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    <TD>Journal of Neuro-Oncology</TD></TR>
  <TR>
    <TD>=A9&nbsp;The Author(s)&nbsp;2008</TD></TR>
  <TR>
    <TD>10.1007/s11060-008-9577-6</TD></TR></TBODY></TABLE><!--Begin =
Abstract-->
<H2 class=3Drubric>lab. Investigation-human/animal tissue</H2>
<DIV class=3DHeading1><A name=3Dtitle></A>Selective cancer-germline gene =
expression=20
in pediatric brain tumors </DIV>
<P class=3DAuthorGroup>Joannes&nbsp;F.&nbsp;M.&nbsp;Jacobs<SUP>1</SUP>,=20
Oliver&nbsp;M.&nbsp;Grauer<SUP>2, 3</SUP>, =
Francis&nbsp;Brasseur<SUP>4</SUP>,=20
Peter&nbsp;M.&nbsp;Hoogerbrugge<SUP>1</SUP>, =
Pieter&nbsp;Wesseling<SUP>5,=20
6</SUP>, Corrie&nbsp;E.&nbsp;Gidding<SUP>1</SUP>,=20
Mandy&nbsp;W.&nbsp;M.&nbsp;M.&nbsp;van de&nbsp;Rakt<SUP>3</SUP>,=20
Carl&nbsp;G.&nbsp;Figdor<SUP>3</SUP>, =
Pierre&nbsp;G.&nbsp;Coulie<SUP>7</SUP>,=20
I.&nbsp;Jolanda&nbsp;M.&nbsp;de&nbsp;Vries<SUP>1, 3</SUP> and=20
Gosse&nbsp;J.&nbsp;Adema<SUP>3&nbsp;<A=20
href=3D"http://www.springerlink.com/content/0wqj8uh583261461/fulltext.htm=
l#ContactOfAuthor11"><IMG=20
alt=3D"Contact Information"=20
src=3D"http://www.springerlink.com/content/0wqj8uh583261461/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>Department of Pediatric Oncology, =
Radboud=20
      University Nijmegen Medical Centre, Nijmegen, The=20
  Netherlands</SPAN></TD></TR></TBODY></TABLE>
<TABLE>
  <TBODY>
  <TR vAlign=3Dtop>
    <TD><SPAN class=3DAffiliation><A =
name=3DAff2></A>(2)&nbsp;</SPAN></TD>
    <TD><SPAN class=3DAffiliation>Department of Neurology, University of =

      Regensburg, Regensburg, Germany</SPAN></TD></TR></TBODY></TABLE>
<TABLE>
  <TBODY>
  <TR vAlign=3Dtop>
    <TD><SPAN class=3DAffiliation><A =
name=3DAff3></A>(3)&nbsp;</SPAN></TD>
    <TD><SPAN class=3DAffiliation>Department of Tumor Immunology, =
Nijmegen=20
      Centre for Molecular Life Sciences/278 TIL, P.O. Box&nbsp;9101, =
6500=20
      HB&nbsp;Nijmegen, The Netherlands</SPAN></TD></TR></TBODY></TABLE>
<TABLE>
  <TBODY>
  <TR vAlign=3Dtop>
    <TD><SPAN class=3DAffiliation><A =
name=3DAff4></A>(4)&nbsp;</SPAN></TD>
    <TD><SPAN class=3DAffiliation>Ludwig Institute for Cancer Research, =
Brussels=20
      Branch, Belgium</SPAN></TD></TR></TBODY></TABLE>
<TABLE>
  <TBODY>
  <TR vAlign=3Dtop>
    <TD><SPAN class=3DAffiliation><A =
name=3DAff5></A>(5)&nbsp;</SPAN></TD>
    <TD><SPAN class=3DAffiliation>Department of Pathology, Radboud =
University=20
      Nijmegen Medical Centre, Nijmegen, The=20
Netherlands</SPAN></TD></TR></TBODY></TABLE>
<TABLE>
  <TBODY>
  <TR vAlign=3Dtop>
    <TD><SPAN class=3DAffiliation><A =
name=3DAff6></A>(6)&nbsp;</SPAN></TD>
    <TD><SPAN class=3DAffiliation>Department of Pathology, =
Canisius-Wilhelmina=20
      Hospital, Nijmegen, The =
Netherlands</SPAN></TD></TR></TBODY></TABLE>
<TABLE>
  <TBODY>
  <TR vAlign=3Dtop>
    <TD><SPAN class=3DAffiliation><A =
name=3DAff7></A>(7)&nbsp;</SPAN></TD>
    <TD><SPAN class=3DAffiliation>Christian de Duve Institute of =
Cellular=20
      Pathology, Universit=E9 Catholique de Louvain, Brussels,=20
  Belgium</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/0wqj8uh583261461/contact.gif" =

      border=3D0></TD>
    =
<TD><STRONG>Gosse&nbsp;</STRONG><STRONG>J.&nbsp;</STRONG><STRONG>Adema</S=
TRONG><STRONG></STRONG><BR><STRONG>Email:=20
      </STRONG><A=20
  =
href=3D"mailto:g.adema@ncmls.ru.nl">g.adema@ncmls.ru.nl</A></TD></TR></TB=
ODY></TABLE>
<P class=3DAffiliation><STRONG>Received:=20
</STRONG>4&nbsp;January&nbsp;2008&nbsp;&nbsp;<STRONG>Accepted:=20
</STRONG>26&nbsp;March&nbsp;2008&nbsp;&nbsp;<STRONG>Published online:=20
</STRONG>9&nbsp;April&nbsp;2008 </P>
<DIV class=3DAbstract><A name=3DAbs1></A><SPAN=20
class=3DAbstractHeading>Abstract&nbsp;&nbsp;</SPAN>Cancer-germline genes =
(CGGs)=20
code for immunogenic antigens that are present in various human tumors =
and can=20
be targeted by immunotherapy. Their expression has been studied in a =
wide range=20
of human tumors in adults. We measured the expression of 12 CGGs in =
pediatric=20
brain tumors, to identify targets for therapeutic cancer vaccines. Real =
Time PCR=20
was used to quantify the expression of genes <I>MAGE-A1</I>, =
<I>MAGE-A2</I>,=20
<I>MAGE-A3</I>, <I>MAGE-A4</I>, <I>MAGE-A6</I>, <I>MAGE-A10</I>,=20
<I>MAGE-A12</I>, <I>MAGE-C2</I>, <I>NY-ESO-1</I> and=20
<I>GAGE-1</I>,<I>2</I>,<I>8</I> in 50 pediatric brain tumors of =
different=20
histological subtypes. Protein expression was examined with=20
immunohistochemistry. Fifty-five percent of the medulloblastomas=20
(<I>n</I>&nbsp;=3D&nbsp;11), 86% of the ependymomas =
(<I>n</I>&nbsp;=3D&nbsp;7), 40%=20
of the choroid plexus tumors (<I>n</I>&nbsp;=3D&nbsp;5) and 67% of =
astrocytic=20
tumors (<I>n</I>&nbsp;=3D&nbsp;27) expressed one or more CGGs. =
Immunohistochemical=20
analysis confirmed qPCR results. With exception of a minority of tumors, =
the=20
overall level of CGG expression in pediatric brain tumors was low. We =
observed a=20
high expression of at least one CGG in 32% of the samples. CGG-encoded =
antigens=20
are therefore suitable targets in a very selected group of pediatric =
patients=20
with a brain tumor. Interestingly, glioblastomas from adult patients =
expressed=20
CGGs more often and at significantly higher levels compared to pediatric =

glioblastomas. This observation is in line with the notion that =
pediatric and=20
adult glioblastomas develop along different genetic pathways. </DIV>
<DIV class=3DAbstractSection><SPAN =
class=3DAbstractSectionHeading>Electronic=20
supplementary material&nbsp;&nbsp;</SPAN><SPAN class=3D"">The online =
version of=20
this article (doi:<A=20
href=3D"http://dx.doi.org/10.1007/s11060-008-9577-6">10.1007/s11060-008-9=
577-6</A>)=20
contains supplementary material, which is available to authorized=20
users.</SPAN></DIV>
<P class=3DKeyword><SPAN =
class=3DKeywordHeading>Keywords&nbsp;&nbsp;</SPAN>Brain=20
tumor&nbsp;-&nbsp;Pediatrics&nbsp;-&nbsp;qPCR&nbsp;-&nbsp;MAGE&nbsp;-&nbs=
p;NY-ESO-1&nbsp;-&nbsp;Immune=20
target </P>
<DIV class=3D""><A name=3DSec1></A>
<HR>

<DIV class=3Dheading2>Introduction</DIV>
<P class=3D"">Despite major advances in the treatment of childhood =
cancer, cancer=20
remains a common cause of death for children &gt;1&nbsp;year of age =
[<CITE><A=20
href=3D"http://www.springerlink.com/content/0wqj8uh583261461/fulltext.htm=
l#CR1">1</A></CITE>].=20
Twenty-two percent of all pediatric cancers are CNS tumors. Pediatric =
patients=20
with relapsed cancers of the CNS have a poor prognosis, and therefore =
novel=20
therapies for these patients are urgently needed. Immunotherapy against =
brain=20
tumors presents unique challenges since the brain is considered an =
immune=20
privileged site. However, recent studies demonstrated that the immune =
cells have=20
access to the brain in spite of the blood-brain barrier [<CITE><A=20
href=3D"http://www.springerlink.com/content/0wqj8uh583261461/fulltext.htm=
l#CR2">2</A></CITE>,=20
<CITE><A=20
href=3D"http://www.springerlink.com/content/0wqj8uh583261461/fulltext.htm=
l#CR3">3</A></CITE>].=20
Within malignant brain tumors the blood-brain barrier is generally =
considered=20
non-functional. Progress in our understanding of immune responses to CNS =
tumors=20
have already led to novel clinical applications [<CITE><A=20
href=3D"http://www.springerlink.com/content/0wqj8uh583261461/fulltext.htm=
l#CR4">4</A></CITE>,=20
<CITE><A=20
href=3D"http://www.springerlink.com/content/0wqj8uh583261461/fulltext.htm=
l#CR5">5</A></CITE>].=20
Most experience has been obtained with immunotherapeutic trials with =
dendritic=20
cell vaccinations, as reviewed by De Vleeschouwer et&nbsp;al. [<CITE><A=20
href=3D"http://www.springerlink.com/content/0wqj8uh583261461/fulltext.htm=
l#CR6">6</A></CITE>].=20
</P>
<P class=3D"">Immunotherapy is an attractive therapeutic option for =
pediatric=20
cancer patients because of its mild toxicity, and because the child=92s =
immune=20
system is more potent and flexible compared to adults [<CITE><A=20
href=3D"http://www.springerlink.com/content/0wqj8uh583261461/fulltext.htm=
l#CR7">7</A></CITE>,=20
<CITE><A=20
href=3D"http://www.springerlink.com/content/0wqj8uh583261461/fulltext.htm=
l#CR8">8</A></CITE>].=20
However, implementation of immunotherapy in pediatric oncology has been =
hampered=20
by the lack of known tumor-specific antigens on pediatric tumors. </P>
<P class=3D"">Cancer-germline genes (CGGs) are expressed in a wide range =
of human=20
tumors and have a highly restricted expression pattern in normal tissues =

[<CITE><A=20
href=3D"http://www.springerlink.com/content/0wqj8uh583261461/fulltext.htm=
l#CR9">9</A></CITE>,=20
<CITE><A=20
href=3D"http://www.springerlink.com/content/0wqj8uh583261461/fulltext.htm=
l#CR10">10</A></CITE>].=20
Antigens encoded by CGGs have been extensively studied because of their=20
immunogenicity, tumor specificity, and their expression in a significant =

proportion of adult tumors of various histological types. Recently, we =
have=20
shown that CGGs are expressed in a large percentage of pediatric =
extra-cranial=20
solid tumors [<CITE><A=20
href=3D"http://www.springerlink.com/content/0wqj8uh583261461/fulltext.htm=
l#CR11">11</A></CITE>].=20
</P>
<P class=3D"">The aim of this study was to analyze CGG expression in =
pediatric=20
brain tumors. We report the results of a quantitative real-time PCR =
analysis of=20
the expression of 12 CGGs in a panel of medulloblastomas, ependymomas, =
tumors of=20
the choroid plexus and astrocytic tumors. </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>Tumor samples</DIV>
<P class=3D"">Fresh-frozen tumor samples were available at the =
Department of=20
Pathology at the Radboud University Nijmegen Medical Centre. All samples =
were=20
from pediatric patients (0=9619-years-old) with a brain tumor diagnosed =
at the=20
Department of Pediatric Hemato-Oncology. Sections of the frozen samples =
were=20
stained with hematoxylin-eosin and reviewed by the pathologist to verify =
tumor=20
histology and to evaluate the percentage of tumor cells. Samples were =
only=20
considered for study if the contents of tumor cells was &#8805;80%. =
</P></DIV>
<DIV class=3D""><A name=3DSec4></A>
<DIV class=3DHeading3>RNA isolation and cDNA synthesis</DIV>
<P class=3D"">Total RNA was isolated with TriZol reagent (Invitrogen, =
Carlsbad,=20
CA) and samples were treated with Deoxyribonuclease&nbsp;I (Invitrogen)=20
according to the manufacturer=92s protocol. To generate cDNA, =
1&nbsp;&#956;g=20
DNase-treated RNA was reverse-transcribed with the SuperScript =
First-Strand=20
Synthesis System for RT-PCR (Invitrogen) using oligo(dT) primer and 50 =
units=20
SuperScript II, according to the manufacturer=92s protocol. After =
first-strand=20
synthesis, samples were diluted to a final volume of 100&nbsp;&#956;l =
with water.=20
</P></DIV>
<DIV class=3D""><A name=3DSec5></A>
<DIV class=3DHeading3>Conventional PCR</DIV>
<P class=3D"">Duplex PCR amplification of &#946;<I>-actin</I> and =
<I>GAPDH</I>=20
transcripts was carried out in a 25-&#956;l reaction volume containing =
2.5&nbsp;&#956;l of=20
cDNA, 1=D7&nbsp;PCR Buffer (10&nbsp;mM Tris=96HCl pH 8.3, 50&nbsp;mM =
KCl,=20
1.5&nbsp;mM MgCl<SUB>2</SUB>), 100&nbsp;&#956;M each dNTP, =
0.4&nbsp;&#956;M each primer,=20
and 0.625 units Taq DNA polymerase (TaKaRa, Shiga, Japan). =
<I>&#946;-actin</I>=20
primers were as described [<CITE><A=20
href=3D"http://www.springerlink.com/content/0wqj8uh583261461/fulltext.htm=
l#CR12">12</A></CITE>].=20
<I>GAPDH</I> primers (originally available from Clontech, Palo Alto, CA; =
kindly=20
provided by Dr. B. Leth=E9) were =
5&#8242;-TgAAggTCggAgTCAACggATTTggT-3&#8242; (sense) and=20
5&#8242;-CATgTgggCCATgAggTCCACCAC-3&#8242; (antisense). Cycling was =
performed in a=20
TRIO-Thermoblock thermocycler (Biometra, G=F6ttingen, Germany) as =
follows: 94=B0C=20
for 4&nbsp;min, followed by 22 cycles of 1&nbsp;min at 94=B0C, =
1&nbsp;min at 65=B0C,=20
and 1&nbsp;min at 72=B0C. Cycling was concluded with a final extension =
step at=20
72=B0C for 15&nbsp;min. PCR products were fractionated in 1.3% agarose =
gel and=20
visualized by ethidium bromide fluorescence <I>(&#946;-actin</I>, =
626&nbsp;bp;=20
<I>GAPDH</I>, 983&nbsp;bp). PCR amplification of <I>MAGE-A</I> =
transcripts was=20
carried out with the primer pair designed by Zammatteo et&nbsp;al. =
[<CITE><A=20
href=3D"http://www.springerlink.com/content/0wqj8uh583261461/fulltext.htm=
l#CR13">13</A></CITE>].=20
These primers derive from a consensus nucleotide sequence for the last =
exon of=20
the 12 <I>MAGE-A</I> genes and give amplicons of ~539&nbsp;bp. PCR =
conditions=20
were as described above, except that PCR was performed for 30 cycles. =
</P></DIV>
<DIV class=3D""><A name=3DSec6></A>
<DIV class=3DHeading3>Quantitative real-time PCR</DIV>
<DIV class=3DPara>
<DIV class=3D"">Expression of CGGs and of the reference gene =
&#946;-actin, was measured=20
by quantitative PCR, based on TaqMan methodology, using the ABI PRISM =
7700=20
Sequence Detection System (Applied Biosystems, Warrington, UK). PCR =
reactions=20
were prepared with the qPCR Core Kit w/o dUTP reagents (Eurogentec, =
Seraing,=20
Belgium). Each reaction (25&nbsp;&#956;l) contained 2.5&nbsp;&#956;l of =
cDNA, 1=D7&nbsp;PCR=20
buffer containing the passive reference dye ROX, 5&nbsp;mM =
MgCl<SUB>2</SUB>,=20
200&nbsp;&#956;M each dNTP, 200&nbsp;nM each primer, 100&nbsp;nM probe, =
and 0.625=20
units DNA polymerase. Primers, probes and thermal cycling conditions are =
given=20
in Table&nbsp;<A=20
href=3D"http://www.springerlink.com/content/0wqj8uh583261461/fulltext.htm=
l#Tab1">1</A>=20
[<CITE><A=20
href=3D"http://www.springerlink.com/content/0wqj8uh583261461/fulltext.htm=
l#CR14">14</A></CITE>,=20
<CITE><A=20
href=3D"http://www.springerlink.com/content/0wqj8uh583261461/fulltext.htm=
l#CR15">15</A></CITE>].=20
Probes with 6FAM and TAMRA labels were from Eurogentec. Probes with 6FAM =
and=20
MGB-NFQ labels (for MAGE-A3, MAGE-A6 and MAGE-A12) were from Applied =
Biosystems.=20
Quantification of the samples was achieved by extrapolation from a =
standard=20
curve of serial dilution points of cDNA of the relevant gene =
(Supplementary=20
Figure&nbsp;1). Samples and standard dilution points were assayed in =
duplicate=20
or triplicate. Standard calibration curves for &#946;-actin and all CGGs =
were linear=20
over 4 (CGGs) or 5 (=DF-actin) orders of magnitude and had similar PCR=20
efficiencies (slope from &#8722;3.45 to &#8722;3.77). Differences in =
sensitivity between the=20
assays for the various genes (<I>y</I>-intercept from 38.7 to 41.6) were =
due in=20
part to differences in the actual cDNA copy number in the standard =
dilutions.=20
cDNA copy numbers in the standards were verified by testing minimally 12 =

replicates of the 1-copy dilution in each qPCR run. If needed, copy =
numbers of=20
the test samples were corrected by a factor calculated on the basis of =
the=20
results for the 1-copy dilution. Normalization of samples was achieved =
by=20
dividing the copy number of CGG by that of the reference gene, =
&#946;-actin.<A=20
name=3DTab1></A>
<DIV class=3DCapt><SPAN class=3DCaptNr>Table&nbsp;1&nbsp;</SPAN>Primers, =
probes and=20
thermal cycling conditions of qPCR </DIV>
<TABLE border=3D1>
  <COLGROUP>
  <COL align=3Dleft>
  <COL align=3Dleft>
  <COL align=3Dleft>
  <COL align=3Dleft>
  <COL align=3Dleft></COLGROUP>
  <THEAD>
  <TR class=3Dheader>
    <TH align=3Dleft>
      <P class=3D"">Gene</P></TH>
    <TH align=3Dleft>
      <P class=3D"">Forward primer =
(5&#8242;&nbsp;&#8594;&nbsp;3&#8242;)</P></TH>
    <TH align=3Dleft>
      <P class=3D"">Reverse primer =
(5&#8242;&nbsp;&#8594;&nbsp;3&#8242;)</P></TH>
    <TH align=3Dleft>
      <P class=3D"">Probe (5&#8242;&nbsp;&#8594;&nbsp;3&#8242;)</P></TH>
    <TH align=3Dleft>
      <P class=3D"">Annealing-extenstion</P></TH></TR></THEAD>
  <TBODY>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">MAGEA1 [<CITE><A=20
      =
href=3D"http://www.springerlink.com/content/0wqj8uh583261461/fulltext.htm=
l#CR15">15</A></CITE>]=20
      </P></TD>
    <TD align=3Dleft>
      <P class=3D"">gCC gAA ggA ACC TgA CC</P></TD>
    <TD align=3Dleft>
      <P class=3D"">ACT ggg TTg CCT CTg TCg</P></TD>
    <TD align=3Dleft>
      <P class=3D"">TgT gTg CAg gCT gCC ACC TCC T</P></TD>
    <TD align=3Dleft>
      <P class=3D"">90&nbsp;s, 65=B0C</P></TD></TR>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">MAGEA2 [<CITE><A=20
      =
href=3D"http://www.springerlink.com/content/0wqj8uh583261461/fulltext.htm=
l#CR15">15</A></CITE>]=20
      </P></TD>
    <TD align=3Dleft>
      <P class=3D"">AAg TAg gAC CCg Agg CAC Tg</P></TD>
    <TD align=3Dleft>
      <P class=3D"">gAA gAg gAA gAA gCg gTC Tg</P></TD>
    <TD align=3Dleft>
      <P class=3D"">CAT TgA Agg AgA AgA TCT gCC TgT ggg TCT TC</P></TD>
    <TD align=3Dleft>
      <P class=3D"">1&nbsp;min, 60=B0C</P></TD></TR>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">MAGEA3 [<CITE><A=20
      =
href=3D"http://www.springerlink.com/content/0wqj8uh583261461/fulltext.htm=
l#CR14">14</A></CITE>]=20
      </P></TD>
    <TD align=3Dleft>
      <P class=3D"">gTC gTC ggA AAT Tgg CAg TAT</P></TD>
    <TD align=3Dleft>
      <P class=3D"">gCA ggT ggC AAA gAT gTA CAA</P></TD>
    <TD align=3Dleft>
      <P class=3D"">AAA gCT TCC AgT TCC TT</P></TD>
    <TD align=3Dleft>
      <P class=3D"">1&nbsp;min, 62=B0C</P></TD></TR>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">MAGEA4 [<CITE><A=20
      =
href=3D"http://www.springerlink.com/content/0wqj8uh583261461/fulltext.htm=
l#CR15">15</A></CITE>]=20
      </P></TD>
    <TD align=3Dleft>
      <P class=3D"">CCA CTA CCA TCA gCT TCA CTT gC</P></TD>
    <TD align=3Dleft>
      <P class=3D"">CTT CTC ggA ACA Agg ACT CTg C</P></TD>
    <TD align=3Dleft>
      <P class=3D"">Agg CAA CCC AAT gAg ggT TCC AgC</P></TD>
    <TD align=3Dleft>
      <P class=3D"">1&nbsp;min, 63=B0C</P></TD></TR>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">MAGEA6</P></TD>
    <TD align=3Dleft>
      <P class=3D"">gTC gTC ggA AAT Tgg CAg T</P></TD>
    <TD align=3Dleft>
      <P class=3D"">gCA ggT ggC AAA gAT gTA CAC</P></TD>
    <TD align=3Dleft>
      <P class=3D"">TgC AAg gAA TCg gAA gC</P></TD>
    <TD align=3Dleft>
      <P class=3D"">1&nbsp;min, 65=B0C</P></TD></TR>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">MAGEA10</P></TD>
    <TD align=3Dleft>
      <P class=3D"">TAC TgC ACC CCT gAg gAg gTC</P></TD>
    <TD align=3Dleft>
      <P class=3D"">TgT ggT ggC AAT TCT gTC CTg</P></TD>
    <TD align=3Dleft>
      <P class=3D"">AAA Tgg gAg TgA TCC AAg ATC CTT CCC AC</P></TD>
    <TD align=3Dleft>
      <P class=3D"">1&nbsp;min, 64=B0C</P></TD></TR>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">MAGEA12</P></TD>
    <TD align=3Dleft>
      <P class=3D"">ggT ggA AgT ggT CCg CAT Cg</P></TD>
    <TD align=3Dleft>
      <P class=3D"">gCC CTC CAC TgA TCT TTA gCA A</P></TD>
    <TD align=3Dleft>
      <P class=3D"">Agg CAT CTg ATg ggA gg</P></TD>
    <TD align=3Dleft>
      <P class=3D"">1&nbsp;min, 60=B0C</P></TD></TR>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">MAGEC2</P></TD>
    <TD align=3Dleft>
      <P class=3D"">ggg AAT CTg ACg gAT Cgg A</P></TD>
    <TD align=3Dleft>
      <P class=3D"">ggA ATg gAA CgC CTg gAA C</P></TD>
    <TD align=3Dleft>
      <P class=3D"">TgC TCC TgA AgA AgT CgT CAT gCC TCC</P></TD>
    <TD align=3Dleft>
      <P class=3D"">1&nbsp;min, 64=B0C</P></TD></TR>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">GAGE1,2,8<SUP>a</SUP> </P></TD>
    <TD align=3Dleft>
      <P class=3D"">CTA gAC CAA gAC gCT ACg TAg A</P></TD>
    <TD align=3Dleft>
      <P class=3D"">CCC ATC Agg ACC ATC TTC ACA</P></TD>
    <TD align=3Dleft>
      <P class=3D"">CCT ATg Cgg CCC gAg CAg TTC Ag</P></TD>
    <TD align=3Dleft>
      <P class=3D"">1&nbsp;min, 62=B0C</P></TD></TR>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">LAGE2/NY-ESO-1 [<CITE><A=20
      =
href=3D"http://www.springerlink.com/content/0wqj8uh583261461/fulltext.htm=
l#CR15">15</A></CITE>]=20
      </P></TD>
    <TD align=3Dleft>
      <P class=3D"">CgC CTg CTT gAg TTC TAC C</P></TD>
    <TD align=3Dleft>
      <P class=3D"">CAC TgC gTg ATC CAC ATC AAC A</P></TD>
    <TD align=3Dleft>
      <P class=3D"">TCA gTA TgT TgC Cgg ACA CAg TgA ACT C</P></TD>
    <TD align=3Dleft>
      <P class=3D"">1&nbsp;min, 62=B0C</P></TD></TR>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">ACTB [<CITE><A=20
      =
href=3D"http://www.springerlink.com/content/0wqj8uh583261461/fulltext.htm=
l#CR14">14</A></CITE>]=20
      </P></TD>
    <TD align=3Dleft>
      <P class=3D"">ATT gCC gAC Agg ATg CAg AA</P></TD>
    <TD align=3Dleft>
      <P class=3D"">gTC ATA CTC CTg CTT gCT gA</P></TD>
    <TD align=3Dleft>
      <P class=3D"">TCA AgA TCA TTg CTC CTC CTg AgC gC</P></TD>
    <TD align=3Dleft>
      <P class=3D"">1&nbsp;min, 60=B0C</P></TD></TR></TBODY></TABLE>
<DIV class=3DCapt>
<DIV class=3DCaptCont>
<DIV class=3D""><SUP>a</SUP>This assay detects GAGE1, GAGE2 and GAGE8=20
</DIV></DIV></DIV></DIV></DIV></DIV>
<DIV class=3D""><A name=3DSec7></A>
<DIV class=3DHeading3>Immunohistochemistry</DIV>
<P class=3D"">Immunochemistry was performed on 4&nbsp;&#956;m tissue =
sections of=20
formalin-fixed paraffin-embedded tissue blocks. Sections were heated for =

20&nbsp;min in citrate buffer (10&nbsp;mM, pH 6.0) for antigen =
retrieval. The=20
following mouse IgG1 monoclonal antibodies (mAb) were used: E978=20
(anti-<I>NY-ESO-1</I>) [<CITE><A=20
href=3D"http://www.springerlink.com/content/0wqj8uh583261461/fulltext.htm=
l#CR16">16</A></CITE>]=20
(Zymed, San Francisco, CA), MA454 (anti-<I>MAGE-A1</I>) [<CITE><A=20
href=3D"http://www.springerlink.com/content/0wqj8uh583261461/fulltext.htm=
l#CR17">17</A></CITE>]=20
(Zymed, San Francisco, CA), and 57B (anti-<I>MAGE-A4</I>) [<CITE><A=20
href=3D"http://www.springerlink.com/content/0wqj8uh583261461/fulltext.htm=
l#CR18">18</A></CITE>,=20
<CITE><A=20
href=3D"http://www.springerlink.com/content/0wqj8uh583261461/fulltext.htm=
l#CR19">19</A></CITE>]=20
(kindly provided by Dr. G.C. Spagnoli, University Hospital Basel, =
Switzerland).=20
Testis tissue with intact spermatogenesis was used as positive control. =
Tissue=20
sections were incubated with mAb diluted in PBA: E978 =
(2.5&nbsp;&#956;g/ml), MA454=20
(1&nbsp;&#956;g/ml), or 57B (5&nbsp;&#956;g/ml), or with IgG1 negative =
control antibody,=20
at room temperature for 1&nbsp;h. Binding sites of primary antibodies =
were then=20
detected by a biotinylated horse-antimouse secondary reagent (Vector=20
Laboratories, Burlingame, CA) followed by an avidin=96biotin complex =
system (ABC=20
Elite, Vector Laboratories). Diaminobenzidine tetrachloride served as a=20
chromogen. Immunoreactivity was assessed blindly with respect to the mAb =
used.=20
</P></DIV>
<DIV class=3D""><A name=3DSec8></A>
<DIV class=3DHeading3>Statistical analysis</DIV>
<DIV class=3DPara>
<DIV class=3D"">Normalized CGG values are presented as =
means&nbsp;=B1&nbsp;standard=20
deviation (SD). The SD of the normalized CGG values was calculated from =
the SD=20
of the CGG and the &#946;-actin values using the following formula:=20
CV&nbsp;=3D&nbsp;SQRT [CV<SUP>2</SUP>=20
<SUB>&#946;-actin</SUB>&nbsp;+&nbsp;CV<SUB>CGG</SUB><SUP>2</SUP>], where =

CV&nbsp;=3D&nbsp;SD/mean value (as described in the Sequence Detection =
System User=20
Bulletin 2, 1997, Applied Biosystems). Differences in mRNA expression =
levels=20
between pediatric and adult glioblastomas are calculated with the =
Spearman rank=20
correlation. All statistical tests were two-sided, significance was =
determined=20
as <I>P</I>&nbsp;&lt;&nbsp;0.05. </DIV></DIV></DIV></DIV>
<DIV class=3D""><A name=3DSec9></A>
<HR>

<DIV class=3Dheading2>Results</DIV>
<DIV class=3D""><A name=3DSec10></A>
<DIV class=3DHeading3>Study population</DIV>
<DIV class=3DPara>
<DIV class=3D"">We analyzed cancer-germline gene (CGG) expression in 50=20
fresh-frozen tumors by reverse transcription and polymerase chain =
reaction (PCR)=20
amplification. All samples were histologically proven brain tumors from=20
pediatric patients, &#8804;19-years old at the time of tumor resection =
and classified=20
according to the WHO 2007 classification [<CITE><A=20
href=3D"http://www.springerlink.com/content/0wqj8uh583261461/fulltext.htm=
l#CR20">20</A></CITE>]=20
(Table&nbsp;<A=20
href=3D"http://www.springerlink.com/content/0wqj8uh583261461/fulltext.htm=
l#Tab2">2</A>).=20
An extra-cohort of nine glioblastoma samples from adult patients was =
also=20
analyzed, for comparison to pediatric glioblastoma. Integrity of cDNA =
samples=20
was verified by conventional, 22-cycle PCR amplification of a =
626&nbsp;bp=20
<I>&#946;-actin</I> and a 983&nbsp;bp <I>GAPDH</I> product (data not =
shown). Samples=20
were subjected to conventional PCR amplification with consensus primers =
for the=20
12 genes of the <I>MAGE-A</I> family. Twenty-seven samples were =
positive,=20
indicating that, depending on the tumor type, 33=9664% of the =
investigated=20
pediatric tumors expressed at least one <I>MAGE-A</I> gene =
(Table&nbsp;<A=20
href=3D"http://www.springerlink.com/content/0wqj8uh583261461/fulltext.htm=
l#Tab2">2</A>).<A=20
name=3DTab2></A>
<DIV class=3DCapt><SPAN class=3DCaptNr>Table&nbsp;2&nbsp;</SPAN>Study =
group </DIV>
<TABLE border=3D1>
  <COLGROUP>
  <COL align=3Dleft>
  <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"">Tumor type<SUP>a</SUP> </P></TH>
    <TH align=3Dleft char=3D".">
      <P class=3D"">Number of patients</P></TH>
    <TH align=3Dleft char=3D"(">
      <P class=3D"">Average age (range)</P></TH>
    <TH align=3Dleft char=3D".">
      <P class=3D"">% of <I>MAGE-A-</I>positive tumors<SUP>b</SUP>=20
  </P></TH></TR></THEAD>
  <TBODY>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">Medulloblastoma</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">11</P></TD>
    <TD align=3Dchar char=3D"(">
      <P class=3D"">12&nbsp;years (4=9619)</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">55</P></TD></TR>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">Ependymoma</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">7</P></TD>
    <TD align=3Dchar char=3D"(">
      <P class=3D"">7&nbsp;years (1=9612)</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">43</P></TD></TR>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">Choroid plexus tumor</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">5</P></TD>
    <TD align=3Dchar char=3D"(">
      <P class=3D"">4&nbsp;years (1=9610)</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">40</P></TD></TR>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">Pilocytic astrocytoma (WHO grade&nbsp;I)</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">14</P></TD>
    <TD align=3Dchar char=3D"(">
      <P class=3D"">6&nbsp;years (3=9615)</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">64</P></TD></TR>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">Diffuse astrocytoma (WHO grade&nbsp;II)</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">3</P></TD>
    <TD align=3Dchar char=3D"(">
      <P class=3D"">10&nbsp;years (5=9618)</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">33</P></TD></TR>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">Anaplastic astrocytoma (WHO grade&nbsp;III)</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">5</P></TD>
    <TD align=3Dchar char=3D"(">
      <P class=3D"">13 years (7=9619)</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">60</P></TD></TR>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">Glioblastoma (WHO grade&nbsp;IV)</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">5</P></TD>
    <TD align=3Dchar char=3D"(">
      <P class=3D"">13 years (3=9619)</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">60</P></TD></TR>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">Glioblastoma (WHO grade&nbsp;IV)<SUP>c</SUP> =
</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">9</P></TD>
    <TD align=3Dchar char=3D"(">
      <P class=3D"">59 years (31=9674)</P></TD>
    <TD align=3Dchar char=3D".">
      <P class=3D"">100</P></TD></TR></TBODY></TABLE>
<DIV class=3DCapt>
<DIV class=3DCaptCont>
<DIV class=3D""><SUP>a</SUP>According to WHO 2007 classification =
[<CITE><A=20
href=3D"http://www.springerlink.com/content/0wqj8uh583261461/fulltext.htm=
l#CR20">20</A></CITE>]=20
</DIV></DIV>
<DIV class=3DCaptCont>
<DIV class=3D""><SUP>b</SUP>Gene expression was determined by =
conventional PCR=20
with consensus primers for the 12 genes of the <I>MAGE-A</I> family =
</DIV></DIV>
<DIV class=3DCaptCont>
<DIV class=3D""><SUP>c</SUP>An extra cohort of adult patients with =
glioblastomas=20
was studied to compare to pediatric glioblastomas (Fig.&nbsp;<A=20
href=3D"http://www.springerlink.com/content/0wqj8uh583261461/fulltext.htm=
l#Fig2">2</A>)=20
</DIV></DIV></DIV></DIV></DIV></DIV>
<DIV class=3D""><A name=3DSec11></A>
<DIV class=3DHeading3>CGG expression in pediatric brain tumors</DIV>
<DIV class=3DPara>
<DIV class=3D"">The 27 <I>MAGE</I>-<I>A</I>-positive samples were =
subsequently=20
analyzed by quantitative real-time PCR (qPCR), using the TaqMan =
methodology, to=20
measure the expression of the individual MAGE genes, <I>MAGE-A1</I>, =
<I>A2</I>,=20
<I>A3</I>, <I>A4</I>, <I>A6</I>, <I>A10</I>, and <I>A12</I>. In =
addition, all 50=20
samples were tested by qPCR for expression of genes <I>MAGE-C2</I>,=20
<I>NY-ESO-1</I> and <I>GAGE-1</I>,<I>2</I>,<I>8</I> (Table&nbsp;<A=20
href=3D"http://www.springerlink.com/content/0wqj8uh583261461/fulltext.htm=
l#Tab2">2</A>)<I>.</I>=20
CGG expression levels were normalized to those of the =
<I>&#946;-actin</I> gene=20
(Fig.&nbsp;<A=20
href=3D"http://www.springerlink.com/content/0wqj8uh583261461/fulltext.htm=
l#Fig1">1</A>).=20

<DIV class=3DFigure><A name=3DFig1></A><IMG=20
alt=3DMediaObjects/11060_2008_9577_Fig1_HTML.gif=20
src=3D"http://www.springerlink.com/content/0wqj8uh583261461/MediaObjects/=
11060_2008_9577_Fig1_HTML.gif"></DIV>
<DIV class=3DCapt><SPAN =
class=3DCaptNr>Fig.&nbsp;1&nbsp;</SPAN>Cancer-germline gene=20
(CGG) expression in pediatric brain tumors measured by reverse =
transcription and=20
quantitative real-time PCR. Each graph shows the results for one CGG in =
50=20
different tumor samples. Samples are arranged in the same order in all =
graphs.=20
The horizontal axis indicates the tumor type (Medullobl., =
medulloblastoma;=20
Ependy., ependymoma; Plexus, plexus choroideus; Ast., astrocytic tumors=20
grade&nbsp;I to IV). The bars represent normalized CGG expression values =

(CGG/<I>=DF-actin</I> ratios) </DIV>
<HR>
</DIV></DIV>
<DIV class=3DPara>
<DIV class=3D""><I>Medulloblastoma</I>. Six of the 11 medulloblastoma =
tumors=20
expressed at least 1 of the 10 CGGs that were analyzed. The expression =
of CGGs=20
in medulloblastomas was highly clustered (Table&nbsp;<A=20
href=3D"http://www.springerlink.com/content/0wqj8uh583261461/fulltext.htm=
l#Tab3">3</A>).=20
One sample expressed high levels (CGG/<I>=DF-actin</I>=20
ratios&nbsp;&gt;&nbsp;10<SUP>&#8722;2</SUP>) of 6 CGG genes. The other =
medulloblastoma=20
samples only sporadically expressed CGGs, with CGG/<I>=DF-actin</I> =
ratios above=20
10<SUP>&#8722;4</SUP>.<A name=3DTab3></A>
<DIV class=3DCapt><SPAN =
class=3DCaptNr>Table&nbsp;3&nbsp;</SPAN>Clustered pattern of=20
cancer-germline gene expression in medulloblastomas </DIV>
<TABLE border=3D1>
  <COLGROUP>
  <COL align=3Dleft>
  <COL align=3Dleft>
  <COL align=3Dleft>
  <COL align=3Dleft>
  <COL align=3Dleft>
  <COL align=3Dleft>
  <COL align=3Dleft>
  <COL align=3Dleft>
  <COL align=3Dleft>
  <COL align=3Dleft>
  <COL align=3Dleft></COLGROUP>
  <THEAD>
  <TR class=3Dheader>
    <TH align=3Dleft>
      <P class=3D"">Sample</P></TH>
    <TH align=3Dleft>
      <P class=3D"">MAGEA1</P></TH>
    <TH align=3Dleft>
      <P class=3D"">MAGEA2</P></TH>
    <TH align=3Dleft>
      <P class=3D"">MAGEA3</P></TH>
    <TH align=3Dleft>
      <P class=3D"">MAGEA4</P></TH>
    <TH align=3Dleft>
      <P class=3D"">MAGEA6</P></TH>
    <TH align=3Dleft>
      <P class=3D"">MAGEA10</P></TH>
    <TH align=3Dleft>
      <P class=3D"">MAGEA12</P></TH>
    <TH align=3Dleft>
      <P class=3D"">MAGEC2</P></TH>
    <TH align=3Dleft>
      <P class=3D"">NY-ESO-1</P></TH>
    <TH align=3Dleft>
      <P class=3D"">GAGE1,2,8</P></TH></TR></THEAD>
  <TBODY>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">1</P></TD>
    <TD align=3Dleft>
      <P class=3D"">++</P></TD>
    <TD align=3Dleft>
      <P class=3D"">++++</P></TD>
    <TD align=3Dleft>
      <P class=3D"">++++</P></TD>
    <TD align=3Dleft>
      <P class=3D"">&#8722;</P></TD>
    <TD align=3Dleft>
      <P class=3D"">++++</P></TD>
    <TD align=3Dleft>
      <P class=3D"">++++</P></TD>
    <TD align=3Dleft>
      <P class=3D"">++++</P></TD>
    <TD align=3Dleft>
      <P class=3D"">&#8722;</P></TD>
    <TD align=3Dleft>
      <P class=3D"">++++</P></TD>
    <TD align=3Dleft>
      <P class=3D"">+</P></TD></TR>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">2</P></TD>
    <TD align=3Dleft>
      <P class=3D"">&#8722;</P></TD>
    <TD align=3Dleft>
      <P class=3D"">++</P></TD>
    <TD align=3Dleft>
      <P class=3D"">+</P></TD>
    <TD align=3Dleft>
      <P class=3D"">+</P></TD>
    <TD align=3Dleft>
      <P class=3D"">+</P></TD>
    <TD align=3Dleft>
      <P class=3D"">&#8722;</P></TD>
    <TD align=3Dleft>
      <P class=3D"">+</P></TD>
    <TD align=3Dleft>
      <P class=3D"">&#8722;</P></TD>
    <TD align=3Dleft>
      <P class=3D"">&#8722;</P></TD>
    <TD align=3Dleft>
      <P class=3D"">&#8722;</P></TD></TR>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">3</P></TD>
    <TD align=3Dleft>
      <P class=3D"">&#8722;</P></TD>
    <TD align=3Dleft>
      <P class=3D"">&#8722;</P></TD>
    <TD align=3Dleft>
      <P class=3D"">&#8722;</P></TD>
    <TD align=3Dleft>
      <P class=3D"">&#8722;</P></TD>
    <TD align=3Dleft>
      <P class=3D"">+</P></TD>
    <TD align=3Dleft>
      <P class=3D"">&#8722;</P></TD>
    <TD align=3Dleft>
      <P class=3D"">++</P></TD>
    <TD align=3Dleft>
      <P class=3D"">&#8722;</P></TD>
    <TD align=3Dleft>
      <P class=3D"">&#8722;</P></TD>
    <TD align=3Dleft>
      <P class=3D"">++</P></TD></TR>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">4</P></TD>
    <TD align=3Dleft>
      <P class=3D"">&#8722;</P></TD>
    <TD align=3Dleft>
      <P class=3D"">&#8722;</P></TD>
    <TD align=3Dleft>
      <P class=3D"">&#8722;</P></TD>
    <TD align=3Dleft>
      <P class=3D"">&#8722;</P></TD>
    <TD align=3Dleft>
      <P class=3D"">&#8722;</P></TD>
    <TD align=3Dleft>
      <P class=3D"">&#8722;</P></TD>
    <TD align=3Dleft>
      <P class=3D"">+</P></TD>
    <TD align=3Dleft>
      <P class=3D"">&#8722;</P></TD>
    <TD align=3Dleft>
      <P class=3D"">&#8722;</P></TD>
    <TD align=3Dleft>
      <P class=3D"">&#8722;</P></TD></TR>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">5</P></TD>
    <TD align=3Dleft>
      <P class=3D"">&#8722;</P></TD>
    <TD align=3Dleft>
      <P class=3D"">&#8722;</P></TD>
    <TD align=3Dleft>
      <P class=3D"">&#8722;</P></TD>
    <TD align=3Dleft>
      <P class=3D"">&#8722;</P></TD>
    <TD align=3Dleft>
      <P class=3D"">&#8722;</P></TD>
    <TD align=3Dleft>
      <P class=3D"">&#8722;</P></TD>
    <TD align=3Dleft>
      <P class=3D"">+</P></TD>
    <TD align=3Dleft>
      <P class=3D"">&#8722;</P></TD>
    <TD align=3Dleft>
      <P class=3D"">&#8722;</P></TD>
    <TD align=3Dleft>
      <P class=3D"">&#8722;</P></TD></TR>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">6</P></TD>
    <TD align=3Dleft>
      <P class=3D"">&#8722;</P></TD>
    <TD align=3Dleft>
      <P class=3D"">&#8722;</P></TD>
    <TD align=3Dleft>
      <P class=3D"">&#8722;</P></TD>
    <TD align=3Dleft>
      <P class=3D"">+</P></TD>
    <TD align=3Dleft>
      <P class=3D"">&#8722;</P></TD>
    <TD align=3Dleft>
      <P class=3D"">&#8722;</P></TD>
    <TD align=3Dleft>
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    <TD align=3Dleft>
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    <TD align=3Dleft>
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    <TD align=3Dleft>
      <P class=3D"">&#8722;</P></TD></TR>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">7</P></TD>
    <TD align=3Dleft>
      <P class=3D"">&#8722;</P></TD>
    <TD align=3Dleft>
      <P class=3D"">&#8722;</P></TD>
    <TD align=3Dleft>
      <P class=3D"">&#8722;</P></TD>
    <TD align=3Dleft>
      <P class=3D"">&#8722;</P></TD>
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    <TD align=3Dleft>
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    <TD align=3Dleft>
      <P class=3D"">&#8722;</P></TD></TR>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">8</P></TD>
    <TD align=3Dleft>
      <P class=3D"">&#8722;</P></TD>
    <TD align=3Dleft>
      <P class=3D"">&#8722;</P></TD>
    <TD align=3Dleft>
      <P class=3D"">&#8722;</P></TD>
    <TD align=3Dleft>
      <P class=3D"">&#8722;</P></TD>
    <TD align=3Dleft>
      <P class=3D"">&#8722;</P></TD>
    <TD align=3Dleft>
      <P class=3D"">&#8722;</P></TD>
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      <P class=3D"">&#8722;</P></TD>
    <TD align=3Dleft>
      <P class=3D"">&#8722;</P></TD></TR>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">9</P></TD>
    <TD align=3Dleft>
      <P class=3D"">&#8722;</P></TD>
    <TD align=3Dleft>
      <P class=3D"">&#8722;</P></TD>
    <TD align=3Dleft>
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      <P class=3D"">&#8722;</P></TD></TR>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">10</P></TD>
    <TD align=3Dleft>
      <P class=3D"">&#8722;</P></TD>
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      <P class=3D"">&#8722;</P></TD>
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      <P class=3D"">&#8722;</P></TD></TR>
  <TR class=3Dnoclass>
    <TD align=3Dleft>
      <P class=3D"">11</P></TD>
    <TD align=3Dleft>
      <P class=3D"">&#8722;</P></TD>
    <TD align=3Dleft>
      <P class=3D"">&#8722;</P></TD>
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      <P class=3D"">&#8722;</P></TD>
    <TD align=3Dleft>
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    <TD align=3Dleft>
      <P class=3D"">&#8722;</P></TD></TR></TBODY></TABLE>
<DIV class=3DCapt>
<DIV class=3DCaptCont>
<DIV class=3D"">The signs +, ++, +++, and ++++ represent CGG/=DF-actin =
ratios=20
ranging between 10<SUP>&#8722;5</SUP> and 10<SUP>&#8722;4</SUP>, =
10<SUP>&#8722;4</SUP> and=20
10<SUP>&#8722;3</SUP>, 10<SUP>&#8722;3</SUP> and 10<SUP>&#8722;2</SUP>, =
and &gt;10<SUP>&#8722;2</SUP>=20
(see Fig.&nbsp;<A=20
href=3D"http://www.springerlink.com/content/0wqj8uh583261461/fulltext.htm=
l#Fig1">1</A>).=20
&#8722;, no CGG expression was detected </DIV></DIV></DIV></DIV></DIV>
<P class=3D""><I>Ependymoma</I>. Six out of seven ependymoma samples =
expressed at=20
least 1 CGG. None expressed more than 3. Here the expression was =
therefore not=20
clustered. Most positive samples expressed <I>NY-ESO-1</I> and/or=20
<I>GAGE-1</I>,<I>2</I>,<I>8.</I> The overall levels of CGG expression in =

ependymoma samples were low, with CGG/<I>&#946;-actin</I> ratios below=20
1&nbsp;=D7&nbsp;10<SUP>&#8722;3</SUP>. </P>
<P class=3D""><I>Choroid plexus tumor</I>. Of the five choroid plexus =
tumors=20
analyzed, three showed no detectable CGG expression. Samples #1 and #2 =
expressed=20
at least three CGGs. Only in one sample the CGG/<I>&#946;-actin</I> =
ratio exceeded=20
1&nbsp;=D7&nbsp;10<SUP>&#8722;3</SUP>. </P>
<P class=3D""><I>Pilocytic astrocytoma</I>. Nine out of 14 pilocytic =
astrocytomas=20
expressed at least one CGG. The overall expression was low, one low =
pilocytic=20
astrocytoma had a CGG/<I>&#946;-actin</I> ratio =
&gt;1&nbsp;=D7&nbsp;10<SUP>&#8722;4</SUP>.=20
</P>
<P class=3D""><I>Diffuse astrocytoma</I>. Two out of three diffuse =
astrocytomas=20
expressed at least one CGG. The overall expression level of CGGs on =
diffuse=20
astrocytomas was low. </P>
<P class=3D""><I>High grade astrocytoma</I>. Eight out of 10 anaplastic=20
astrocytomas and glioblastomas expressed at least one CGG. =
CGG/<I>&#946;-actin</I>=20
ratio in high grade astrocytomas did not exceed =
1&nbsp;=D7&nbsp;10<SUP>&#8722;3</SUP>.=20
</P></DIV>
<DIV class=3D""><A name=3DSec12></A>
<DIV class=3DHeading3>CGG expression in pediatric versus adult =
glioblastomas</DIV>
<DIV class=3DPara>
<DIV class=3D"">The relatively low expression of MAGE genes in high =
grade=20
astrocytomas was unexpected since <I>MAGE</I> expression has been =
reported in=20
almost all adult glioblastomas [<CITE><A=20
href=3D"http://www.springerlink.com/content/0wqj8uh583261461/fulltext.htm=
l#CR21">21</A></CITE>].=20
In these studies different methods were used, immunohistochemistry =
[<CITE><A=20
href=3D"http://www.springerlink.com/content/0wqj8uh583261461/fulltext.htm=
l#CR21">21</A></CITE>=96<CITE><A=20
href=3D"http://www.springerlink.com/content/0wqj8uh583261461/fulltext.htm=
l#CR24">24</A></CITE>]=20
and conventional, non-quantitative PCR [<CITE><A=20
href=3D"http://www.springerlink.com/content/0wqj8uh583261461/fulltext.htm=
l#CR22">22</A></CITE>=96<CITE><A=20
href=3D"http://www.springerlink.com/content/0wqj8uh583261461/fulltext.htm=
l#CR24">24</A></CITE>].=20
Therefore we decided to select nine new adult glioblastoma samples for=20
<I>MAGE</I> qPCR analysis. All samples expressed at least five =
<I>MAGE-A</I>=20
genes at high levels. The difference between the levels of <I>MAGE-A</I> =
genes=20
expression in pediatric versus adult glioblastomas is shown in =
Fig.&nbsp;<A=20
href=3D"http://www.springerlink.com/content/0wqj8uh583261461/fulltext.htm=
l#Fig2">2</A>=20
(<I>P</I>&nbsp;=3D&nbsp;6.1&nbsp;=D7&nbsp;10<SUP>&#8722;6</SUP>).=20
<DIV class=3DFigure><A name=3DFig2></A><IMG=20
alt=3DMediaObjects/11060_2008_9577_Fig2_HTML.gif=20
src=3D"http://www.springerlink.com/content/0wqj8uh583261461/MediaObjects/=
11060_2008_9577_Fig2_HTML.gif"></DIV>
<DIV class=3DCapt><SPAN class=3DCaptNr>Fig.&nbsp;2&nbsp;</SPAN>MAGE-A =
expression in=20
pediatric and adult glioblastomas. Normalized MAGE-A expression levels=20
(MAGE-A/<I>=DF-actin</I> ratios) of seven MAGE-A genes are shown. =
Horizontal bars=20
represent mean relative MAGE expression. Pediatric glioblastomas =
(<I>closed=20
circles</I>) express significantly lower levels of MAGE-A compared to =
adult=20
glioblastomas (<I>open circles</I>),=20
<I>P</I>&nbsp;=3D&nbsp;6.1&nbsp;=D7&nbsp;10<SUP>&#8722;6</SUP> =
calculated with Spearman=20
rank correlation </DIV>
<HR>
</DIV></DIV></DIV>
<DIV class=3D""><A name=3DSec13></A>
<DIV class=3DHeading3>Cancer-germline protein distribution</DIV>
<DIV class=3DPara>
<DIV class=3D"">Immunohistochemistry with monoclonal antibodies (mAbs) =
E978=20
(anti-NY-ESO=961), MA454 (anti-MAGE-A1) and 57B (anti-MAGE-A4) was =
performed on=20
available paraffin-embedded tissues. Sections from normal testis tissue =
were=20
used as positive controls. The intensity of the stainings correlated =
well with=20
the level of CGG expression, as shown in Fig.&nbsp;<A=20
href=3D"http://www.springerlink.com/content/0wqj8uh583261461/fulltext.htm=
l#Fig3">3</A>a,=20
b. The MAGE-A1, MAGE-A4 and NY-ESO-1 proteins were distributed =
homogenously=20
throughout the tissues. However, a minority of the tissues had a =
heterogeneous=20
staining pattern such as the MAGE-A4 expression in medulloblastoma =
sample #1=20
(Fig.&nbsp;<A=20
href=3D"http://www.springerlink.com/content/0wqj8uh583261461/fulltext.htm=
l#Fig3">3</A>c).=20
No staining could be observed for samples that scored either negative or =
with=20
CGG/<I>&#946;-actin</I> ratios below 10<SUP>&#8722;4</SUP>.=20
<DIV class=3DFigure><A name=3DFig3></A><IMG=20
alt=3DMediaObjects/11060_2008_9577_Fig3_HTML.gif=20
src=3D"http://www.springerlink.com/content/0wqj8uh583261461/MediaObjects/=
11060_2008_9577_Fig3_HTML.gif"></DIV>
<DIV class=3DCapt><SPAN =
class=3DCaptNr>Fig.&nbsp;3&nbsp;</SPAN>(<B>a</B>)=20
Amplification plots of three pilocytic astrocytomas in duplicate showing =
high=20
numbers (H), low numbers (L) or no (N) MAGE-A4 cDNA copies. (<B>b</B>)=20
Immunohistochemistry with mAb 57B (anti-MAGE-A4) of the pilocytic =
astrocytomas=20
shown in <B>a</B>. A section of normal testis is used as a positive =
control. The=20
intensity of the staining correlates with the amount of MAGE-A4 copies.=20
(<B>c</B>) Immunohistochemistry with mAbs E978 (anti-<I>NY-ESO-1</I>), =
MA454=20
(anti-<I>MAGE-A1</I>), 57B (anti-<I>MAGE-A4</I>) and the IgG isotype =
negative=20
control antibody on sections of medulloblastoma sample 1 (see =
Fig.&nbsp;<A=20
href=3D"http://www.springerlink.com/content/0wqj8uh583261461/fulltext.htm=
l#Fig1">1</A>=20
for relative mRNA expression). Original magnification 63=D7. This sample =
was=20
chosen because of the heterogeneous expression of the MAGE-A4 protein =
</DIV>
<HR>
</DIV></DIV></DIV>
<DIV class=3D""><A name=3DSec14></A>
<DIV class=3DHeading3>CGG expression and tumor stage</DIV>
<P class=3D"">The number of patients in each specific group of tumors =
was too=20
small to try to establish a correlation between CGG expression and=20
clinicopathological parameters. Stratification of the pediatric =
astrocytic=20
tumors in pilocytic astrocytoma (grade&nbsp;I), diffuse astrocytoma=20
(grade&nbsp;II) and high grade anaplastic astrocytomas and glioblastomas =

(grade&nbsp;III and IV, respectively), revealed that the level of CGG =
expression=20
was not significantly different between groups. These data suggest that =
CGG=20
expression in astrocytic tumors is not correlated with the grade of the=20
astrocytic tumor in pediatric patients. </P></DIV></DIV>
<DIV class=3D""><A name=3DSec15></A>
<HR>

<DIV class=3Dheading2>Discussion</DIV>
<P class=3D"">Antibodies and T cells can be identified that recognize =
antigenic=20
fragments derived from gene products expressed by tumors [<CITE><A=20
href=3D"http://www.springerlink.com/content/0wqj8uh583261461/fulltext.htm=
l#CR10">10</A></CITE>,=20
<CITE><A=20
href=3D"http://www.springerlink.com/content/0wqj8uh583261461/fulltext.htm=
l#CR25">25</A></CITE>,=20
<CITE><A=20
href=3D"http://www.springerlink.com/content/0wqj8uh583261461/fulltext.htm=
l#CR26">26</A></CITE>].=20
A critical role for these anti-tumor immune mechanisms in the =
eradication of=20
cancer has been demonstrated in numerous animal models and some clinical =
trials=20
[<CITE><A=20
href=3D"http://www.springerlink.com/content/0wqj8uh583261461/fulltext.htm=
l#CR27">27</A></CITE>,=20
<CITE><A=20
href=3D"http://www.springerlink.com/content/0wqj8uh583261461/fulltext.htm=
l#CR28">28</A></CITE>].=20
In the last decennia promising new strategies for the development of =
antibodies=20
and activated T cells against tumor associated antigens have been =
developed=20
[<CITE><A=20
href=3D"http://www.springerlink.com/content/0wqj8uh583261461/fulltext.htm=
l#CR29">29</A></CITE>,=20
<CITE><A=20
href=3D"http://www.springerlink.com/content/0wqj8uh583261461/fulltext.htm=
l#CR30">30</A></CITE>].=20
In spite of the blood brain barrier, there is accumulating evidence that =
even=20
brain tumors can cause immune activation and are amenable for =
immunotherapy=20
[<CITE><A=20
href=3D"http://www.springerlink.com/content/0wqj8uh583261461/fulltext.htm=
l#CR2">2</A></CITE>,=20
<CITE><A=20
href=3D"http://www.springerlink.com/content/0wqj8uh583261461/fulltext.htm=
l#CR4">4</A></CITE>,=20
<CITE><A=20
href=3D"http://www.springerlink.com/content/0wqj8uh583261461/fulltext.htm=
l#CR31">31</A></CITE>].=20
</P>
<P class=3D"">The identification of immunogenic tumor associated =
antigens is an=20
essential step in the development of rational cancer vaccines. The =
potential of=20
CGGs as vaccine targets has led to detailed studies of their expression =
in=20
various malignancies in adult patients [<CITE><A=20
href=3D"http://www.springerlink.com/content/0wqj8uh583261461/fulltext.htm=
l#CR10">10</A></CITE>,=20
<CITE><A=20
href=3D"http://www.springerlink.com/content/0wqj8uh583261461/fulltext.htm=
l#CR32">32</A></CITE>].=20
Previously, we reported on the expression of CGGs in pediatric =
extra-cranial=20
tumors [<CITE><A=20
href=3D"http://www.springerlink.com/content/0wqj8uh583261461/fulltext.htm=
l#CR11">11</A></CITE>].=20
Here we report that, in our cohort of 50 pediatric brain tumors, 68% =
expressed=20
one or more CGGs. Immunohistochemical data correlated well with the qPCR =

results. Apart from a few exceptions, the overall level of CGG =
expression on=20
pediatric brain tumors is low. Preliminary data from cytotoxicity assays =

indicated that glioblastoma cell lines that express low levels of CGGs=20
(CGG/<I>&#946;-actin</I> =
ratio&nbsp;&lt;&nbsp;1&nbsp;=D7&nbsp;10<SUP>&#8722;4</SUP>) are not=20
specifically lysed by HLA-matched anti-MAGE A3 CTL cell lines (data not =
shown).=20
</P>
<P class=3D"">In contrast to the limited CGG expression found in =
pediatric brain=20
tumors, glioblastomas from adult patients express significantly higher =
levels of=20
CGGs (<I>P</I>&nbsp;&lt;&nbsp;0,001). This finding is in line with =
reported=20
differences between adult and pediatric glioblastomas in p53 and EGFR =
expression=20
[<CITE><A=20
href=3D"http://www.springerlink.com/content/0wqj8uh583261461/fulltext.htm=
l#CR33">33</A></CITE>,=20
<CITE><A=20
href=3D"http://www.springerlink.com/content/0wqj8uh583261461/fulltext.htm=
l#CR34">34</A></CITE>].=20
The differences in protein expression may be explained by chromosomal=20
aberrations and differences in microsatellite stability between adult=20
glioblastomas and pediatric glioblastomas [<CITE><A=20
href=3D"http://www.springerlink.com/content/0wqj8uh583261461/fulltext.htm=
l#CR35">35</A></CITE>,=20
<CITE><A=20
href=3D"http://www.springerlink.com/content/0wqj8uh583261461/fulltext.htm=
l#CR36">36</A></CITE>].=20
The significant difference in MAGE-A expression between adult and =
pediatric=20
glioblastomas supports the view that these tumors develop along distinct =
genetic=20
pathways. </P>
<P class=3D"">In conclusion, we report limited CGG expression in =
pediatric brain=20
tumors. Only a small percentage of brain tumors express high levels of =
CGGs.=20
These data indicate that CGGs can only be used as immune target in a =
selected=20
group of pediatric brain tumors. </P></DIV>
<DIV class=3DAcknowledgments><SPAN=20
class=3DAcknowledgmentsHeading>Acknowledgements&nbsp;&nbsp;</SPAN><SPAN=20
class=3D"">The authors wish to thank Dr. B. Leth=E9 for providing the =
reagents for=20
LAGE-2/NY-ESO, GAGE-1,2,8 and ACTB quantitative PCR. Dr. E. De Plaen for =
the=20
reagents for MAGEA2 and MAGEA12 quantitative PCR. Riki Willems for =
assistance=20
with the pathology database. Th=E9r=E8se Aerts and Madeleine Swinarska =
for technical=20
assistance. This work was supported by grants from =93The Quality of =
Life Gala=94=20
and =93Stichting Vrienden van het Kinderoncologisch Centrum Zuid-Oost=20
Nederland=94.</SPAN>
<DIV class=3DFormalPara>
<DIV class=3D""><SPAN style=3D"FONT-STYLE: italic; TEXT-DECORATION: =
none">Open=20
Access</SPAN>&nbsp;&nbsp; This article is distributed under the terms of =
the=20
Creative Commons Attribution Noncommercial License which permits any=20
noncommercial use, distribution, and reproduction in any medium, =
provided the=20
original author(s) and source are credited. </DIV></DIV></DIV>
<DIV class=3DAppendix><A name=3DAppESM1></A>
<DIV class=3D""><A name=3DSecESM1></A>
<HR>

<DIV class=3Dheading2>Electronic Supplementary Material</DIV>
<DIV class=3DPara>
<DIV class=3D"">
<DIV><A=20
href=3D"http://www.springerlink.com/content/0wqj8uh583261461/MediaObjects=
/11060_2008_9577_MOESM1_ESM.pdf">
<DIV class=3DCapt>(PDF 1216 kb)</DIV></A></DIV></DIV></DIV></DIV></DIV>
<P></P>
<HR>

<H2><A name=3DBib1></A>References </H2>
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  <TR>
    <TD>&nbsp;</TD></TR></TBODY></TABLE></BODY></HTML>

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