3DFulltext>
<DIV class=3D""><A name=3DSec1></A>
<HR>

<DIV class=3Dheading2>Introduction</DIV>
<P class=3D"">Survival of patients with glioblastoma (GBM) remains poor. =
Current=20
therapy for gliomas is typically based on cytoreductive strategies, such =
as=20
surgery and radiotherapy, which target the bulk of the tumor mass rather =
than=20
specific cell types [<CITE><A=20
href=3D"http://www.springerlink.com/content/l26153725wvl0625/fulltext.htm=
l#CR1">1</A></CITE>].=20
Recently it has been suggested that a minority of the cells in the =
tumor,=20
so-called cancer stem cells, account for most of the proliferation, =
growth and=20
radioresistance [<CITE><A=20
href=3D"http://www.springerlink.com/content/l26153725wvl0625/fulltext.htm=
l#CR2">2</A></CITE>;=20
reviewed in <CITE><A=20
href=3D"http://www.springerlink.com/content/l26153725wvl0625/fulltext.htm=
l#CR3">3</A></CITE>].=20
These cells can also propagate tumors in vitro. Therefore the =
elimination of=20
these cancer stem cells may prove necessary to effect a cure for glioma =
patients=20
[<CITE><A=20
href=3D"http://www.springerlink.com/content/l26153725wvl0625/fulltext.htm=
l#CR4">4</A></CITE>].=20
The subventricular zone (SVZ), which lines the ventricles, maintains the =
ability=20
to generate neurons and glia throughout adulthood, and in animal models, =
gliomas=20
can be generated from this cell population. It has been suggested that =
the SVZ=20
is a source for cancer stem cells that initiate gliomagenesis [<CITE><A=20
href=3D"http://www.springerlink.com/content/l26153725wvl0625/fulltext.htm=
l#CR5">5</A></CITE>].=20
</P>
<P class=3D"">GBM are heterogeneous tumors, histopathologically, =
radiographically=20
and genetically [<CITE><A=20
href=3D"http://www.springerlink.com/content/l26153725wvl0625/fulltext.htm=
l#CR6">6</A></CITE>].=20
Several genetic subtypes have been characterized that are associated =
with=20
varying survival rates. For instance, an oligodendroglioma-like genetic=20
signature is associated with longer survival [<CITE><A=20
href=3D"http://www.springerlink.com/content/l26153725wvl0625/fulltext.htm=
l#CR7">7</A></CITE>,=20
<CITE><A=20
href=3D"http://www.springerlink.com/content/l26153725wvl0625/fulltext.htm=
l#CR8">8</A></CITE>].=20
Survival also may be impacted by the tumor=92s relationship to the SVZ =
[<CITE><A=20
href=3D"http://www.springerlink.com/content/l26153725wvl0625/fulltext.htm=
l#CR9">9</A></CITE>,=20
<CITE><A=20
href=3D"http://www.springerlink.com/content/l26153725wvl0625/fulltext.htm=
l#CR10">10</A></CITE>].=20
Lim et al. [<CITE><A=20
href=3D"http://www.springerlink.com/content/l26153725wvl0625/fulltext.htm=
l#CR9">9</A></CITE>]=20
have suggested that GBM contacting the SVZ are more likely to recur as=20
multifocal disease. And previously it has been shown that multifocal =
disease is=20
associated with shortened survival [<CITE><A=20
href=3D"http://www.springerlink.com/content/l26153725wvl0625/fulltext.htm=
l#CR11">11</A></CITE>].=20
It also has been proposed that GBM contacting the SVZ may be more likely =
to be=20
stem-cell-derived, with potential impact on treatment decisions =
[<CITE><A=20
href=3D"http://www.springerlink.com/content/l26153725wvl0625/fulltext.htm=
l#CR9">9</A></CITE>].=20
</P>
<P class=3D"">Recently several stem cell marker proteins have been =
identified. The=20
most well-characterized, CD133, is associated with shortened survival =
[<CITE><A=20
href=3D"http://www.springerlink.com/content/l26153725wvl0625/fulltext.htm=
l#CR12">12</A></CITE>].=20
However, to date, there has been no analysis of gene expression and =
survival in=20
tumors based on contact with the SVZ. Therefore, we used microarrays to =
analyze=20
the relationship between tumors associated with the SVZ, expression of =
cancer=20
stem cell and proliferative genes, and survival. </P></DIV>
<DIV class=3D""><A name=3DSec2></A>
<HR>

<DIV class=3Dheading2>Materials and methods</DIV>
<DIV class=3D""><A name=3DSec3></A>
<DIV class=3DHeading3>Patient selection</DIV>
<P class=3D"">All patients participating in this study signed =
institutional review=20
board-approved informed consent. Data acquisition was performed in =
compliance=20
with all applicable Health Insurance Portability and Accountability Act =
(HIPAA)=20
regulations. Patients were retrospectively selected from our =
institution=92s=20
neuro-oncology database. All patients (<I>n</I>&nbsp;=3D&nbsp;47) who =
met the=20
following criteria were selected: (1) pathology confirmed GBM (2) =
baseline=20
(pre-surgical) MRI (3) fresh-frozen tissue adequate for microarray =
analysis. All=20
patients were treated with radiation therapy (RT; 6000 =B1 500&nbsp;cGy) =
and=20
subtotal/total resection at time of initial tumor presentation. Accrual =
dates=20
for this study were October 1999 to January 2004. A total of 26/47 =
(55.3%)=20
patients received adjuvant chemotherapy, most commonly temozolomide =
(15/47,=20
31.9%). Some patients also received salvage chemotherapy. </P></DIV>
<DIV class=3D""><A name=3DSec4></A>
<DIV class=3DHeading3>Imaging</DIV>
<DIV class=3DPara>
<DIV class=3D"">MRI sequences were acquired on a 1.5T scanner and =
typically=20
included axial T1 weighted (TR 400, TE 15, slice thickness 5&nbsp;mm), =
T2=20
weighted fast spin-echo (TR 4000, TE 126-130, slice thickness =
5&nbsp;mm),=20
diffusion-weighted and gadopentetate dimeglumine enhanced (Magnevist; =
Berlex,=20
Wayne, NJ; 0.1&nbsp;mmol/kg) axial and coronal T1 weighted images (TR =
400, TE=20
15, slice thickness 3&nbsp;mm), with a field of view of 24&nbsp;cm and a =
matrix=20
size of 256&nbsp;=D7&nbsp;256. Post-contrast images were acquired =
immediately=20
after contrast injection. The contrast-enhancing tumor was assessed for =
contact=20
with the SVZ and cortex as described in [<CITE><A=20
href=3D"http://www.springerlink.com/content/l26153725wvl0625/fulltext.htm=
l#CR9">9</A></CITE>].=20
Tumors were segregated into 4 groups by a neuroradiologist (W.P.): group =
I,=20
tumor contacting SVZ and infiltrating cortex 9 =
(<I>n</I>&nbsp;=3D&nbsp;13); group=20
II, tumor contacting SVZ but not involving the cortex =
(<I>n</I>&nbsp;=3D&nbsp;12);=20
group III, tumor involving cortex but not contacting the SVZ=20
(<I>n</I>&nbsp;=3D&nbsp;16); group IV, tumor involving neither SVZ nor =
cortex=20
(<I>n</I>&nbsp;=3D&nbsp;6; Fig.&nbsp;<A=20
href=3D"http://www.springerlink.com/content/l26153725wvl0625/fulltext.htm=
l#Fig1">1</A>).=20
A minority of patients had multifocal disease at initial presentation =
(7/47,=20
15%). For these patients, the scans were graded according to the =
dominant tumor=20
nodule.=20
<DIV class=3DFigure><A name=3DFig1></A><IMG=20
alt=3DMediaObjects/11060_2009_9983_Fig1_HTML.jpg=20
src=3D"http://www.springerlink.com/content/l26153725wvl0625/MediaObjects/=
11060_2009_9983_Fig1_HTML.jpg"></DIV>
<DIV class=3DCapt><SPAN class=3DCaptNr>Fig.&nbsp;1&nbsp;</SPAN>Tumor =
grading. Group=20
I (<B>a</B>), tumor contacting SVZ and infiltrating cortex=20
(<I>n</I>&nbsp;=3D&nbsp;13); group II (<B>b</B>), tumor contacting SVZ =
but not=20
involving the cortex (<I>n</I>&nbsp;=3D&nbsp;12); group III (<B>c</B>), =
tumor=20
involving cortex but not contacting the SVZ (<I>n</I>&nbsp;=3D&nbsp;16); =
group IV=20
(<B>d</B>), tumor involving neither SVZ nor cortex =
(<I>n</I>&nbsp;=3D&nbsp;6)=20
</DIV>
<HR>
</DIV></DIV></DIV>
<DIV class=3D""><A name=3DSec5></A>
<DIV class=3DHeading3>Microarray data</DIV>
<P class=3D"">RNA was purified from fresh frozen tumor biopsies and was =
hybridized=20
to U133A and U133 Plus 2.0 Arrays at our DNA Microarray Facility. The =
data files=20
generated were normalized using RMA from Bioconductor (for R, version =
2.6; <A=20
href=3D"http://cran.r-project.org/bin/windows/base/">http://cran.r-projec=
t.org/bin/windows/base/</A>),=20
relative to other Affymetrix microarrays of the same platform using the =
Celsius=20
Database (<A=20
href=3D"http://www.ncbi.nlm.nih.gov/pubmed/17570842">http://www.ncbi.nlm.=
nih.gov/pubmed/17570842</A>).=20
Only probes from the U133A portion that were retained in U133 Plus 2.0 =
arrays=20
were analyzed for each sample. RMA normalized data was imported into =
dChip=20
[<CITE><A=20
href=3D"http://www.springerlink.com/content/l26153725wvl0625/fulltext.htm=
l#CR12">12</A></CITE>]=20
for differential expression analysis among genes in different SVZ =
groups. We=20
compared the fold change for the mean of each gene in the different SVZ =
groups,=20
and used a <I>t</I>-test to assess the significance. Significant genes =
were=20
further analyzed by the DAVID bioinformatics tool (The US National =
Institutes of=20
Health (NIH) Database for Annotation, Visualization and Integrated =
Discovery; <A=20
href=3D"http://david.abcc.ncifcrf.gov/">http://david.abcc.ncifcrf.gov/</A=
>;=20
[<CITE><A=20
href=3D"http://www.springerlink.com/content/l26153725wvl0625/fulltext.htm=
l#CR13">13</A></CITE>,=20
<CITE><A=20
href=3D"http://www.springerlink.com/content/l26153725wvl0625/fulltext.htm=
l#CR14">14</A></CITE>]),=20
to identify enriched biological groups [<CITE><A=20
href=3D"http://www.springerlink.com/content/l26153725wvl0625/fulltext.htm=
l#CR15">15</A></CITE>].=20
</P></DIV>
<DIV class=3D""><A name=3DSec6></A>
<DIV class=3DHeading3>Heat map generation</DIV>
<P class=3D"">Gene expression was visualized through heat maps generated =
by dChip.=20
Differentially expressed genes identified among the SVZ groups were=20
hierarchically clustered, while the samples were arranged by SVZ group.=20
</P></DIV>
<DIV class=3D""><A name=3DSec7></A>
<DIV class=3DHeading3>Statistical methods</DIV>
<P class=3D"">The survival library from R (version 2.6; <A=20
href=3D"http://cran.r-project.org/">http://cran.r-project.org/</A>) and =
the Cox=20
proportional hazards model were used to analyze the relationship between =
gene=20
expression and survival. Results for all genes were obtained and the =
values for=20
the stem cell genes of interest, and the differentially expressed genes =
for the=20
SVZ groups were extracted. Cox model regression analysis results were =
obtained=20
for each of the genes and a transformed hazard ratio (<I>Z</I> score, =
the ratio=20
of the regression coefficient to the standard error) was generated. A =
positive=20
score <I>Z</I> coefficient indicates higher expression corresponding to =
higher=20
hazard rate and shorter survival, and lower expression corresponding to =
a lower=20
hazard rate and longer survival. Kaplan=96Meier curves with log-rank =
test were=20
used to analyze survivability patterns between SVZ grades and gene =
expression.=20
Expression fold change between tumor groups was computed with a fold =
change=20
threshold of 1.2 with a lower 90% confidence bound, and a <I>t</I>-test =
<I>P</I>=20
value of 0.05. For most analyses, a <I>P</I> value of &lt;0.05 was =
accepted as=20
significant. Some analyses were performed with a Bonferroni correction =
for=20
multiple testing. </P></DIV></DIV>
<DIV class=3D""><A name=3DSec8></A>
<HR>

<DIV class=3Dheading2>Results</DIV>
<DIV class=3D""><A name=3DSec9></A>
<DIV class=3DHeading3>Poor correlation of stem cell gene =
expression</DIV>
<DIV class=3DPara>
<DIV class=3D"">A total of 7 genes of interest related to cancer stem =
cells or=20
tumor proliferation, based on previous literature [<CITE><A=20
href=3D"http://www.springerlink.com/content/l26153725wvl0625/fulltext.htm=
l#CR12">12</A></CITE>,=20
<CITE><A=20
href=3D"http://www.springerlink.com/content/l26153725wvl0625/fulltext.htm=
l#CR16">16</A></CITE>=96<CITE><A=20
href=3D"http://www.springerlink.com/content/l26153725wvl0625/fulltext.htm=
l#CR20">20</A></CITE>],=20
were selected for analysis. The genes were CD133 (prominin1), maternal =
embryonic=20
leucine zipper kinase (MELK), bone morphogenetic protein 4 (BMP 4), E2F=20
transcription factor 2 (E2F 2), mitogen-activated protein kinase 8 (MAPK =
8; also=20
known as Jun NH2-terminal kinase), OLIG2, and nestin (NES). We attempted =
to find=20
a stem cell signature which consisted of a number of genes whose =
expression=20
correlated highly with one another or over expression of stem cells =
genes in a=20
specific tumor group, but found none. The correlation matrix showed very =
low=20
correlation between any of the stem cell genes of interest =
(Table&nbsp;<A=20
href=3D"http://www.springerlink.com/content/l26153725wvl0625/fulltext.htm=
l#Tab1">1</A>).=20
Lack of correlation is confirmed on the heat map (Fig.&nbsp;<A=20
href=3D"http://www.springerlink.com/content/l26153725wvl0625/fulltext.htm=
l#Fig2">2</A>).<A=20
name=3DTab1></A>
<DIV class=3DCapt><SPAN =
class=3DCaptNr>Table&nbsp;1&nbsp;</SPAN>Correlation of stem=20
cell gene expression in 47 patients with GBM </DIV>
<TABLE border=3D1>
  <COLGROUP>
  <COL align=3Dleft>
  <COL align=3Dchar char=3D".">
  <COL align=3Dchar char=3D".">
  <COL align=3Dchar char=3D".">
  <COL align=3Dchar char=3D".">
  <COL align=3Dchar char=3D".">
  <COL align=3Dchar char=3D".">
  <COL align=3Dchar char=3D".">
  <COL align=3Dchar char=3D".">
  <COL align=3Dchar char=3D"."></COLGROUP>
  <THEAD>
  <TR class=3Dheader>
    <TH align=3Dleft>&nbsp;</TH>
    <TH align=3Dleft char=3D".">
      <P class=3D"">CD133</P></TH>
    <TH align=3Dleft char=3D".">
      <P class=3D"">MELK</P></TH>
    <TH align=3Dleft char=3D".">
      <P class=3D"">E2F2</P></TH>
    <TH align=3Dleft char=3D".">
      <P class=3D"">MAPK8 (P 1<SUP>a</SUP>) </P></TH>
    <TH align=3Dleft char=3D".">
      <P class=3D"">MAPK8 (P 2)</P></TH>
    <TH align=3Dleft char=3D".">
      <P class=3D"">BMP4</P></TH>
    <TH align=3Dleft char=3D".">
      <P class=3D"">OLIG2 (P 1)</P></TH>
    <TH align=3Dleft char=3D".">
      <P class=3D"">OLIG2 (P 2)</P></TH>
    <TH align=3Dleft char=3D".">
      <P class=3D"">NES</P></TH></TR></THEAD>
  <TBODY>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">CD133</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">=96</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">0.22</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">&#8722;0.17</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">&#8722;0.21</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">&#8722;0.11</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">&#8722;0.24</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">&#8722;0.09</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">&#8722;0.06</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">0.04</P></TD></TR>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">MELK</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">0.22</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">=96</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">0.22</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">&#8722;0.03</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">0.06</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">0.04</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">&#8722;0.14</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">&#8722;0.07</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">0.36</P></TD></TR>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">E2F2</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">&#8722;0.17</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">0.22</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">=96</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">0.53</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">0.52</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">0.53</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">&#8722;0.16</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">&#8722;0.20</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">0.06</P></TD></TR>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">MAPK8 (PS 1)</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">&#8722;0.21</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">&#8722;0.03</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">0.53</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">=96</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">0.81</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">0.43</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">0.13</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">0.22</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">&#8722;0.02</P></TD></TR>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">MAPK8 (PS 2)</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">&#8722;0.11</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">0.06</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">0.52</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">0.81</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">=96</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">0.27</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">0.17</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">0.13</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">&#8722;0.01</P></TD></TR>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">BMP4</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">&#8722;0.24</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">0.04</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">0.53</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">0.43</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">0.27</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">=96</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">&#8722;0.17</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">&#8722;0.13</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">0.15</P></TD></TR>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">OLIG2 (PS 1)</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">&#8722;0.09</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">&#8722;0.14</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">&#8722;0.16</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">0.13</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">0.17</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">&#8722;0.17</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">=96</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">0.77</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">&#8722;0.05</P></TD></TR>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">OLIG2 (PS 2)</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">&#8722;0.06</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">&#8722;0.07</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">&#8722;0.20</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">0.22</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">0.13</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">&#8722;0.13</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">0.77</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">=96</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">&#8722;0.11</P></TD></TR>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">NES</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">0.04</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">0.36</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">0.06</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">&#8722;0.02</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">&#8722;0.01</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">0.15</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">&#8722;0.05</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">&#8722;0.11</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">=96</P></TD></TR></TBODY></TABLE>
<DIV class=3DCapt>
<DIV class=3DCaptCont>
<DIV class=3D""><SUP>a</SUP> <I>P</I> probe. Both MAPK8 and OLIG2 had 2 =
probes=20
available for analysis </DIV></DIV></DIV>
<DIV class=3DFigure><A name=3DFig2></A><IMG=20
alt=3DMediaObjects/11060_2009_9983_Fig2_HTML.gif=20
src=3D"http://www.springerlink.com/content/l26153725wvl0625/MediaObjects/=
11060_2009_9983_Fig2_HTML.gif"></DIV>
<DIV class=3DCapt><SPAN class=3DCaptNr>Fig.&nbsp;2&nbsp;</SPAN>Heat map =
of stem cell=20
probes across group I=96IV. Red corresponds to higher gene expression =
levels.=20
Patient samples noted along top row </DIV>
<HR>
</DIV></DIV></DIV>
<DIV class=3D""><A name=3DSec10></A>
<DIV class=3DHeading3>Stem cell genes associated with survival</DIV>
<DIV class=3DPara>
<DIV class=3D"">In the survival analysis (Cox Model), 6 of the 7 genes =
of interest=20
had at least one probe with a <I>P</I> value significant at the 0.05 =
level. With=20
a multiple testing correction for the 9 probes, 2 of these genes (MAPK8 =
and=20
MELK) are significantly associated with survival at a Bonferroni =
adjusted=20
<I>P</I> value of 0.006 (Table&nbsp;<A=20
href=3D"http://www.springerlink.com/content/l26153725wvl0625/fulltext.htm=
l#Tab2">2</A>).=20
Increased expression levels of CD133 and MELK were associated with =
shorter=20
survival, whereas increased expression levels of nestin, OLIG2, BMP4, =
ETF2 and=20
MAPK8 were associated with longer survival.<A name=3DTab2></A>
<DIV class=3DCapt><SPAN class=3DCaptNr>Table&nbsp;2&nbsp;</SPAN>Stem=20
cell/proliferative gene expression and survival </DIV>
<TABLE border=3D1>
  <COLGROUP>
  <COL align=3Dleft>
  <COL align=3Dleft>
  <COL align=3Dchar char=3D".">
  <COL align=3Dchar char=3D"."></COLGROUP>
  <THEAD>
  <TR class=3Dheader>
    <TH align=3Dleft colSpan=3D2>
      <P class=3D"">Gene</P></TH>
    <TH align=3Dleft rowSpan=3D2 char=3D".">
      <P class=3D"">Z<SUP>a</SUP> </P></TH>
    <TH align=3Dleft rowSpan=3D2 char=3D".">
      <P class=3D""><I>P</I> value<SUP>b,c</SUP> </P></TH></TR>
  <TR class=3Dheader>
    <TH align=3Dleft>
      <P class=3D"">Gene symbol</P></TH>
    <TH align=3Dleft>
      <P class=3D"">Probe</P></TH></TR></THEAD>
  <TBODY>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">MAPK8</P></TD>
    <TD align=3Dleft>
      <P class=3D"">210477_x_at</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">&#8722;3.17</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D""><B>0.002</B> </P></TD></TR>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">MAPK8</P></TD>
    <TD align=3Dleft>
      <P class=3D"">210671_x_at</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">&#8722;2.46</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D""><B>0.014</B> </P></TD></TR>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">BMP4</P></TD>
    <TD align=3Dleft>
      <P class=3D"">211518_s_at</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">&#8722;2.11</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D""><B>0.035</B> </P></TD></TR>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">NES</P></TD>
    <TD align=3Dleft>
      <P class=3D"">218678_at</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">&#8722;2.11</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D""><B>0.035</B> </P></TD></TR>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">OLIG2</P></TD>
    <TD align=3Dleft>
      <P class=3D"">213825_at</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">&#8722;1.98</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D""><B>0.048</B> </P></TD></TR>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">OLIG2</P></TD>
    <TD align=3Dleft>
      <P class=3D"">213824_at</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">&#8722;1.51</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">0.130</P></TD></TR>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">E2F2</P></TD>
    <TD align=3Dleft>
      <P class=3D"">207042_at</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">&#8722;1.36</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">0.170</P></TD></TR>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">CD133</P></TD>
    <TD align=3Dleft>
      <P class=3D"">204304_s_at</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">2.65</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D""><B>0.008</B> </P></TD></TR>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">MELK</P></TD>
    <TD align=3Dleft>
      <P class=3D"">204825_at</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">2.79</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D""><B>0.005</B> </P></TD></TR></TBODY></TABLE>
<DIV class=3DCapt>
<DIV class=3DCaptCont>
<DIV class=3D""><SUP>a</SUP> <I>Z</I> is the transformed hazard ratio. A =
negative=20
<I>Z</I> score indicates higher expression levels are correlated with =
longer=20
survival, and a positive <I>Z</I> score indicates higher expression is=20
correlated with shorter survival </DIV></DIV>
<DIV class=3DCaptCont>
<DIV class=3D""><SUP>b</SUP> <I>P</I> value from Cox model </DIV></DIV>
<DIV class=3DCaptCont>
<DIV class=3D""><SUP>c</SUP>Values &lt;0.05 bolded=20
</DIV></DIV></DIV></DIV></DIV></DIV>
<DIV class=3D""><A name=3DSec11></A>
<DIV class=3DHeading3>Differentially expressed genes in group II tumors: =

association with immune system not cancer stem cells</DIV>
<DIV class=3DPara>
<DIV class=3D"">Although we did not find that stem cell gene expression =
was=20
increased in tumors contacting the SVZ, several other genes were =
over-expressed=20
in tumors contacting the SVZ. The list of differentially expressed genes =
was=20
submitted to the DAVID bioinformatics tool in order to look at over =
represented=20
functional groups and to determine general function. Of the 33 genes (36 =
probes)=20
over-expressed in group II tumors, 7 showed a Gene Ontology (GO) term of =
the=20
immune system process (<I>P</I> value&nbsp;=3D&nbsp;0.03). These non =
stem-cell=20
genes were: FCGR3A, HLA-DRB5, BCL6, FCGR3B, MAFB, HLA-DRA, HLA-E. These =
genes=20
all had higher expression in the group II, with a fold change ranging =
from 1.48=20
to 2.43 (Table&nbsp;<A=20
href=3D"http://www.springerlink.com/content/l26153725wvl0625/fulltext.htm=
l#Tab3">3</A>).=20
The average fold change of the 7 immune system differentially expressed =
genes=20
was 1.82. Figure&nbsp;<A=20
href=3D"http://www.springerlink.com/content/l26153725wvl0625/fulltext.htm=
l#Fig3">3</A>=20
is a heat map showing gene expression by SVZ grade. Note the high =
expression of=20
these immune-related genes in group II tumors. We also assessed the =
expression=20
of these genes in group IV versus groups I and III, since group IV =
tumors also=20
lack cortical involvement. Except for BCL6, the immune genes were all=20
over-expressed in group IV tumors with fold changes ranging from 1.31 to =
2.31.<A=20
name=3DTab3></A>
<DIV class=3DCapt><SPAN class=3DCaptNr>Table&nbsp;3&nbsp;</SPAN>Immune =
system genes=20
over-expressed in group II tumors </DIV>
<TABLE border=3D1>
  <COLGROUP>
  <COL align=3Dleft>
  <COL align=3Dchar char=3D".">
  <COL align=3Dchar char=3D"=96">
  <COL align=3Dchar char=3D"."></COLGROUP>
  <THEAD>
  <TR class=3Dheader>
    <TH align=3Dleft>
      <P class=3D"">Gene symbol</P></TH>
    <TH align=3Dleft char=3D".">
      <P class=3D"">Fold change</P></TH>
    <TH align=3Dleft char=3D"=96">
      <P class=3D"">90% CI</P></TH>
    <TH align=3Dleft char=3D".">
      <P class=3D""><I>P</I> value<SUP>a,b</SUP> </P></TH></TR></THEAD>
  <TBODY>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">FCGR3A</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">2.43</P></TD>
    <TD align=3Dchar char=3D"=96">
      <P class=3D"">1.49=963.73</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D""><B>0.020</B> </P></TD></TR>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">BCL6</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">2.05</P></TD>
    <TD align=3Dchar char=3D"=96">
      <P class=3D"">1.43=962.80</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D""><B>0.013</B> </P></TD></TR>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">HLA-DRA</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">1.79</P></TD>
    <TD align=3Dchar char=3D"=96">
      <P class=3D"">1.20=962.51</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D""><B>0.039</B> </P></TD></TR>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">HLA-DRB5 (PS 2)</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">1.78</P></TD>
    <TD align=3Dchar char=3D"=96">
      <P class=3D"">1.20=962.54</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D""><B>0.037</B> </P></TD></TR>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">HLA-DRB5 (PS 1)</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">1.77</P></TD>
    <TD align=3Dchar char=3D"=96">
      <P class=3D"">1.21=962.48</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D""><B>0.033</B> </P></TD></TR>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">MAFB</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">1.67</P></TD>
    <TD align=3Dchar char=3D"=96">
      <P class=3D"">1.25=962.23</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D""><B>0.011</B> </P></TD></TR>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">HLA-E (PS 1)</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">1.56</P></TD>
    <TD align=3Dchar char=3D"=96">
      <P class=3D"">1.27=961.93</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D""><B>0.002</B> </P></TD></TR>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">HLA-E (PS 2)</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">1.48</P></TD>
    <TD align=3Dchar char=3D"=96">
      <P class=3D"">1.24=961.76</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D""><B>0.002</B> </P></TD></TR></TBODY></TABLE>
<DIV class=3DCapt>
<DIV class=3DCaptCont>
<DIV class=3D""><I>PS</I> probes </DIV></DIV>
<DIV class=3DCaptCont>
<DIV class=3D""><SUP>a</SUP> <I>P</I> value from Cox model </DIV></DIV>
<DIV class=3DCaptCont>
<DIV class=3D""><SUP>b</SUP>Values &lt;0.05 bolded </DIV></DIV></DIV>
<DIV class=3DFigure><A name=3DFig3></A><IMG=20
alt=3DMediaObjects/11060_2009_9983_Fig3_HTML.gif=20
src=3D"http://www.springerlink.com/content/l26153725wvl0625/MediaObjects/=
11060_2009_9983_Fig3_HTML.gif"></DIV>
<DIV class=3DCapt><SPAN class=3DCaptNr>Fig.&nbsp;3&nbsp;</SPAN>Heat map =
of=20
differentially expressed genes between group II tumors versus the =
remainders.=20
Note that several immune related genes (blue) show higher expression in =
group 2=20
tumors. <I>Red</I> corresponds to higher gene expression levels </DIV>
<HR>
</DIV></DIV></DIV>
<DIV class=3D""><A name=3DSec12></A>
<DIV class=3DHeading3>Association of genes overexpressed in =
SVZ-contacting tumors=20
and survival</DIV>
<DIV class=3DPara>
<DIV class=3D"">We also analyzed the relationship between genes =
over-expressed in=20
group II tumors and survival (Table&nbsp;<A=20
href=3D"http://www.springerlink.com/content/l26153725wvl0625/fulltext.htm=
l#Tab4">4</A>).=20
Expression of 5 genes (either increased or decreased) correlated with =
shorter=20
survival. Comparing gene expression in tumors grade I and II versus III =
and IV=20
(i.e., all tumors that contact the SVZ versus all that do not), led to =
the=20
identification of CARM1 which is highly expressed in SVZ-contacting =
tumors and=20
also is highly correlated with survival (<I>P</I>&nbsp;=3D&nbsp;0.005). =
Thus this=20
analytic approach may be of use in screening for genes that may impact =
the=20
relationship between SVZ contact and survival.<A name=3DTab4></A>
<DIV class=3DCapt><SPAN class=3DCaptNr>Table&nbsp;4&nbsp;</SPAN>Genes =
over-expressed=20
in group II tumors that correlate with survival </DIV>
<TABLE border=3D1>
  <COLGROUP>
  <COL align=3Dleft>
  <COL align=3Dchar char=3D".">
  <COL align=3Dchar char=3D".">
  <COL align=3Dchar char=3D".">
  <COL align=3Dleft>
  <COL align=3Dchar char=3D"."></COLGROUP>
  <THEAD>
  <TR class=3Dheader>
    <TH align=3Dleft>
      <P class=3D"">Gene symbol</P></TH>
    <TH align=3Dleft char=3D".">
      <P class=3D"">Z<SUP>a</SUP> </P></TH>
    <TH align=3Dleft char=3D".">
      <P class=3D""><I>P</I> value </P></TH>
    <TH align=3Dleft char=3D".">
      <P class=3D"">Fold change</P></TH>
    <TH align=3Dleft>
      <P class=3D"">90% CI<SUP>b</SUP> </P></TH>
    <TH align=3Dleft char=3D".">
      <P class=3D""><I>P</I> value FC<SUP>c,d,e</SUP> =
</P></TH></TR></THEAD>
  <TBODY>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">COL5A2</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">3.15</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D""><B>0.002</B> </P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">2.03</P></TD>
    <TD align=3Dchar char=3D"=96">
      <P class=3D"">1.30=963.11</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D""><B>0.026</B> </P></TD></TR>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">LOXL2</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">3.10</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D""><B>0.002</B> </P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">2.29</P></TD>
    <TD align=3Dchar char=3D"=96">
      <P class=3D"">1.31=963.57</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D""><B>0.046</B> </P></TD></TR>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">SLIT2</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">2.42</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D""><B>0.016</B> </P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">2.04</P></TD>
    <TD align=3Dchar char=3D"=96">
      <P class=3D"">1.30=962.99</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D""><B>0.034</B> </P></TD></TR>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">SOX11</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">2.37</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D""><B>0.018</B> </P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">&#8722;2.74</P></TD>
    <TD align=3Dchar char=3D"=96">
      <P class=3D"">&#8722;1.37=968.14</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D""><B>0.024</B> </P></TD></TR>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">TTK</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">2.32</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D""><B>0.020</B> </P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">&#8722;1.61</P></TD>
    <TD align=3Dchar char=3D"=96">
      <P class=3D"">&#8722;1.23=962.07</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D""><B>0.010</B> </P></TD></TR></TBODY></TABLE>
<DIV class=3DCapt>
<DIV class=3DCaptCont>
<DIV class=3D""><SUP>a</SUP> <I>Z</I> is the transformed hazard ratio. A =
negative=20
<I>Z</I> score indicates higher expression levels are correlated with =
longer=20
survival, and a positive <I>Z</I> score indicates higher expression is=20
correlated with shorter survival </DIV></DIV>
<DIV class=3DCaptCont>
<DIV class=3D""><SUP>b</SUP> <I>CI</I> Confidence interval </DIV></DIV>
<DIV class=3DCaptCont>
<DIV class=3D""><SUP>c</SUP> <I>FC</I> fold change </DIV></DIV>
<DIV class=3DCaptCont>
<DIV class=3D""><SUP>d</SUP> <I>P</I> value from Cox model </DIV></DIV>
<DIV class=3DCaptCont>
<DIV class=3D""><SUP>e</SUP>Values &lt;0.05 bolded=20
</DIV></DIV></DIV></DIV></DIV></DIV>
<DIV class=3D""><A name=3DSec13></A>
<DIV class=3DHeading3>Kaplan=96Meier analysis</DIV>
<DIV class=3DPara>
<DIV class=3D"">We generated Kaplan=96Meier curves to compare survival =
between=20
tumors in group I and II versus III and IV. There was a trend towards =
shorter=20
survival (median survival 358 versus 654&nbsp;days, =
<I>P</I>&nbsp;=3D&nbsp;0.066;=20
Fig.&nbsp;<A=20
href=3D"http://www.springerlink.com/content/l26153725wvl0625/fulltext.htm=
l#Fig4">4</A>).=20
There was no significant difference in survival curves for group II =
versus I,=20
III, IV (Fig.&nbsp;<A=20
href=3D"http://www.springerlink.com/content/l26153725wvl0625/fulltext.htm=
l#Fig4">4</A>).=20
There was no statistically significant difference between rates of =
multifocal=20
disease, at presentation and recurrence, between groups. However, there =
was a=20
significant difference between gross total resection rates in group I =
versus=20
remainder tumors (Table&nbsp;<A=20
href=3D"http://www.springerlink.com/content/l26153725wvl0625/fulltext.htm=
l#Tab5">5</A>).=20
Figure&nbsp;<A=20
href=3D"http://www.springerlink.com/content/l26153725wvl0625/fulltext.htm=
l#Fig5">5</A>=20
shows Kaplan=96Meier curves for patients grouped by gene expression.=20
<DIV class=3DFigure><A name=3DFig4></A><IMG=20
alt=3DMediaObjects/11060_2009_9983_Fig4_HTML.gif=20
src=3D"http://www.springerlink.com/content/l26153725wvl0625/MediaObjects/=
11060_2009_9983_Fig4_HTML.gif"></DIV>
<DIV class=3DCapt><SPAN =
class=3DCaptNr>Fig.&nbsp;4&nbsp;</SPAN>Kaplan=96Meier curves=20
of group II versus groups I, III and IV combined (<B>a</B>) and =
Kaplan=96Meier=20
curves of group I and II combined versus groups III and IV combined =
(<B>b</B>)=20
</DIV>
<HR>
<A name=3DTab5></A>
<DIV class=3DCapt><SPAN class=3DCaptNr>Table&nbsp;5&nbsp;</SPAN>Clinical =
data by=20
tumor group </DIV>
<TABLE border=3D1>
  <COLGROUP>
  <COL align=3Dleft>
  <COL align=3Dchar char=3D"(">
  <COL align=3Dchar char=3D"(">
  <COL align=3Dchar char=3D"(">
  <COL align=3Dchar char=3D"("></COLGROUP>
  <THEAD>
  <TR class=3Dheader>
    <TH align=3Dleft>
      <P class=3D"">Group</P></TH>
    <TH align=3Dleft char=3D"(">
      <P class=3D"">I</P></TH>
    <TH align=3Dleft char=3D"(">
      <P class=3D"">II</P></TH>
    <TH align=3Dleft char=3D"(">
      <P class=3D"">III</P></TH>
    <TH align=3Dleft char=3D"(">
      <P class=3D"">IV</P></TH></TR></THEAD>
  <TBODY>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D""><I>n</I> </P></TD>
    <TD align=3Dchar char=3D"(">
      <P class=3D"">13</P></TD>
    <TD align=3Dchar char=3D"(">
      <P class=3D"">12</P></TD>
    <TD align=3Dchar char=3D"(">
      <P class=3D"">16</P></TD>
    <TD align=3Dchar char=3D"(">
      <P class=3D"">6</P></TD></TR>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">Age (SE<SUP>a</SUP>) </P></TD>
    <TD align=3Dchar char=3D"(">
      <P class=3D"">54 (4.5)</P></TD>
    <TD align=3Dchar char=3D"(">
      <P class=3D"">48.8 (2.5)</P></TD>
    <TD align=3Dchar char=3D"(">
      <P class=3D"">50.6 (3.7)</P></TD>
    <TD align=3Dchar char=3D"(">
      <P class=3D"">44 (4.5)</P></TD></TR>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">KPS<SUP>b</SUP> (SE) </P></TD>
    <TD align=3Dchar char=3D"(">
      <P class=3D"">83.8 (4.6)</P></TD>
    <TD align=3Dchar char=3D"(">
      <P class=3D"">80.8 (5.1)</P></TD>
    <TD align=3Dchar char=3D"(">
      <P class=3D"">85 (4.2)</P></TD>
    <TD align=3Dchar char=3D"(">
      <P class=3D"">88 (7.3)</P></TD></TR>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">Adj chemo<SUP>c</SUP> (%) </P></TD>
    <TD align=3Dchar char=3D"(">
      <P class=3D"">6/13 (46.2)</P></TD>
    <TD align=3Dchar char=3D"(">
      <P class=3D"">8/12 (66.7)</P></TD>
    <TD align=3Dchar char=3D"(">
      <P class=3D"">9/16 (56.3)</P></TD>
    <TD align=3Dchar char=3D"(">
      <P class=3D"">3/6 (50.0)</P></TD></TR>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">Temozolomide<SUP>d</SUP> (%) </P></TD>
    <TD align=3Dchar char=3D"(">
      <P class=3D"">2/13 (15.4)</P></TD>
    <TD align=3Dchar char=3D"(">
      <P class=3D"">4/12 (33.3)</P></TD>
    <TD align=3Dchar char=3D"(">
      <P class=3D"">7/16 (43.8)</P></TD>
    <TD align=3Dchar char=3D"(">
      <P class=3D"">2/6 (33.3)</P></TD></TR>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">GTR<SUP>e,f</SUP> (%) </P></TD>
    <TD align=3Dchar char=3D"(">
      <P class=3D"">2/13 (15.4)</P></TD>
    <TD align=3Dchar char=3D"(">
      <P class=3D"">6/12 (50.0)</P></TD>
    <TD align=3Dchar char=3D"(">
      <P class=3D"">7/16 (43.8)</P></TD>
    <TD align=3Dchar char=3D"(">
      <P class=3D"">4/6 (66.7)</P></TD></TR>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">Multifocal, initial (%)</P></TD>
    <TD align=3Dchar char=3D"(">
      <P class=3D"">1/13 (7.7)</P></TD>
    <TD align=3Dchar char=3D"(">
      <P class=3D"">1/12 (8.3)</P></TD>
    <TD align=3Dchar char=3D"(">
      <P class=3D"">4/16 (25.0)</P></TD>
    <TD align=3Dchar char=3D"(">
      <P class=3D"">1/6 (16.7)</P></TD></TR>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">Multifocal, relapse<SUP>g</SUP> (%) </P></TD>
    <TD align=3Dchar char=3D"(">
      <P class=3D"">3/11 (27.3)</P></TD>
    <TD align=3Dchar char=3D"(">
      <P class=3D"">1/8 (12.5)</P></TD>
    <TD align=3Dchar char=3D"(">
      <P class=3D"">4/13 (30.8)</P></TD>
    <TD align=3Dchar char=3D"(">
      <P class=3D"">1/5 (20.0)</P></TD></TR></TBODY></TABLE>
<DIV class=3DCapt>
<DIV class=3DCaptCont>
<DIV class=3D""><SUP>a</SUP> <I>SE</I> standard error </DIV></DIV>
<DIV class=3DCaptCont>
<DIV class=3D""><SUP>b</SUP> <I>KPS</I> Karnosfsky performance status at =

presentation </DIV></DIV>
<DIV class=3DCaptCont>
<DIV class=3D""><SUP>c</SUP> <I>adj chemo</I> adjuvant chemotherapy of =
any kind=20
</DIV></DIV>
<DIV class=3DCaptCont>
<DIV class=3D""><SUP>d</SUP> <I>temozolomide</I> adjuvant chemotherapy =
with=20
temozolomide </DIV></DIV>
<DIV class=3DCaptCont>
<DIV class=3D""><SUP>e</SUP> <I>GTR</I> gross total resection =
</DIV></DIV>
<DIV class=3DCaptCont>
<DIV class=3D""><SUP>f</SUP> <I>P</I>&nbsp;=3D&nbsp;0.03 for group I =
versus other=20
tumors, chi-square test </DIV></DIV>
<DIV class=3DCaptCont>
<DIV class=3D""><SUP>g</SUP>Six patients have yet to progress, and =
follow-up scans=20
were not available for 4 patients, accounting for reduced patient number =
for=20
this variable </DIV></DIV></DIV>
<DIV class=3DFigure><A name=3DFig5></A><IMG=20
alt=3DMediaObjects/11060_2009_9983_Fig5_HTML.gif=20
src=3D"http://www.springerlink.com/content/l26153725wvl0625/MediaObjects/=
11060_2009_9983_Fig5_HTML.gif"></DIV>
<DIV class=3DCapt><SPAN =
class=3DCaptNr>Fig.&nbsp;5&nbsp;</SPAN>Kaplan=96Meier curves=20
for selected genes: <B>a</B> MAPK8, <B>b</B> CD133, <B>c</B> BMP-4, =
<B>d</B>=20
MELK, <B>e</B> CARM1 and <B>f</B> LOXL2. For all graphs the <I>dashed =
line</I>=20
indicates survival data for patients with greater than median gene =
expression=20
levels, and the solid line is for patients with less than median gene =
expression=20
levels. Note that increased levels of MAPK and BMP-4 expression are =
associated=20
with longer survival, whereas increased CD133 and MELK expression levels =

correlate with shorter survival. Increased CARM1 and LOXL2 (enriched in=20
SVZ-contacting tumors) also are correlated with poorer survival </DIV>
<HR>
</DIV></DIV></DIV></DIV>
<DIV class=3D""><A name=3DSec14></A>
<HR>

<DIV class=3Dheading2>Discussion</DIV>
<P class=3D"">It has been hypothesized that gliomas arise from stem =
cells=20
originating in the SVZ. Previously it has been reported that more =
aggressive=20
patterns of GBM recurrence are associated with tumors that contact the =
SVZ, and=20
that these tumors may express a =93stem-cell-derived=94 phenotype that =
could impact=20
treatment decisions [<CITE><A=20
href=3D"http://www.springerlink.com/content/l26153725wvl0625/fulltext.htm=
l#CR9">9</A></CITE>].=20
Therefore we analyzed the relationship between expression of cancer =
stem-cell=20
genes, contact of tumor with the SVZ and patient survival. </P>
<P class=3D"">Similar to previous work [<CITE><A=20
href=3D"http://www.springerlink.com/content/l26153725wvl0625/fulltext.htm=
l#CR9">9</A></CITE>,=20
<CITE><A=20
href=3D"http://www.springerlink.com/content/l26153725wvl0625/fulltext.htm=
l#CR10">10</A></CITE>],=20
we found that patients with GBM which contacted the SVZ trended with =
shorter=20
survival. One possibility is that shortened survival may be related to =
tumor=20
size, as tumors that contact both the cortex and SVZ tend to be large. =
However,=20
previous reports have shown little correlation between GBM size and =
survival=20
[<CITE><A=20
href=3D"http://www.springerlink.com/content/l26153725wvl0625/fulltext.htm=
l#CR11">11</A></CITE>].=20
Additionally, Barami et al. [<CITE><A=20
href=3D"http://www.springerlink.com/content/l26153725wvl0625/fulltext.htm=
l#CR21">21</A></CITE>]=20
found that contact with the ventricular wall was independent of size. =
</P>
<P class=3D"">Another possibility is that tumors that contact the SVZ =
have higher=20
rates of multifocal or subependymal/leptomeningeal spread. We did not =
find a=20
significant difference in rates of non-local recurrence between tumor =
groups=20
based on SVZ contact. And only one patient had leptomeningeal =
recurrence. Unlike=20
Lim et al. [<CITE><A=20
href=3D"http://www.springerlink.com/content/l26153725wvl0625/fulltext.htm=
l#CR9">9</A></CITE>]=20
we found that group III tumors had the highest rates of multifocal =
disease, both=20
at initial presentation and at recurrence. Thus it is unclear if tumors =
that=20
contact the SVZ have more aggressive patterns of recurrence. Small =
sample sizes=20
may account for these discordant results. </P>
<P class=3D"">There was a difference in gross total resection rates =
between=20
groups. Group I tumors (those that contact both the ventricle and =
cortex) had a=20
lower gross total resection rate than other tumors. Since complete =
resection is=20
associated with better survival [<CITE><A=20
href=3D"http://www.springerlink.com/content/l26153725wvl0625/fulltext.htm=
l#CR22">22</A></CITE>],=20
this could be a potential confound for the survival data. Additionally, =
deeper=20
tumors may have more impact on critical structures, increasing their =
impact on=20
disability. Thus we cannot say whether tumors that contact the SVZ are=20
intrinsically more malignant than those that do not. </P>
<P class=3D"">We found no relationship between stem-cell gene expression =
and SVZ=20
grade. Specifically there was no correlation in expression between the =
selected=20
stem cell genes, and there was no enrichment of stem cell genes in =
tumors that=20
contacted the SVZ. Although not correlated with tumor location, =
expression of=20
several stem cell genes, including CD133, was associated with shortened=20
survival, similar to previous reports [<CITE><A=20
href=3D"http://www.springerlink.com/content/l26153725wvl0625/fulltext.htm=
l#CR12">12</A></CITE>,=20
<CITE><A=20
href=3D"http://www.springerlink.com/content/l26153725wvl0625/fulltext.htm=
l#CR23">23</A></CITE>].=20
For instance, Zhang et al. [<CITE><A=20
href=3D"http://www.springerlink.com/content/l26153725wvl0625/fulltext.htm=
l#CR23">23</A></CITE>]=20
found that CD133 expression was negatively correlated with both overall =
survival=20
and progression-free survival. CD133 expression has been shown to be =
associated=20
with resistance to radiation and chemotherapy [<CITE><A=20
href=3D"http://www.springerlink.com/content/l26153725wvl0625/fulltext.htm=
l#CR24">24</A></CITE>].=20
Similar results have been demonstrated in mice models of GBM [<CITE><A=20
href=3D"http://www.springerlink.com/content/l26153725wvl0625/fulltext.htm=
l#CR25">25</A></CITE>].=20
Interestingly, BMP4 has been hypothesized to down-regulate CD133 and =
increase=20
survival in a mouse model [<CITE><A=20
href=3D"http://www.springerlink.com/content/l26153725wvl0625/fulltext.htm=
l#CR26">26</A></CITE>].=20
Although there was no significant correlation between CD133 and BMP4 =
expression,=20
increased BMP4 expression was associated with longer survival. </P>
<P class=3D"">Unlike the report of Zhang et al. [<CITE><A=20
href=3D"http://www.springerlink.com/content/l26153725wvl0625/fulltext.htm=
l#CR23">23</A></CITE>]=20
we found that higher nestin expression was associated with longer =
survival. Some=20
differences between the two experiments are notable. Zhang measured =
protein=20
level with immunohistochemistry, whereas we examined gene expression. =
Zhang also=20
looked at grade II=96IV tumors, whereas we restricted our analysis to =
grade IV=20
tumors (GBM). Another group [<CITE><A=20
href=3D"http://www.springerlink.com/content/l26153725wvl0625/fulltext.htm=
l#CR27">27</A></CITE>]=20
found no difference in survival based on nestin expression levels. =
Similarly,=20
increased OLIG2 expression was associated with longer survival. OLIG2 is =

associated with oligodendrogliomal lineage development [<CITE><A=20
href=3D"http://www.springerlink.com/content/l26153725wvl0625/fulltext.htm=
l#CR17">17</A></CITE>],=20
and an oligodendroglioma component and genetic signature have previously =
been=20
shown to be associated with longer survival [<CITE><A=20
href=3D"http://www.springerlink.com/content/l26153725wvl0625/fulltext.htm=
l#CR28">28</A></CITE>].=20
Another gene whose expression was associated with longer survival was =
MAPK8=20
(also known as JNK). Previously it has been shown that in cultured =
glioma cells,=20
inhibition of this kinase reduces VEGF secretion [<CITE><A=20
href=3D"http://www.springerlink.com/content/l26153725wvl0625/fulltext.htm=
l#CR29">29</A></CITE>].=20
Since VEGF is associated with malignancy, this may explain why patients =
with=20
tumors with lower MAPK8 gene expression had longer survival. </P>
<P class=3D"">Since patients in group I&nbsp;+&nbsp;II trended with =
shorter=20
survival, we also analyzed the expression of genes in SVZ-contacting =
tumors=20
compared to those that did not contact the SVZ in order to look for =
candidate=20
genes that may be involved with shorter survival. Several genes, =
including CARM1=20
and LOXL2, over-expressed in SVZ-contacting tumors, were associated with =
poor=20
survival. LOXL2 has been shown to promote cell migration in breast =
cancer=20
[<CITE><A=20
href=3D"http://www.springerlink.com/content/l26153725wvl0625/fulltext.htm=
l#CR30">30</A></CITE>],=20
and is associated with a poor prognosis in squamous cell carcinomas =
[<CITE><A=20
href=3D"http://www.springerlink.com/content/l26153725wvl0625/fulltext.htm=
l#CR31">31</A></CITE>],=20
but this is the first report of an association with poor survival in =
GBM. CARM1=20
promotes breast cancer proliferation through an estrogen-dependent =
mechanism=20
[<CITE><A=20
href=3D"http://www.springerlink.com/content/l26153725wvl0625/fulltext.htm=
l#CR32">32</A></CITE>],=20
but has no known role in glioma malignancy. </P>
<P class=3D"">Interestingly, although no stem-cell gene expression =
signature was=20
associated with SVZ group II, many genes associated with the immune =
system were=20
over-expressed in group II, including several MHC genes. This is of =
potential=20
importance as MHC expression modulates response to dendrictic cell =
therapy=20
currently being developed to treat GBM, and thus tumors that contact the =
SVZ may=20
respond differently to this treatment compared to other tumors [<CITE><A =

href=3D"http://www.springerlink.com/content/l26153725wvl0625/fulltext.htm=
l#CR33">33</A></CITE>].=20
Additionally, it has been shown that migrating glioma cells =
down-regulate MHC=20
class II genes. If the SVZ is the origin for gliomagenic cells, this =
suggests=20
that high expression of these genes may account for the lack of spread =
of group=20
II tumor progenitor cells away from the SVZ prior to tumor formation =
[<CITE><A=20
href=3D"http://www.springerlink.com/content/l26153725wvl0625/fulltext.htm=
l#CR34">34</A></CITE>].=20
</P>
<P class=3D"">In conclusion, we found no evidence that tumors contacting =
the SVZ=20
were more likely, based on gene expression, to be =
=93stem-cell-derived=94, than=20
tumors not contacting the SVZ. Two possible explanations are: (1) the =
SVZ is not=20
the origin for tumor producing cells; or (2) cells responsible for =
gliomagenesis=20
are produced in the SVZ but migrate towards the cortex before =
tumorigenesis. The=20
second hypothesis is supported by animal studies which have shown that =
gliomas=20
arising from the periventricular region can grow along white matter =
tracts,=20
losing their connection to the SVZ [<CITE><A=20
href=3D"http://www.springerlink.com/content/l26153725wvl0625/fulltext.htm=
l#CR35">35</A></CITE>].=20
We did find, however, that SVZ-contacting cells over-expressed several =
genes=20
associated with patient survival and with the immune system. The =
expression of=20
immune-related genes could impact response to treatment, and may mediate =
cell=20
migration away from the SVZ. Thus, more research is required to clarify =
the=20
relationship between migration of SVZ derived cancer stem cells and the =
temporal=20
relationship to gliomagenesis. </P></DIV>
<DIV class=3DAcknowledgments><SPAN class=3DAcknowledgmentsHeading>Open=20
Access&nbsp;&nbsp;</SPAN><SPAN class=3D"">This article is distributed =
under the=20
terms of the Creative Commons Attribution Noncommercial License which =
permits=20
any noncommercial use, distribution, and reproduction in any medium, =
provided=20
the original author(s) and source are credited.</SPAN></DIV>
<P></P>
<HR>

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