10.1007/s00401-008-0422-y</TITLE= > <META http-equiv=3DContent-Type content=3D"text/html; = charset=3DISO-8859-1"><LINK=20 href=3D"http://www.springerlink.com/config/css/A++_common.css" = type=3Dtext/css=20 rel=3DstyleSheet><LINK=20 href=3D"http://www.springerlink.com/config/css/content_A++_SpringerLink.c= ss"=20 type=3Dtext/css rel=3DstyleSheet> <META content=3D"MSHTML 6.00.6000.16705" name=3DGENERATOR></HEAD> <BODY class=3Dfor-print> <TABLE cellPadding=3D2 border=3D1> <TBODY> <TR class=3Dheader> <TD>Acta Neuropathologica</TD></TR> <TR> <TD>=A9 The Author(s) 2008</TD></TR> <TR> <TD>10.1007/s00401-008-0422-y</TD></TR></TBODY></TABLE> <H2 class=3Drubric>Original Paper</H2> <DIV class=3DHeading1><A name=3Dtitle></A>Accumulation of genomic = aberrations during=20 clinical progression of medulloblastoma </DIV> Andrey Korshunov1, 2 <A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#ContactOfAuthor1"><IMG=20 alt=3D"Contact Information"=20 src=3D"http://www.springerlink.com/content/tt4661418w621n24/contact.gif" = border=3D0></A>, Axel Benner3, = Marc Remke4,=20 Peter Lichter4, Andreas von Deimling1, = 2=20 and Stefan Pfister4, 5 <TABLE> <TBODY> <TR vAlign=3Dtop> <TD><A = name=3DAff1></A>(1) </TD> <TD>Clinical Cooperation Unit = Neuropathology,=20 German Cancer Research Center, Im Neuenheimer Feld 280,=20 69120 Heidelberg, Germany</TD></TR></TBODY></TABLE> <TABLE> <TBODY> <TR vAlign=3Dtop> <TD><A = name=3DAff2></A>(2) </TD> <TD>Department of Neuropathology, = University of=20 Heidelberg, Im Neuenheimer Feld 220/221, 69120 Heidelberg,=20 Germany</TD></TR></TBODY></TABLE> <TABLE> <TBODY> <TR vAlign=3Dtop> <TD><A = name=3DAff3></A>(3) </TD> <TD>Division Biostatistics, German Cancer = Research=20 Center, Im Neuenheimer Feld 280, 69120 Heidelberg,=20 Germany</TD></TR></TBODY></TABLE> <TABLE> <TBODY> <TR vAlign=3Dtop> <TD><A = name=3DAff4></A>(4) </TD> <TD>Division Molecular Genetics, German = Cancer=20 Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg,=20 Germany</TD></TR></TBODY></TABLE> <TABLE> <TBODY> <TR vAlign=3Dtop> <TD><A = name=3DAff5></A>(5) </TD> <TD>Department of Pediatric Oncology, = Hematology=20 & Immunology, University of Heidelberg, Im Neuenheimer Feld = 153,=20 69120 Heidelberg, Germany</TD></TR></TBODY></TABLE> <A name=3DContactOfAuthor1></A> <TABLE class=3DContact> <TBODY> <TR> <TD vAlign=3Dtop><IMG alt=3D"Contact Information"=20 = src=3D"http://www.springerlink.com/content/tt4661418w621n24/contact.gif" = border=3D0></TD> = <TD>Andrey Korshunov</STRO= NG> Email:=20 <A=20 = href=3D"mailto:Andrey.Korshunov@med.uni-heidelberg.de">Andrey.Korshunov@m= ed.uni-heidelberg.de</A></TD></TR></TBODY></TABLE> Received:=20 14 July 2008 Revised:=20 6 August 2008 Accepted:=20 6 August 2008 Published online:=20 15 August 2008 <DIV class=3DAbstract><A name=3DAbs1></A><SPAN=20 class=3DAbstractHeading>Abstract Medulloblastomas = comprise the=20 most frequent malignant brain tumor in childhood and one of the biggest=20 challenges in pediatric oncology. The current concept suggests that = these tumors=20 may undergo stepwise progression as it has been shown for other brain = tumors.=20 However, conclusive evidence of molecular progression over time has not = been=20 demonstrated yet for medulloblastoma. In the present study, 28 pairs of=20 medulloblastoma at primary diagnosis and at the time of recurrence, = either=20 occurring as local tumor regrowth or tumor dissemination, were=20 histopathologically and molecularly analyzed. Cytogenetic analysis = included=20 interphase fluorescence in situ hybridization for five genomic loci = (MYC,=20 MYCN, 17p, 17q, 6q) that have previously been identified as = prognostic=20 markers in primary tumors. Of 16 tumors showing early recurrence=20 (<4 years after first diagnosis), only one showed increased = histological=20 anaplasia in the secondary lesion (6%), and two acquired genomic lesions = indicative for a more malignant phenotype (13%). In contrast to this, of = 12=20 tumors with a time to recurrence of 4 years or more, nine tumors = (75%)=20 showed a more malignant phenotype either reflected by increased = anaplasia alone=20 or by both increased anaplasia and acquirement of genomic aberrations = known to=20 be associated with inferior patient outcome. These results suggest that = early=20 recurrence in medulloblastoma mainly occurs in tumors with a highly = malignant=20 genotype and phenotype per se, whereas late recurrence is often = dependent on=20 tumor evolution toward a more malignant biology. Therefore, biopsy of = recurrent=20 tumors should be performed to assess the biologic properties of the = relapsed=20 tumor, especially when targeted therapy approaches are considered. = </DIV> <SPAN=20 class=3DKeywordHeading>Keywords Medulloblastoma -&= nbsp;Progression - Metastasis - Genomic=20 aberration - Anaplasia - Chromosome 17q <DIV class=3D""><A name=3DSec1></A> <HR> <DIV class=3Dheading2>Introduction</DIV> Brain tumors are the leading cause of cancer-related = mortality in=20 pediatric patients, with medulloblastoma (MB) being the most common=20 representative in this age group. Considerable advances have been made = in the=20 treatment of MB in the last three decades by using multimodal = therapeutic=20 regimens involving surgery, radiotherapy, and chemotherapy. About = 10=9615% of=20 patients die from disease within 2 years after diagnosis, whereas=20 approximately 60% of patients can nowadays be cured [<CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR9">9</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR11">11</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR22">22</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR23">23</A></CITE>].=20 However, the predictive performance of current risk stratification, = which is=20 entirely based on clinical variables, needs to be improved because of = the dismal=20 prognosis of children with tumor relapse, and because of relevant=20 treatment-induced long-term effects of survivors [<CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR21">21</A></CITE>].=20 Clinical staging systems based on age, metastatic stage at diagnosis = and, in=20 some studies, extent of surgical resection have so far been the most = useful=20 methods for the stratification of patients into standard and high risk = therapy=20 groups [<CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR1">1</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR26">26</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR27">27</A></CITE>].=20 Additionally, after refinement of the histological classification of MB = in the=20 2007 WHO classification, the histological subtype is of potential = prognostic=20 relevance: large cell/anaplastic MB behaves more aggressively, while=20 nodular/desmoplastic variants show a more favorable clinical course as = compared=20 to the large group of classic MB [<CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR3">3</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR5">5</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR7">7</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR10">10</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR13">13</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR14">14</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR17">17</A></CITE>].=20 Some recent studies have extended the concept of anaplasia in MB and = suggested=20 that this phenomenon reflects malignant progression of a tumor and may = occur in=20 various MB subtypes independent of large cell histology [<CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR3">3</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR4">4</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR12">12</A></CITE>].=20 Progression over time from nonanaplastic to anaplastic MB has been = described in=20 cases with local tumor recurrence [<CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR3">3</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR12">12</A></CITE>]=20 as well as in secondary metastatic lesions with or without local = recurrence=20 [<CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR4">4</A></CITE>].=20 To date, numerous molecular studies have highlighted = various=20 chromosomal aberrations in MB, and some of them were found to be = associated with=20 clinical outcome. Cytogenetic aberrations in MB involving chromosome 17 = as well=20 as amplifications of the MYC or MYCN oncogenes have = previously=20 been associated with large cell histology and poor patient survival in = different=20 studies [<CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR6">6</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR7">7</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR9">9</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR11">11</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR13">13</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR15">15</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR17">17</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR18">18</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR23">23</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR24">24</A></CITE>].=20 In contrast, several recent reports have identified monosomy of = chromosome 6 to=20 be associated with consecutive WNT pathway activation and favorable = clinical=20 outcome [<CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR2">2</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR8">8</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR25">25</A></CITE>].=20 Nevertheless, in contrast to astrocytomas and = oligodendrogliomas,=20 molecular progression of MB has only been suspected, but has not been=20 unanimously proven. A stepwise progression of genetic abnormalities in = these=20 tumors was identified in a few cases only, when comparing primary and = recurrent=20 tumor specimens [<CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR3">3</A></CITE>].=20 Here, we performed comparative histopathologic and = cytogenetic=20 analyses of 28 MB cases, for which samples from primary manifestation = and tumor=20 recurrence were available. The aim of this study was to determine which=20 histopathological features and chromosome abnormalities may be = associated with=20 MB molecular progression over time. Interphase fluorescence in situ=20 hybridization (FISH) was carried out for five regions of interest, = namely=20 MYC, MYCN, chromosome 6q, and chromosomal arms 17p and = 17q.=20 </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 population</DIV> <DIV class=3DPara> <DIV class=3D"">Specimens from 28 MB patients who experienced = disease=20 recurrence were analyzed in this study. All these patients had received=20 reoperations at secondary diagnosis and specimens from primary and = subsequent=20 interventions were available for comparative histopathological and = molecular=20 analyses. Patient details are outlined in Table <A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#Tab1">1</A>.=20 There were 21 males and 7 females, and age ranged from 3 to = 37 years=20 (median 14 years). Sixteen patients experienced early recurrence=20 (<4 years after first diagnosis), and 12 patients had a time to=20 recurrence of 48 months or more (late recurrence). Staging = procedures=20 included MRI before and immediately after neurosurgery. Metastatic = disease was=20 detected by craniospinal imaging and analysis of the cerebrospinal fluid = following the Chang classification. Most of the patients (26) had no=20 leptomeningeal tumor dissemination (i.e. M0 stage) at the time of = primary=20 diagnosis. All patients were treated according to a standard treatment = protocol,=20 consisting of surgical tumor resection, followed by craniospinal = irradiation=20 with 36 Gy and a boost to the posterior fossa adding up to a total = dose of=20 53=9656 Gy, and eight cycles of vincristin, lomustine, and = cisplatin. Gross=20 total tumor resection was achieved in 23 patients (82%) as assessed by = early=20 postoperative MRI. Tumors progressed as the following (Table <A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#Tab2">2</A>):=20 (1) isolated local recurrence (13 cases); (2) local recurrence in = combination=20 with leptomeningeal dissemination (six cases); among the latter, in one = patient,=20 tumor progressed as local recurrence combined with leptomeningeal = dissemination=20 and seedings outside the CNS (bone marrow and soft tissues); (3) = isolated=20 leptomeningeal dissemination in nine cases. Specimens from secondary = tumors were=20 obtained either after removal of local progression = (n =3D 18) or=20 after resection/biopsy of tumor metastases = (n =3D 10).<A=20 name=3DTab1></A> <DIV class=3DCapt>Table 1 Clinical = characteristics of medulloblastoma patients with early progression=20 (n =3D 16) and late progression = (n =3D 12) </DIV> <TABLE border=3D1> <COLGROUP> <COL align=3Dleft> <COL align=3Dleft> <COL align=3Dleft> <COL align=3Dleft></COLGROUP> <THEAD> <TR class=3Dheader> <TH align=3Dleft> Variable</TH> <TH align=3Dleft> Early recurrence (<48 months)</TH> <TH align=3Dleft> Late recurrence (≥48 months)</TH> <TH align=3Dleft> Total</TH></TR></THEAD> <TBODY> <TR class=3Dnoclass> <TD align=3Dleft colSpan=3D4> Agea </TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> Median age (years)</TD> <TD align=3Dleft> 11.5</TD> <TD align=3Dleft> 15</TD> <TD align=3Dleft> 14</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> Range (years)</TD> <TD align=3Dleft> 3=9622</TD> <TD align=3Dleft> 7=9637</TD> <TD align=3Dleft> 3=9637</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft colSpan=3D4> Gender</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> Female</TD> <TD align=3Dleft> 5 (31%)</TD> <TD align=3Dleft> 2 (17%)</TD> <TD align=3Dleft> 7 (25%)</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> Male</TD> <TD align=3Dleft> 11 (69%)</TD> <TD align=3Dleft> 10 (83%)</TD> <TD align=3Dleft> 21 (75%)</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft colSpan=3D4> Chang stage</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> M0 </TD> <TD align=3Dleft> 14 (87%)</TD> <TD align=3Dleft> 12 (100%)</TD> <TD align=3Dleft> 26 (93%)</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> M1=963 </TD> <TD align=3Dleft> 2 (13%)</TD> <TD align=3Dleft> 0 (0%)</TD> <TD align=3Dleft> 2 (7%)</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft colSpan=3D4> Level of resection at primary = diagnosis</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> Gross total resection</TD> <TD align=3Dleft> 12 (75%)</TD> <TD align=3Dleft> 11 (92%)</TD> <TD align=3Dleft> </TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> Macroscopic tumor residue</TD> <TD align=3Dleft> 4 (25%)</TD> <TD align=3Dleft> 1 (8%)</TD> <TD align=3Dleft> </TD></TR> <TR class=3Dnoclass> <TD align=3Dleft colSpan=3D4> Type of progression</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> Local progression</TD> <TD align=3Dleft> 5 (31%)</TD> <TD align=3Dleft> 8 (67%)</TD> <TD align=3Dleft> 13 (46%)</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> Dissemination</TD> <TD align=3Dleft> 6 (38%)</TD> <TD align=3Dleft> 3 (25%)</TD> <TD align=3Dleft> 9 (32%)</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> Local progression and dissemination</TD> <TD align=3Dleft> 5 (31%)</TD> <TD align=3Dleft> 1 (8%)</TD> <TD align=3Dleft> 6 (21%) </TD></TR> <TR class=3Dnoclass> <TD align=3Dleft colSpan=3D4> Time to progression</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> Median (months)</TD> <TD align=3Dleft> 16</TD> <TD align=3Dleft> 70</TD> <TD align=3Dleft> 34</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> Range (months)</TD> <TD align=3Dleft> 4=9639</TD> <TD align=3Dleft> 48=96138</TD> <TD align=3Dleft> 4=96138</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> Total</TD> <TD align=3Dleft> 16</TD> <TD align=3Dleft> 12</TD> <TD align=3Dleft> 28</TD></TR></TBODY></TABLE> <DIV class=3DCapt> <DIV class=3DCaptCont> <DIV class=3D"">aAt initial diagnosis </DIV></DIV></DIV><A=20 name=3DTab2></A> <DIV class=3DCapt>Table 2 Histopathological=20 and molecular characteristics of medulloblastoma patients at primary = diagnosis=20 and at the time of disease progression </DIV> <TABLE border=3D1> <COLGROUP> <COL align=3Dleft> <COL align=3Dchar char=3D"."> <COL align=3Dleft> <COL align=3Dleft> <COL align=3Dleft> <COL align=3Dleft> <COL align=3Dleft></COLGROUP> <THEAD> <TR class=3Dheader> <TH align=3Dleft rowSpan=3D2> Number</TH> <TH align=3Dleft rowSpan=3D2 char=3D"."> Time to progression (months)</TH> <TH align=3Dleft rowSpan=3D2> Type of progression</TH> <TH align=3Dleft colSpan=3D2> Histology</TH> <TH align=3Dleft colSpan=3D2> Molecular markers</TH></TR> <TR class=3Dheader> <TH align=3Dleft> Primary diagnosis</TH> <TH align=3Dleft> Progression</TH> <TH align=3Dleft> Primary diagnosis</TH> <TH align=3Dleft> Progression</TH></TR></THEAD> <TBODY> <TR class=3Dnoclass> <TD align=3Dleft> 1</TD> <TD align=3Dchar char=3D"."> 4</TD> <TD align=3Dleft> mts</TD> <TD align=3Dleft> LCA</TD> <TD align=3Dleft> LCA</TD> <TD align=3Dleft> i17q</TD> <TD align=3Dleft> i17q</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> 2</TD> <TD align=3Dchar char=3D"."> 6</TD> <TD align=3Dleft> Local</TD> <TD align=3Dleft> LCA</TD> <TD align=3Dleft> LCA</TD> <TD align=3Dleft> MYC; i17q; gain 6q</TD> <TD align=3Dleft> MYC; i17q; gain 6q</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> 3</TD> <TD align=3Dchar char=3D"."> 8</TD> <TD align=3Dleft> Local/mts</TD> <TD align=3Dleft> LCA</TD> <TD align=3Dleft> LCA</TD> <TD align=3Dleft> MYCN; gain 17q</TD> <TD align=3Dleft> MYCN; gain 17q</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> 4</TD> <TD align=3Dchar char=3D"."> 8</TD> <TD align=3Dleft> mts</TD> <TD align=3Dleft> Classic NA</TD> <TD align=3Dleft> Classic NA</TD> <TD align=3Dleft> i17q</TD> <TD align=3Dleft> i17q</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> 5</TD> <TD align=3Dchar char=3D"."> 9</TD> <TD align=3Dleft> Local/mts</TD> <TD align=3Dleft> Classic NA</TD> <TD align=3Dleft> Classic NA</TD> <TD align=3Dleft> i17q</TD> <TD align=3Dleft> i17q</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> 6</TD> <TD align=3Dchar char=3D"."> 11</TD> <TD align=3Dleft> Local</TD> <TD align=3Dleft> Classic MA</TD> <TD align=3Dleft> Classic MA</TD> <TD align=3Dleft> Balanced </TD> <TD align=3Dleft> gain 17q </TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> 7</TD> <TD align=3Dchar char=3D"."> 12</TD> <TD align=3Dleft> Local/mts</TD> <TD align=3Dleft> Classic MA</TD> <TD align=3Dleft> Classic MA</TD> <TD align=3Dleft> i17q</TD> <TD align=3Dleft> i17q</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> 8a </TD> <TD align=3Dchar char=3D"."> 15</TD> <TD align=3Dleft> Local/mts</TD> <TD align=3Dleft> LCA</TD> <TD align=3Dleft> LCA</TD> <TD align=3Dleft> MYC; i17q; gain 6q</TD> <TD align=3Dleft> MYC; i17q; gain 6q</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> 9</TD> <TD align=3Dchar char=3D"."> 17</TD> <TD align=3Dleft> Local</TD> <TD align=3Dleft> Classic MA</TD> <TD align=3Dleft> Classic MA</TD> <TD align=3Dleft> Balanced</TD> <TD align=3Dleft> Balanced</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> 10</TD> <TD align=3Dchar char=3D"."> 18</TD> <TD align=3Dleft> Local/mts</TD> <TD align=3Dleft> Classic MA</TD> <TD align=3Dleft> Classic MA</TD> <TD align=3Dleft> Balanced </TD> <TD align=3Dleft> gain 17q </TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> 11</TD> <TD align=3Dchar char=3D"."> 18</TD> <TD align=3Dleft> Local</TD> <TD align=3Dleft> Classic NA</TD> <TD align=3Dleft> Classic NA</TD> <TD align=3Dleft> gain 17q</TD> <TD align=3Dleft> gain 17q</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> 12</TD> <TD align=3Dchar char=3D"."> 21</TD> <TD align=3Dleft> mts</TD> <TD align=3Dleft> Classic MA</TD> <TD align=3Dleft> Classic MA</TD> <TD align=3Dleft> MYCN; i17q </TD> <TD align=3Dleft> i17q </TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> 13</TD> <TD align=3Dchar char=3D"."> 27</TD> <TD align=3Dleft> mts</TD> <TD align=3Dleft> Classic NA</TD> <TD align=3Dleft> Classic NA</TD> <TD align=3Dleft> MYCN; i17q </TD> <TD align=3Dleft> i17q </TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> 14</TD> <TD align=3Dchar char=3D"."> 30</TD> <TD align=3Dleft> mts</TD> <TD align=3Dleft> Classic NA </TD> <TD align=3Dleft> Classic MA </TD> <TD align=3Dleft> i17q</TD> <TD align=3Dleft> i17q</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> 15</TD> <TD align=3Dchar char=3D"."> 38</TD> <TD align=3Dleft> Local</TD> <TD align=3Dleft> Classic NA</TD> <TD align=3Dleft> Classic NA</TD> <TD align=3Dleft> Balanced</TD> <TD align=3Dleft> Balanced</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> 16</TD> <TD align=3Dchar char=3D"."> 39</TD> <TD align=3Dleft> mts</TD> <TD align=3Dleft> Classic NA</TD> <TD align=3Dleft> Classic NA</TD> <TD align=3Dleft> i17q</TD> <TD align=3Dleft> i17q</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> 17</TD> <TD align=3Dchar char=3D"."> 48</TD> <TD align=3Dleft> Local</TD> <TD align=3Dleft> Classic NA</TD> <TD align=3Dleft> Classic NA</TD> <TD align=3Dleft> Balanced</TD> <TD align=3Dleft> Balanced</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> 18</TD> <TD align=3Dchar char=3D"."> 57</TD> <TD align=3Dleft> Local</TD> <TD align=3Dleft> Classic MA </TD> <TD align=3Dleft> LCA </TD> <TD align=3Dleft> Balanced </TD> <TD align=3Dleft> MYCN </TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> 19</TD> <TD align=3Dchar char=3D"."> 58</TD> <TD align=3Dleft> mts</TD> <TD align=3Dleft> Classic NA </TD> <TD align=3Dleft> Classic MA </TD> <TD align=3Dleft> Balanced</TD> <TD align=3Dleft> balanced</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> 20</TD> <TD align=3Dchar char=3D"."> 61</TD> <TD align=3Dleft> mts</TD> <TD align=3Dleft> Classic MA </TD> <TD align=3Dleft> LCA </TD> <TD align=3Dleft> i17q</TD> <TD align=3Dleft> i17q</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> 21</TD> <TD align=3Dchar char=3D"."> 62</TD> <TD align=3Dleft> Local</TD> <TD align=3Dleft> Classic NA </TD> <TD align=3Dleft> Classic MA </TD> <TD align=3Dleft> i17q</TD> <TD align=3Dleft> i17q</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> 22</TD> <TD align=3Dchar char=3D"."> 67</TD> <TD align=3Dleft> Local</TD> <TD align=3Dleft> Classic NA</TD> <TD align=3Dleft> Classic NA</TD> <TD align=3Dleft> Balanced</TD> <TD align=3Dleft> Balanced</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> 23</TD> <TD align=3Dchar char=3D"."> 73</TD> <TD align=3Dleft> mts</TD> <TD align=3Dleft> Classic MA </TD> <TD align=3Dleft> LCA </TD> <TD align=3Dleft> Balanced </TD> <TD align=3Dleft> i17q </TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> 24</TD> <TD align=3Dchar char=3D"."> 78</TD> <TD align=3Dleft> Local</TD> <TD align=3Dleft> Classic NA </TD> <TD align=3Dleft> Classic MA </TD> <TD align=3Dleft> Balanced </TD> <TD align=3Dleft> gain 17q; gain 6q </TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> 25</TD> <TD align=3Dchar char=3D"."> 91</TD> <TD align=3Dleft> Local</TD> <TD align=3Dleft> Classic NA </TD> <TD align=3Dleft> LCA </TD> <TD align=3Dleft> Balanced </TD> <TD align=3Dleft> MYCN </TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> 26</TD> <TD align=3Dchar char=3D"."> 96</TD> <TD align=3Dleft> Local/mts</TD> <TD align=3Dleft> Classic NA </TD> <TD align=3Dleft> LCA </TD> <TD align=3Dleft> Balanced </TD> <TD align=3Dleft> gain 17q; gain 6q </TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> 27</TD> <TD align=3Dchar char=3D"."> 132</TD> <TD align=3Dleft> Local</TD> <TD align=3Dleft> Classic NA </TD> <TD align=3Dleft> Classic MA </TD> <TD align=3Dleft> Balanced</TD> <TD align=3Dleft> Balanced</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> 28</TD> <TD align=3Dchar char=3D"."> 138</TD> <TD align=3Dleft> Local</TD> <TD align=3Dleft> Classic MA</TD> <TD align=3Dleft> Classic MA</TD> <TD align=3Dleft> i17q</TD> <TD align=3Dleft> i17q</TD></TR></TBODY></TABLE> <DIV class=3DCapt> <DIV class=3DCaptCont> <DIV class=3D"">Cases with accumulation of histological and molecular = changes are=20 indicated in bold</DIV></DIV> <DIV class=3DCaptCont> <DIV class=3D"">mts metastatic disease, LCA large = cell/anaplastic=20 histology, NA no signs of anaplasia, MA moderate anaplasia = </DIV></DIV> <DIV class=3DCaptCont> <DIV class=3D"">aCase 8 also showed metastasis outside the = CNS (soft=20 tissue and bone marrow) </DIV></DIV></DIV></DIV></DIV></DIV> <DIV class=3D""><A name=3DSec4></A> <DIV class=3DHeading3>Fluorescence in situ hybridization</DIV> Multicolor interphase FISH analysis of 5-μm paraffin = sections was=20 performed using the following three commercial probe sets delineating = the loci=20 of interest (Vysis, USA): (1) dual-color probe set containing a = centromere=20 2p11-q11 probe (spectrum orange) and a locus-specific 2p24/MYCN = probe=20 (spectrum green); (2) dual-color probe set containing a centromere = 8p11-q11=20 probe (spectrum green) and a locus-specific 8q24/MYC probe = (spectrum=20 orange); (3) multicolor probe set containing locus-specific = 17p13.3/LIS1=20 (spectrum orange), 17q21/RARA (spectrum green), and = 6q23/MYB=20 (spectrum aqua) probes. All FISH experiments were performed in = duplicate. Pretreatment of slides, hybridization, posthybridization = processing,=20 and signal detection were performed as previously reported [<CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR15">15</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR20">20</A></CITE>].=20 Briefly, the samples showing sufficient FISH efficiency (>90% nuclei = with=20 signals) were evaluated by two independent investigators. Signals were = scored in=20 at least 200 nonoverlapping, intact nuclei. Non-neoplastic cerebellar = specimens=20 (n =3D 10) were used as a control for each probe set. = Specimens=20 were considered amplified for MYCN and MYC, when more than = 10% of=20 tumor cells exhibited either more than eight signals of the respective = oncogene=20 probe with the reference/control ratio >2.0 or innumerable tight = clusters of=20 signals of the locus-specific probe. Gains of 6q and/or 17q were defined = as=20 >10% of nuclei containing three or more signals for the respective = probes, if=20 no such findings were detected for the 17p locus (to rule out = polyploidy).=20 Hemizygous deletions of 6q and 17p were defined as >50% of nuclei=20 (mean =B1 three standard deviations in neoplastic controls) = containing=20 one signal either for the 17p or for the 6q probes and ≥2 signals = of the 17q=20 probe. No homozygous deletions were identified for any of the = investigated loci=20 in this study. </DIV></DIV> <DIV class=3D""><A name=3DSec5></A> <HR> <DIV class=3Dheading2>Results</DIV> <DIV class=3D""><A name=3DSec6></A> <DIV class=3DHeading3>Pathological and clinical findings</DIV> On histopathological examination of primary samples, 15 = tumors were=20 diagnosed as classic MB without anaplastic features (Table <A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#Tab2">2</A>).=20 In addition, nine classic MB showed patterns of =93mild=94 anaplasia, = according to=20 Eberhart et al. [<CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR3">3</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR5">5</A></CITE>].=20 In this small set of samples, there was no significant difference = concerning=20 progression-free survival (PFS) and overall survival (OS) between these = two=20 subgroups of classic MB. The remaining four samples were identified as = large=20 cell/anaplastic MB. Latter showed significantly shorter median PFS=20 (7 months; P < 0.001) compared to classic MB = with=20 =93mild=94 anaplasia and those without anaplastic patterns, whereas = there were no=20 differences in median PFS for classic MB with =93mild=94 anaplasia and = for those=20 without anaplastic patterns (21 and 48 months, respectively;=20 P =3D 0.78). <DIV class=3DPara> <DIV class=3D"">Eighteen MB recurrences (14 with classic and 4 with = large cell=20 histology) exactly resembled the histopathological features of their=20 corresponding primaries. In contrast, the remaining ten samples with = classic=20 histology in the primary tumor clearly showed anaplastic features such = as=20 increasing nuclear pleomorphism toward either dark polygonal or pale = round=20 nuclei, cell =93wrapping,=94 elevated number of mitoses and apoptotic = bodies, and=20 appearance of necroses (Fig. <A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#Fig1">1</A>).=20 In two of these samples, microvascular proliferation was observed in the = recurrent tumor. We estimated the cumulative incidence functions from = competing=20 risks with respect to the acquisition of such anaplastic patterns. Time = to=20 progression for tumors with such acquired anaplastic patterns was longer = when=20 compared with samples that showed unchanged histological appearance=20 (Fig. <A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#Fig2">2</A>).=20 <DIV class=3DFigure><A name=3DFig1></A><IMG=20 alt=3DMediaObjects/401_2008_422_Fig1_HTML.jpg=20 src=3D"http://www.springerlink.com/content/tt4661418w621n24/MediaObjects/= 401_2008_422_Fig1_HTML.jpg"></DIV> <DIV class=3DCapt>Fig. 1 Examples = of=20 increasing anaplasia in recurrent MB (H&E, =D7400). a Primary = tumor=20 with Homer=96Wright pseudorosettes and no signs of anaplasia (Case 24). = b=20 Local recurrence of this tumor after 78 months with signs of = moderate=20 anaplasia. c Primary tumor with moderate anaplasia (Case 20). = d=20 Cranial metastasis of this tumor after 61 months with severe = cellular=20 anaplasia. e Primary tumor with signs of focal moderate anaplasia = (Case=20 18). f Local recurrence of this tumor after 57 months with = severe=20 diffuse anaplasia </DIV> <HR> <DIV class=3DFigure><A name=3DFig2></A><IMG=20 alt=3DMediaObjects/401_2008_422_Fig2_HTML.gif=20 src=3D"http://www.springerlink.com/content/tt4661418w621n24/MediaObjects/= 401_2008_422_Fig2_HTML.gif"></DIV> <DIV class=3DCapt>Fig. 2 Cumulative incidence=20 of tumor progression for tumors with acquired anaplastic features in the = recurrent tumor and for those without. Time to progression was = considerably=20 longer for patients whose recurrent tumors showed acquired anaplastic = features=20 </DIV> <HR> </DIV></DIV></DIV> <DIV class=3D""><A name=3DSec7></A> <DIV class=3DHeading3>Cytogenetic aberrations in primary and recurrent=20 tumors</DIV> <DIV class=3DPara> <DIV class=3D"">Totally, 84 multicolor FISH hybridizations were = performed=20 (Table <A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#Tab2">2</A>,=20 Fig. <A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#Fig3">3</A>).=20 Among the primary tumors, 13 samples (46%) exhibited initially balanced=20 cytogenetic profiles for all tested markers, whereas the remaining 15 = tumors=20 (54%) showed one or more aberrations at the loci of interest. Consistent = with=20 previously published results, aberrations of chromosome 17 were the most = common=20 finding affecting 15 (54%) of the primary tumors. Concomitant loss 17p = and gain=20 17q indicative for isochromosome 17 [i(17q)] was detected in 13 cases, = while=20 isolated gain of 17q was found in two tumors. In addition, five samples = showed=20 either MYC or MYCN amplification, and two tumors disclosed = 6q=20 gain. There were no tumors with 6q loss in this series of tumors with=20 recurrence. Comparing cytogenetic findings with histopathological data, = we found=20 that 3/4 tumors with large cell histology showed either MYC or=20 MYCN amplifications and all of them carried i(17q). Median OS for = patients, whose tumors initially showed genomic aberrations, was shorter = than=20 that for patients whose primaries showed balanced profiles (43 and=20 128 months, respectively, P =3D 0.18). Similar = results were=20 obtained for PFS. The estimated median PFS for patients, whose tumors = initially=20 showed genomic aberrations, was 18 months, whereas the median PFS = for=20 patients whose primaries showed balanced profiles, was 58 months=20 (P =3D 0.09).=20 <DIV class=3DFigure><A name=3DFig3></A><IMG=20 alt=3DMediaObjects/401_2008_422_Fig3_HTML.jpg=20 src=3D"http://www.springerlink.com/content/tt4661418w621n24/MediaObjects/= 401_2008_422_Fig3_HTML.jpg"></DIV> <DIV class=3DCapt>Fig. 3 Acquired = cytogenetic=20 aberrations in progressed MB detected by FISH analysis. a Case = 26,=20 primary tumor: balanced profile for chromosomal loci 17p13 (red), = 17q21=20 (green), and 6q23 (blue) with two signals for each probe. = b=20 Local progression of this tumor after 96 months: appearance of = 17q21 and 6q=20 gains (three and more signals). c Case 25, primary tumor: = balanced=20 profile for chromosomal loci 2p24/MYCN (green) and = 2p11-q11=20 (red) with two signals for each probe. d Local progression = of this=20 tumor after 91 months: appearance of MYCN oncogene = amplification=20 (numerous green signals and signal clusters) </DIV> <HR> </DIV></DIV> Analysis of samples from recurrent tumors disclosed an = unchanged=20 cytogenetic profile for the investigated loci in 19 cases (68%). Among = these,=20 six tumors were initially balanced, and 13 samples already showed = chromosomal=20 aberrations in their primaries. In the latter 13 cases, we found no = differences=20 in the extent of cytogenetic abnormalities when primary and recurrent = specimens=20 were compared. Seven recurrent tumors with initially balanced profile = showed=20 appearance of acquired cytogenetic abnormalities involving various loci = of=20 interest. Among them, five tumors showed acquired abnormalities of 17q=20 accompanied by gain of 6q in two cases. Two other tumors with initially = balanced=20 profile disclosed appearance of MYCN oncogene amplification. = Besides=20 this, analysis of two intracranial metastases, which developed from = primaries=20 with concomitant MYCN amplification and i(17q), revealed that = tumor cells=20 bearing oncogene amplification were not detected in secondary lesions, = whereas=20 cells with i(17q) were retained. </DIV></DIV> <DIV class=3D""><A name=3DSec8></A> <HR> <DIV class=3Dheading2>Discussion</DIV> The concept of anaplastic MB has been expanded for = clinical purposes=20 hypothesizing that even a lower degree of anaplasia and/or focal = anaplasia may=20 affect patient outcome. Most studies on the role of gradual anaplasia = revealed=20 that even focal anaplasia had a significant negative effect on = progression-free=20 and overall survival [<CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR3">3</A></CITE>=96<CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR7">7</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR10">10</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR13">13</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR14">14</A></CITE>].=20 However, some other studies showed no association between the degree of=20 anaplasia and MB prognosis [<CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR12">12</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR16">16</A></CITE>].=20 The current concept of the molecular pathogenesis of MB = suggests=20 that these tumors have the ability to undergo stepwise progression as it = has=20 been demonstrated for other tumors, including neuroectodermal neoplasms=20 [<CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR3">3</A></CITE>].=20 The hypothetic model of MB pathogenesis includes primary =93initiation = events=94=20 (i.e. Hedgehog, NOTCH, and WNT activation) and subsequent =93progression = events=94=20 (i.e. chromosome 17 aberrations, MYC/MYCN amplifications, and = others).=20 Nevertheless, this has been a hypothesis only, because conclusive = evidence of=20 temporal molecular MB progression has not been presented yet. Only one = study=20 reported on CGH analyses of a single pair of primary MB and its = recurrence=20 [<CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR6">6</A></CITE>].=20 In that case, an increased number of overall chromosomal aberrations was = found=20 in the recurrent tumor, one of the acquired lesions being a deletion of = 17p.=20 In the current study investigating 28 pairs of primary MB = and their=20 recurrences, we found an increase in anaplastic features by = histopathological=20 assessment in approximately 40% of tumors. Notably, the time to = progression was=20 longer for tumors with increasing degree of anaplasia in the recurrence, = suggesting that a certain interval is required for gradual accumulation = of such=20 changes within the histological appearance. To test the likelihood of = molecular=20 progression in MB, we analyzed five chromosomal loci that have been = reported to=20 be crucial for MB biology and prognosis [<CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR2">2</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR6">6</A></CITE>=96<CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR9">9</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR11">11</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR13">13</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR15">15</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR17">17</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR18">18</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR23">23</A></CITE>=96<CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR25">25</A></CITE>].=20 We found that patients bearing primary tumors with characteristic = chromosomal=20 aberrations such as MYC or MYCN oncogene amplification, = 17q gain,=20 or 6q gain had a shorter median PFS and OS, suggesting that tumor cells = with=20 these chromosomal abnormalities represent cytological sources for = selective=20 growth advantage underlying further clinical progression per se. These = findings=20 are exactly in line with our results obtained from larger series of MB = [<CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR19">19</A></CITE>].=20 MYCN amplification is a hallmark of tumors with = poor clinical=20 outcome [<CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR13">13</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR19">19</A></CITE>].=20 Intriguingly, two MB showed unexpected loss of cells bearing MYCN = amplification within their intracranial metastatic deposits. Metastatic = lesions=20 have been widely thought to arise from a small selected fraction of = cells in the=20 primary tumor, which share molecular alterations associated with loss of = cell=20 adhesion and increased cell motility. Perhaps, MYCN amplification = has no=20 effects on the capability of MB cells to disseminate throughout the CNS. = However, this alteration seems to induce locally aggressive behavior, as = we=20 found acquired MYCN amplification in two local MB recurrences = that were=20 removed 5 and 7.5 years after the primary operation, respectively. = To our=20 knowledge, this is the first report on postponed oncogene amplifications = in MB=20 progression. Interestingly, both these tumors showed local tumor = progression,=20 but no signs of metastatic disease (Table <A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#Tab2">2</A>).=20 Undoubtedly, the analysis of two cases only allows for careful general=20 conclusions. Nevertheless, these findings appear to be intriguing and = further=20 analysis of genetic alterations associated with MB dissemination is = warranted.=20 Five of 13 tumors (38%), whose primaries disclosed = balanced profiles=20 for all investigated loci, showed acquired anomalies of chromosome 17,=20 accompanied by gains of 6q in two tumors. Notably, cases with isolated = gain of=20 17q (without concomitant deletion of 17p) prevailed in this group (four = out of=20 five), whereas i(17q) comprised the vast majority of abnormalities = detected in=20 primary tumors (13 out of 15 tumors). These findings strongly support = the notion=20 that i(17q) may represent a unique primary chromosomal aberration in a = subset of=20 MB [<CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR3">3</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR15">15</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR18">18</A></CITE>].=20 On the contrary, isolated gain of chromosome 17q appears to be a = centrally=20 important aberration for the development of metastatic disease. This is = in line=20 with our recent demonstration of isolated gain of 17q being associated = with=20 unfavorable prognosis, whereas isolated 17p deletion is not [<CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR19">19</A></CITE>].=20 In general, all our findings indicate that sequential = chromosomal=20 aberrations of the type seen in other neuroectodermal tumors can occur = in MB as=20 well. Nevertheless, it remains uncertain whether these cytogenetic = abnormalities=20 are already intrinsically present in a small (and accordingly = =93invisible=94)=20 fraction of primary tumor cells with their sequential clonal expansion = after=20 treatment or accumulated over time in a stepwise cytogenetic fashion. = In conclusion, we found that MB can undergo temporal = molecular=20 progression with accumulation of =93prognostically unfavorable=94 = cytogenetic=20 changes, including even acquisition of oncogene amplifications. Thus, = treatment=20 failures after standard combined treatment of MB patients may, at least = in part,=20 be caused by acquired and steady progressing changes in tumor biology,=20 especially in tumors showing late recurrence. Particularly, when = considering=20 targeted therapy approaches for relapsed patients, secondary biopsy = should be=20 performed to assess the biologic properties of the recurrent tumor, = which may=20 differ substantially from the primary tumor. </DIV> <DIV class=3DAcknowledgments><SPAN=20 class=3DAcknowledgmentsHeading>Acknowledgments <SPAN=20 class=3D"">This study was supported by a grant from the Deutsche=20 Kinderkrebsstiftung to S. Pfister. <DIV class=3DFormalPara> <DIV class=3D"">Open=20 Access 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> <HR> <H2><A name=3DBib1></A>References </H2> <TABLE> <TBODY class=3DCitation> <TR vAlign=3Dtop> <TD>1.</TD> <TD><A name=3DCR1></A>Albright A, Wisoff J, Zeltzer P, Boyett J, = Rorke L,=20 Stanley P (1996) Effects of medulloblastoma resections on outcome = in=20 children: a report from the Children=92s Cancer Group. = Neurosurgery=20 38(2):265=96271. doi:<A=20 = href=3D"http://dx.doi.org/10.1097/00006123-199602000-00007">10.1097/00006= 123-199602000-00007</A>=20 <A=20 = href=3D"http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=3DRetrieve&= db=3DPubMed&dopt=3DAbstract&list_uids=3D8869053"=20 target=3D_blank><IMG height=3D20 alt=3DPubMed=20 = src=3D"http://www.springerlink.com/content/tt4661418w621n24/pubmed_link.g= if"=20 width=3D65 border=3D0></A> <A=20 href=3D"http://dx.doi.org/10.1097/00006123-199602000-00007"=20 target=3D_blank><IMG height=3D20 alt=3DCrossRef=20 = src=3D"http://www.springerlink.com/content/tt4661418w621n24/crossref_link= .gif"=20 width=3D65 border=3D0></A> <A=20 = href=3D"http://chemport.cas.org/cgi-bin/sdcgi?APP=3Dftslink&action=3D= reflink&origin=3Dspringer&version=3D1.0&coi=3D1%3ASTN%3A280%3= AByiD383mvFc%253D&md5=3D11aadfe149c021ae8e99115dda759d04"=20 target=3D_blank><IMG height=3D20 alt=3DChemPort=20 = src=3D"http://www.springerlink.com/content/tt4661418w621n24/chemport_link= .gif"=20 width=3D65 border=3D0></A> </TD></TR> <TR> <TD> </TD></TR> <TR vAlign=3Dtop> <TD>2.</TD> <TD><A name=3DCR2></A>Clifford S, Lusher M, Lindsey J et al (2006)=20 Wnt/wingless pathway activation and chromosome 6 loss characterise = a=20 distinct molecular sub-group of medulloblastomas associated with a = favourable prognosis. 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From: Subject: 10.1007/s00401-008-0422-y Date: Sun, 31 Aug 2008 11:34:17 +0200 MIME-Version: 1.0 Content-Type: multipart/related; type="text/html"; boundary="----=_NextPart_000_0000_01C90B5D.80E64B10" X-MimeOLE: Produced By Microsoft MimeOLE V6.00.2900.5579 This is a multi-part message in MIME format. ------=_NextPart_000_0000_01C90B5D.80E64B10 Content-Type: text/html; charset="iso-8859-1" Content-Transfer-Encoding: quoted-printable Content-Location: http://www.springerlink.com/content/tt4661418w621n24/fulltext.html 10.1007/s00401-008-0422-y</TITLE= > <META http-equiv=3DContent-Type content=3D"text/html; = charset=3DISO-8859-1"><LINK=20 href=3D"http://www.springerlink.com/config/css/A++_common.css" = type=3Dtext/css=20 rel=3DstyleSheet><LINK=20 href=3D"http://www.springerlink.com/config/css/content_A++_SpringerLink.c= ss"=20 type=3Dtext/css rel=3DstyleSheet> <META content=3D"MSHTML 6.00.6000.16705" name=3DGENERATOR></HEAD> <BODY class=3Dfor-print> <TABLE cellPadding=3D2 border=3D1> <TBODY> <TR class=3Dheader> <TD>Acta Neuropathologica</TD></TR> <TR> <TD>=A9 The Author(s) 2008</TD></TR> <TR> <TD>10.1007/s00401-008-0422-y</TD></TR></TBODY></TABLE> <H2 class=3Drubric>Original Paper</H2> <DIV class=3DHeading1><A name=3Dtitle></A>Accumulation of genomic = aberrations during=20 clinical progression of medulloblastoma </DIV> Andrey Korshunov1, 2 <A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#ContactOfAuthor1"><IMG=20 alt=3D"Contact Information"=20 src=3D"http://www.springerlink.com/content/tt4661418w621n24/contact.gif" = border=3D0></A>, Axel Benner3, = Marc Remke4,=20 Peter Lichter4, Andreas von Deimling1, = 2=20 and Stefan Pfister4, 5 <TABLE> <TBODY> <TR vAlign=3Dtop> <TD><A = name=3DAff1></A>(1) </TD> <TD>Clinical Cooperation Unit = Neuropathology,=20 German Cancer Research Center, Im Neuenheimer Feld 280,=20 69120 Heidelberg, Germany</TD></TR></TBODY></TABLE> <TABLE> <TBODY> <TR vAlign=3Dtop> <TD><A = name=3DAff2></A>(2) </TD> <TD>Department of Neuropathology, = University of=20 Heidelberg, Im Neuenheimer Feld 220/221, 69120 Heidelberg,=20 Germany</TD></TR></TBODY></TABLE> <TABLE> <TBODY> <TR vAlign=3Dtop> <TD><A = name=3DAff3></A>(3) </TD> <TD>Division Biostatistics, German Cancer = Research=20 Center, Im Neuenheimer Feld 280, 69120 Heidelberg,=20 Germany</TD></TR></TBODY></TABLE> <TABLE> <TBODY> <TR vAlign=3Dtop> <TD><A = name=3DAff4></A>(4) </TD> <TD>Division Molecular Genetics, German = Cancer=20 Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg,=20 Germany</TD></TR></TBODY></TABLE> <TABLE> <TBODY> <TR vAlign=3Dtop> <TD><A = name=3DAff5></A>(5) </TD> <TD>Department of Pediatric Oncology, = Hematology=20 & Immunology, University of Heidelberg, Im Neuenheimer Feld = 153,=20 69120 Heidelberg, Germany</TD></TR></TBODY></TABLE> <A name=3DContactOfAuthor1></A> <TABLE class=3DContact> <TBODY> <TR> <TD vAlign=3Dtop><IMG alt=3D"Contact Information"=20 = src=3D"http://www.springerlink.com/content/tt4661418w621n24/contact.gif" = border=3D0></TD> = <TD>Andrey Korshunov</STRO= NG> Email:=20 <A=20 = href=3D"mailto:Andrey.Korshunov@med.uni-heidelberg.de">Andrey.Korshunov@m= ed.uni-heidelberg.de</A></TD></TR></TBODY></TABLE> Received:=20 14 July 2008 Revised:=20 6 August 2008 Accepted:=20 6 August 2008 Published online:=20 15 August 2008 <DIV class=3DAbstract><A name=3DAbs1></A><SPAN=20 class=3DAbstractHeading>Abstract Medulloblastomas = comprise the=20 most frequent malignant brain tumor in childhood and one of the biggest=20 challenges in pediatric oncology. The current concept suggests that = these tumors=20 may undergo stepwise progression as it has been shown for other brain = tumors.=20 However, conclusive evidence of molecular progression over time has not = been=20 demonstrated yet for medulloblastoma. In the present study, 28 pairs of=20 medulloblastoma at primary diagnosis and at the time of recurrence, = either=20 occurring as local tumor regrowth or tumor dissemination, were=20 histopathologically and molecularly analyzed. Cytogenetic analysis = included=20 interphase fluorescence in situ hybridization for five genomic loci = (MYC,=20 MYCN, 17p, 17q, 6q) that have previously been identified as = prognostic=20 markers in primary tumors. Of 16 tumors showing early recurrence=20 (<4 years after first diagnosis), only one showed increased = histological=20 anaplasia in the secondary lesion (6%), and two acquired genomic lesions = indicative for a more malignant phenotype (13%). In contrast to this, of = 12=20 tumors with a time to recurrence of 4 years or more, nine tumors = (75%)=20 showed a more malignant phenotype either reflected by increased = anaplasia alone=20 or by both increased anaplasia and acquirement of genomic aberrations = known to=20 be associated with inferior patient outcome. These results suggest that = early=20 recurrence in medulloblastoma mainly occurs in tumors with a highly = malignant=20 genotype and phenotype per se, whereas late recurrence is often = dependent on=20 tumor evolution toward a more malignant biology. Therefore, biopsy of = recurrent=20 tumors should be performed to assess the biologic properties of the = relapsed=20 tumor, especially when targeted therapy approaches are considered. = </DIV> <SPAN=20 class=3DKeywordHeading>Keywords Medulloblastoma -&= nbsp;Progression - Metastasis - Genomic=20 aberration - Anaplasia - Chromosome 17q <DIV class=3D""><A name=3DSec1></A> <HR> <DIV class=3Dheading2>Introduction</DIV> Brain tumors are the leading cause of cancer-related = mortality in=20 pediatric patients, with medulloblastoma (MB) being the most common=20 representative in this age group. Considerable advances have been made = in the=20 treatment of MB in the last three decades by using multimodal = therapeutic=20 regimens involving surgery, radiotherapy, and chemotherapy. About = 10=9615% of=20 patients die from disease within 2 years after diagnosis, whereas=20 approximately 60% of patients can nowadays be cured [<CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR9">9</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR11">11</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR22">22</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR23">23</A></CITE>].=20 However, the predictive performance of current risk stratification, = which is=20 entirely based on clinical variables, needs to be improved because of = the dismal=20 prognosis of children with tumor relapse, and because of relevant=20 treatment-induced long-term effects of survivors [<CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR21">21</A></CITE>].=20 Clinical staging systems based on age, metastatic stage at diagnosis = and, in=20 some studies, extent of surgical resection have so far been the most = useful=20 methods for the stratification of patients into standard and high risk = therapy=20 groups [<CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR1">1</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR26">26</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR27">27</A></CITE>].=20 Additionally, after refinement of the histological classification of MB = in the=20 2007 WHO classification, the histological subtype is of potential = prognostic=20 relevance: large cell/anaplastic MB behaves more aggressively, while=20 nodular/desmoplastic variants show a more favorable clinical course as = compared=20 to the large group of classic MB [<CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR3">3</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR5">5</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR7">7</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR10">10</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR13">13</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR14">14</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR17">17</A></CITE>].=20 Some recent studies have extended the concept of anaplasia in MB and = suggested=20 that this phenomenon reflects malignant progression of a tumor and may = occur in=20 various MB subtypes independent of large cell histology [<CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR3">3</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR4">4</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR12">12</A></CITE>].=20 Progression over time from nonanaplastic to anaplastic MB has been = described in=20 cases with local tumor recurrence [<CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR3">3</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR12">12</A></CITE>]=20 as well as in secondary metastatic lesions with or without local = recurrence=20 [<CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR4">4</A></CITE>].=20 To date, numerous molecular studies have highlighted = various=20 chromosomal aberrations in MB, and some of them were found to be = associated with=20 clinical outcome. Cytogenetic aberrations in MB involving chromosome 17 = as well=20 as amplifications of the MYC or MYCN oncogenes have = previously=20 been associated with large cell histology and poor patient survival in = different=20 studies [<CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR6">6</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR7">7</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR9">9</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR11">11</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR13">13</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR15">15</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR17">17</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR18">18</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR23">23</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR24">24</A></CITE>].=20 In contrast, several recent reports have identified monosomy of = chromosome 6 to=20 be associated with consecutive WNT pathway activation and favorable = clinical=20 outcome [<CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR2">2</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR8">8</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR25">25</A></CITE>].=20 Nevertheless, in contrast to astrocytomas and = oligodendrogliomas,=20 molecular progression of MB has only been suspected, but has not been=20 unanimously proven. A stepwise progression of genetic abnormalities in = these=20 tumors was identified in a few cases only, when comparing primary and = recurrent=20 tumor specimens [<CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR3">3</A></CITE>].=20 Here, we performed comparative histopathologic and = cytogenetic=20 analyses of 28 MB cases, for which samples from primary manifestation = and tumor=20 recurrence were available. The aim of this study was to determine which=20 histopathological features and chromosome abnormalities may be = associated with=20 MB molecular progression over time. Interphase fluorescence in situ=20 hybridization (FISH) was carried out for five regions of interest, = namely=20 MYC, MYCN, chromosome 6q, and chromosomal arms 17p and = 17q.=20 </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 population</DIV> <DIV class=3DPara> <DIV class=3D"">Specimens from 28 MB patients who experienced = disease=20 recurrence were analyzed in this study. All these patients had received=20 reoperations at secondary diagnosis and specimens from primary and = subsequent=20 interventions were available for comparative histopathological and = molecular=20 analyses. Patient details are outlined in Table <A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#Tab1">1</A>.=20 There were 21 males and 7 females, and age ranged from 3 to = 37 years=20 (median 14 years). Sixteen patients experienced early recurrence=20 (<4 years after first diagnosis), and 12 patients had a time to=20 recurrence of 48 months or more (late recurrence). Staging = procedures=20 included MRI before and immediately after neurosurgery. Metastatic = disease was=20 detected by craniospinal imaging and analysis of the cerebrospinal fluid = following the Chang classification. Most of the patients (26) had no=20 leptomeningeal tumor dissemination (i.e. M0 stage) at the time of = primary=20 diagnosis. All patients were treated according to a standard treatment = protocol,=20 consisting of surgical tumor resection, followed by craniospinal = irradiation=20 with 36 Gy and a boost to the posterior fossa adding up to a total = dose of=20 53=9656 Gy, and eight cycles of vincristin, lomustine, and = cisplatin. Gross=20 total tumor resection was achieved in 23 patients (82%) as assessed by = early=20 postoperative MRI. Tumors progressed as the following (Table <A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#Tab2">2</A>):=20 (1) isolated local recurrence (13 cases); (2) local recurrence in = combination=20 with leptomeningeal dissemination (six cases); among the latter, in one = patient,=20 tumor progressed as local recurrence combined with leptomeningeal = dissemination=20 and seedings outside the CNS (bone marrow and soft tissues); (3) = isolated=20 leptomeningeal dissemination in nine cases. Specimens from secondary = tumors were=20 obtained either after removal of local progression = (n =3D 18) or=20 after resection/biopsy of tumor metastases = (n =3D 10).<A=20 name=3DTab1></A> <DIV class=3DCapt>Table 1 Clinical = characteristics of medulloblastoma patients with early progression=20 (n =3D 16) and late progression = (n =3D 12) </DIV> <TABLE border=3D1> <COLGROUP> <COL align=3Dleft> <COL align=3Dleft> <COL align=3Dleft> <COL align=3Dleft></COLGROUP> <THEAD> <TR class=3Dheader> <TH align=3Dleft> Variable</TH> <TH align=3Dleft> Early recurrence (<48 months)</TH> <TH align=3Dleft> Late recurrence (≥48 months)</TH> <TH align=3Dleft> Total</TH></TR></THEAD> <TBODY> <TR class=3Dnoclass> <TD align=3Dleft colSpan=3D4> Agea </TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> Median age (years)</TD> <TD align=3Dleft> 11.5</TD> <TD align=3Dleft> 15</TD> <TD align=3Dleft> 14</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> Range (years)</TD> <TD align=3Dleft> 3=9622</TD> <TD align=3Dleft> 7=9637</TD> <TD align=3Dleft> 3=9637</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft colSpan=3D4> Gender</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> Female</TD> <TD align=3Dleft> 5 (31%)</TD> <TD align=3Dleft> 2 (17%)</TD> <TD align=3Dleft> 7 (25%)</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> Male</TD> <TD align=3Dleft> 11 (69%)</TD> <TD align=3Dleft> 10 (83%)</TD> <TD align=3Dleft> 21 (75%)</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft colSpan=3D4> Chang stage</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> M0 </TD> <TD align=3Dleft> 14 (87%)</TD> <TD align=3Dleft> 12 (100%)</TD> <TD align=3Dleft> 26 (93%)</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> M1=963 </TD> <TD align=3Dleft> 2 (13%)</TD> <TD align=3Dleft> 0 (0%)</TD> <TD align=3Dleft> 2 (7%)</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft colSpan=3D4> Level of resection at primary = diagnosis</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> Gross total resection</TD> <TD align=3Dleft> 12 (75%)</TD> <TD align=3Dleft> 11 (92%)</TD> <TD align=3Dleft> </TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> Macroscopic tumor residue</TD> <TD align=3Dleft> 4 (25%)</TD> <TD align=3Dleft> 1 (8%)</TD> <TD align=3Dleft> </TD></TR> <TR class=3Dnoclass> <TD align=3Dleft colSpan=3D4> Type of progression</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> Local progression</TD> <TD align=3Dleft> 5 (31%)</TD> <TD align=3Dleft> 8 (67%)</TD> <TD align=3Dleft> 13 (46%)</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> Dissemination</TD> <TD align=3Dleft> 6 (38%)</TD> <TD align=3Dleft> 3 (25%)</TD> <TD align=3Dleft> 9 (32%)</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> Local progression and dissemination</TD> <TD align=3Dleft> 5 (31%)</TD> <TD align=3Dleft> 1 (8%)</TD> <TD align=3Dleft> 6 (21%) </TD></TR> <TR class=3Dnoclass> <TD align=3Dleft colSpan=3D4> Time to progression</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> Median (months)</TD> <TD align=3Dleft> 16</TD> <TD align=3Dleft> 70</TD> <TD align=3Dleft> 34</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> Range (months)</TD> <TD align=3Dleft> 4=9639</TD> <TD align=3Dleft> 48=96138</TD> <TD align=3Dleft> 4=96138</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> Total</TD> <TD align=3Dleft> 16</TD> <TD align=3Dleft> 12</TD> <TD align=3Dleft> 28</TD></TR></TBODY></TABLE> <DIV class=3DCapt> <DIV class=3DCaptCont> <DIV class=3D"">aAt initial diagnosis </DIV></DIV></DIV><A=20 name=3DTab2></A> <DIV class=3DCapt>Table 2 Histopathological=20 and molecular characteristics of medulloblastoma patients at primary = diagnosis=20 and at the time of disease progression </DIV> <TABLE border=3D1> <COLGROUP> <COL align=3Dleft> <COL align=3Dchar char=3D"."> <COL align=3Dleft> <COL align=3Dleft> <COL align=3Dleft> <COL align=3Dleft> <COL align=3Dleft></COLGROUP> <THEAD> <TR class=3Dheader> <TH align=3Dleft rowSpan=3D2> Number</TH> <TH align=3Dleft rowSpan=3D2 char=3D"."> Time to progression (months)</TH> <TH align=3Dleft rowSpan=3D2> Type of progression</TH> <TH align=3Dleft colSpan=3D2> Histology</TH> <TH align=3Dleft colSpan=3D2> Molecular markers</TH></TR> <TR class=3Dheader> <TH align=3Dleft> Primary diagnosis</TH> <TH align=3Dleft> Progression</TH> <TH align=3Dleft> Primary diagnosis</TH> <TH align=3Dleft> Progression</TH></TR></THEAD> <TBODY> <TR class=3Dnoclass> <TD align=3Dleft> 1</TD> <TD align=3Dchar char=3D"."> 4</TD> <TD align=3Dleft> mts</TD> <TD align=3Dleft> LCA</TD> <TD align=3Dleft> LCA</TD> <TD align=3Dleft> i17q</TD> <TD align=3Dleft> i17q</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> 2</TD> <TD align=3Dchar char=3D"."> 6</TD> <TD align=3Dleft> Local</TD> <TD align=3Dleft> LCA</TD> <TD align=3Dleft> LCA</TD> <TD align=3Dleft> MYC; i17q; gain 6q</TD> <TD align=3Dleft> MYC; i17q; gain 6q</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> 3</TD> <TD align=3Dchar char=3D"."> 8</TD> <TD align=3Dleft> Local/mts</TD> <TD align=3Dleft> LCA</TD> <TD align=3Dleft> LCA</TD> <TD align=3Dleft> MYCN; gain 17q</TD> <TD align=3Dleft> MYCN; gain 17q</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> 4</TD> <TD align=3Dchar char=3D"."> 8</TD> <TD align=3Dleft> mts</TD> <TD align=3Dleft> Classic NA</TD> <TD align=3Dleft> Classic NA</TD> <TD align=3Dleft> i17q</TD> <TD align=3Dleft> i17q</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> 5</TD> <TD align=3Dchar char=3D"."> 9</TD> <TD align=3Dleft> Local/mts</TD> <TD align=3Dleft> Classic NA</TD> <TD align=3Dleft> Classic NA</TD> <TD align=3Dleft> i17q</TD> <TD align=3Dleft> i17q</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> 6</TD> <TD align=3Dchar char=3D"."> 11</TD> <TD align=3Dleft> Local</TD> <TD align=3Dleft> Classic MA</TD> <TD align=3Dleft> Classic MA</TD> <TD align=3Dleft> Balanced </TD> <TD align=3Dleft> gain 17q </TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> 7</TD> <TD align=3Dchar char=3D"."> 12</TD> <TD align=3Dleft> Local/mts</TD> <TD align=3Dleft> Classic MA</TD> <TD align=3Dleft> Classic MA</TD> <TD align=3Dleft> i17q</TD> <TD align=3Dleft> i17q</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> 8a </TD> <TD align=3Dchar char=3D"."> 15</TD> <TD align=3Dleft> Local/mts</TD> <TD align=3Dleft> LCA</TD> <TD align=3Dleft> LCA</TD> <TD align=3Dleft> MYC; i17q; gain 6q</TD> <TD align=3Dleft> MYC; i17q; gain 6q</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> 9</TD> <TD align=3Dchar char=3D"."> 17</TD> <TD align=3Dleft> Local</TD> <TD align=3Dleft> Classic MA</TD> <TD align=3Dleft> Classic MA</TD> <TD align=3Dleft> Balanced</TD> <TD align=3Dleft> Balanced</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> 10</TD> <TD align=3Dchar char=3D"."> 18</TD> <TD align=3Dleft> Local/mts</TD> <TD align=3Dleft> Classic MA</TD> <TD align=3Dleft> Classic MA</TD> <TD align=3Dleft> Balanced </TD> <TD align=3Dleft> gain 17q </TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> 11</TD> <TD align=3Dchar char=3D"."> 18</TD> <TD align=3Dleft> Local</TD> <TD align=3Dleft> Classic NA</TD> <TD align=3Dleft> Classic NA</TD> <TD align=3Dleft> gain 17q</TD> <TD align=3Dleft> gain 17q</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> 12</TD> <TD align=3Dchar char=3D"."> 21</TD> <TD align=3Dleft> mts</TD> <TD align=3Dleft> Classic MA</TD> <TD align=3Dleft> Classic MA</TD> <TD align=3Dleft> MYCN; i17q </TD> <TD align=3Dleft> i17q </TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> 13</TD> <TD align=3Dchar char=3D"."> 27</TD> <TD align=3Dleft> mts</TD> <TD align=3Dleft> Classic NA</TD> <TD align=3Dleft> Classic NA</TD> <TD align=3Dleft> MYCN; i17q </TD> <TD align=3Dleft> i17q </TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> 14</TD> <TD align=3Dchar char=3D"."> 30</TD> <TD align=3Dleft> mts</TD> <TD align=3Dleft> Classic NA </TD> <TD align=3Dleft> Classic MA </TD> <TD align=3Dleft> i17q</TD> <TD align=3Dleft> i17q</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> 15</TD> <TD align=3Dchar char=3D"."> 38</TD> <TD align=3Dleft> Local</TD> <TD align=3Dleft> Classic NA</TD> <TD align=3Dleft> Classic NA</TD> <TD align=3Dleft> Balanced</TD> <TD align=3Dleft> Balanced</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> 16</TD> <TD align=3Dchar char=3D"."> 39</TD> <TD align=3Dleft> mts</TD> <TD align=3Dleft> Classic NA</TD> <TD align=3Dleft> Classic NA</TD> <TD align=3Dleft> i17q</TD> <TD align=3Dleft> i17q</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> 17</TD> <TD align=3Dchar char=3D"."> 48</TD> <TD align=3Dleft> Local</TD> <TD align=3Dleft> Classic NA</TD> <TD align=3Dleft> Classic NA</TD> <TD align=3Dleft> Balanced</TD> <TD align=3Dleft> Balanced</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> 18</TD> <TD align=3Dchar char=3D"."> 57</TD> <TD align=3Dleft> Local</TD> <TD align=3Dleft> Classic MA </TD> <TD align=3Dleft> LCA </TD> <TD align=3Dleft> Balanced </TD> <TD align=3Dleft> MYCN </TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> 19</TD> <TD align=3Dchar char=3D"."> 58</TD> <TD align=3Dleft> mts</TD> <TD align=3Dleft> Classic NA </TD> <TD align=3Dleft> Classic MA </TD> <TD align=3Dleft> Balanced</TD> <TD align=3Dleft> balanced</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> 20</TD> <TD align=3Dchar char=3D"."> 61</TD> <TD align=3Dleft> mts</TD> <TD align=3Dleft> Classic MA </TD> <TD align=3Dleft> LCA </TD> <TD align=3Dleft> i17q</TD> <TD align=3Dleft> i17q</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> 21</TD> <TD align=3Dchar char=3D"."> 62</TD> <TD align=3Dleft> Local</TD> <TD align=3Dleft> Classic NA </TD> <TD align=3Dleft> Classic MA </TD> <TD align=3Dleft> i17q</TD> <TD align=3Dleft> i17q</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> 22</TD> <TD align=3Dchar char=3D"."> 67</TD> <TD align=3Dleft> Local</TD> <TD align=3Dleft> Classic NA</TD> <TD align=3Dleft> Classic NA</TD> <TD align=3Dleft> Balanced</TD> <TD align=3Dleft> Balanced</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> 23</TD> <TD align=3Dchar char=3D"."> 73</TD> <TD align=3Dleft> mts</TD> <TD align=3Dleft> Classic MA </TD> <TD align=3Dleft> LCA </TD> <TD align=3Dleft> Balanced </TD> <TD align=3Dleft> i17q </TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> 24</TD> <TD align=3Dchar char=3D"."> 78</TD> <TD align=3Dleft> Local</TD> <TD align=3Dleft> Classic NA </TD> <TD align=3Dleft> Classic MA </TD> <TD align=3Dleft> Balanced </TD> <TD align=3Dleft> gain 17q; gain 6q </TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> 25</TD> <TD align=3Dchar char=3D"."> 91</TD> <TD align=3Dleft> Local</TD> <TD align=3Dleft> Classic NA </TD> <TD align=3Dleft> LCA </TD> <TD align=3Dleft> Balanced </TD> <TD align=3Dleft> MYCN </TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> 26</TD> <TD align=3Dchar char=3D"."> 96</TD> <TD align=3Dleft> Local/mts</TD> <TD align=3Dleft> Classic NA </TD> <TD align=3Dleft> LCA </TD> <TD align=3Dleft> Balanced </TD> <TD align=3Dleft> gain 17q; gain 6q </TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> 27</TD> <TD align=3Dchar char=3D"."> 132</TD> <TD align=3Dleft> Local</TD> <TD align=3Dleft> Classic NA </TD> <TD align=3Dleft> Classic MA </TD> <TD align=3Dleft> Balanced</TD> <TD align=3Dleft> Balanced</TD></TR> <TR class=3Dnoclass> <TD align=3Dleft> 28</TD> <TD align=3Dchar char=3D"."> 138</TD> <TD align=3Dleft> Local</TD> <TD align=3Dleft> Classic MA</TD> <TD align=3Dleft> Classic MA</TD> <TD align=3Dleft> i17q</TD> <TD align=3Dleft> i17q</TD></TR></TBODY></TABLE> <DIV class=3DCapt> <DIV class=3DCaptCont> <DIV class=3D"">Cases with accumulation of histological and molecular = changes are=20 indicated in bold</DIV></DIV> <DIV class=3DCaptCont> <DIV class=3D"">mts metastatic disease, LCA large = cell/anaplastic=20 histology, NA no signs of anaplasia, MA moderate anaplasia = </DIV></DIV> <DIV class=3DCaptCont> <DIV class=3D"">aCase 8 also showed metastasis outside the = CNS (soft=20 tissue and bone marrow) </DIV></DIV></DIV></DIV></DIV></DIV> <DIV class=3D""><A name=3DSec4></A> <DIV class=3DHeading3>Fluorescence in situ hybridization</DIV> Multicolor interphase FISH analysis of 5-μm paraffin = sections was=20 performed using the following three commercial probe sets delineating = the loci=20 of interest (Vysis, USA): (1) dual-color probe set containing a = centromere=20 2p11-q11 probe (spectrum orange) and a locus-specific 2p24/MYCN = probe=20 (spectrum green); (2) dual-color probe set containing a centromere = 8p11-q11=20 probe (spectrum green) and a locus-specific 8q24/MYC probe = (spectrum=20 orange); (3) multicolor probe set containing locus-specific = 17p13.3/LIS1=20 (spectrum orange), 17q21/RARA (spectrum green), and = 6q23/MYB=20 (spectrum aqua) probes. All FISH experiments were performed in = duplicate. Pretreatment of slides, hybridization, posthybridization = processing,=20 and signal detection were performed as previously reported [<CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR15">15</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR20">20</A></CITE>].=20 Briefly, the samples showing sufficient FISH efficiency (>90% nuclei = with=20 signals) were evaluated by two independent investigators. Signals were = scored in=20 at least 200 nonoverlapping, intact nuclei. Non-neoplastic cerebellar = specimens=20 (n =3D 10) were used as a control for each probe set. = Specimens=20 were considered amplified for MYCN and MYC, when more than = 10% of=20 tumor cells exhibited either more than eight signals of the respective = oncogene=20 probe with the reference/control ratio >2.0 or innumerable tight = clusters of=20 signals of the locus-specific probe. Gains of 6q and/or 17q were defined = as=20 >10% of nuclei containing three or more signals for the respective = probes, if=20 no such findings were detected for the 17p locus (to rule out = polyploidy).=20 Hemizygous deletions of 6q and 17p were defined as >50% of nuclei=20 (mean =B1 three standard deviations in neoplastic controls) = containing=20 one signal either for the 17p or for the 6q probes and ≥2 signals = of the 17q=20 probe. No homozygous deletions were identified for any of the = investigated loci=20 in this study. </DIV></DIV> <DIV class=3D""><A name=3DSec5></A> <HR> <DIV class=3Dheading2>Results</DIV> <DIV class=3D""><A name=3DSec6></A> <DIV class=3DHeading3>Pathological and clinical findings</DIV> On histopathological examination of primary samples, 15 = tumors were=20 diagnosed as classic MB without anaplastic features (Table <A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#Tab2">2</A>).=20 In addition, nine classic MB showed patterns of =93mild=94 anaplasia, = according to=20 Eberhart et al. [<CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR3">3</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR5">5</A></CITE>].=20 In this small set of samples, there was no significant difference = concerning=20 progression-free survival (PFS) and overall survival (OS) between these = two=20 subgroups of classic MB. The remaining four samples were identified as = large=20 cell/anaplastic MB. Latter showed significantly shorter median PFS=20 (7 months; P < 0.001) compared to classic MB = with=20 =93mild=94 anaplasia and those without anaplastic patterns, whereas = there were no=20 differences in median PFS for classic MB with =93mild=94 anaplasia and = for those=20 without anaplastic patterns (21 and 48 months, respectively;=20 P =3D 0.78). <DIV class=3DPara> <DIV class=3D"">Eighteen MB recurrences (14 with classic and 4 with = large cell=20 histology) exactly resembled the histopathological features of their=20 corresponding primaries. In contrast, the remaining ten samples with = classic=20 histology in the primary tumor clearly showed anaplastic features such = as=20 increasing nuclear pleomorphism toward either dark polygonal or pale = round=20 nuclei, cell =93wrapping,=94 elevated number of mitoses and apoptotic = bodies, and=20 appearance of necroses (Fig. <A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#Fig1">1</A>).=20 In two of these samples, microvascular proliferation was observed in the = recurrent tumor. We estimated the cumulative incidence functions from = competing=20 risks with respect to the acquisition of such anaplastic patterns. Time = to=20 progression for tumors with such acquired anaplastic patterns was longer = when=20 compared with samples that showed unchanged histological appearance=20 (Fig. <A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#Fig2">2</A>).=20 <DIV class=3DFigure><A name=3DFig1></A><IMG=20 alt=3DMediaObjects/401_2008_422_Fig1_HTML.jpg=20 src=3D"http://www.springerlink.com/content/tt4661418w621n24/MediaObjects/= 401_2008_422_Fig1_HTML.jpg"></DIV> <DIV class=3DCapt>Fig. 1 Examples = of=20 increasing anaplasia in recurrent MB (H&E, =D7400). a Primary = tumor=20 with Homer=96Wright pseudorosettes and no signs of anaplasia (Case 24). = b=20 Local recurrence of this tumor after 78 months with signs of = moderate=20 anaplasia. c Primary tumor with moderate anaplasia (Case 20). = d=20 Cranial metastasis of this tumor after 61 months with severe = cellular=20 anaplasia. e Primary tumor with signs of focal moderate anaplasia = (Case=20 18). f Local recurrence of this tumor after 57 months with = severe=20 diffuse anaplasia </DIV> <HR> <DIV class=3DFigure><A name=3DFig2></A><IMG=20 alt=3DMediaObjects/401_2008_422_Fig2_HTML.gif=20 src=3D"http://www.springerlink.com/content/tt4661418w621n24/MediaObjects/= 401_2008_422_Fig2_HTML.gif"></DIV> <DIV class=3DCapt>Fig. 2 Cumulative incidence=20 of tumor progression for tumors with acquired anaplastic features in the = recurrent tumor and for those without. Time to progression was = considerably=20 longer for patients whose recurrent tumors showed acquired anaplastic = features=20 </DIV> <HR> </DIV></DIV></DIV> <DIV class=3D""><A name=3DSec7></A> <DIV class=3DHeading3>Cytogenetic aberrations in primary and recurrent=20 tumors</DIV> <DIV class=3DPara> <DIV class=3D"">Totally, 84 multicolor FISH hybridizations were = performed=20 (Table <A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#Tab2">2</A>,=20 Fig. <A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#Fig3">3</A>).=20 Among the primary tumors, 13 samples (46%) exhibited initially balanced=20 cytogenetic profiles for all tested markers, whereas the remaining 15 = tumors=20 (54%) showed one or more aberrations at the loci of interest. Consistent = with=20 previously published results, aberrations of chromosome 17 were the most = common=20 finding affecting 15 (54%) of the primary tumors. Concomitant loss 17p = and gain=20 17q indicative for isochromosome 17 [i(17q)] was detected in 13 cases, = while=20 isolated gain of 17q was found in two tumors. In addition, five samples = showed=20 either MYC or MYCN amplification, and two tumors disclosed = 6q=20 gain. There were no tumors with 6q loss in this series of tumors with=20 recurrence. Comparing cytogenetic findings with histopathological data, = we found=20 that 3/4 tumors with large cell histology showed either MYC or=20 MYCN amplifications and all of them carried i(17q). Median OS for = patients, whose tumors initially showed genomic aberrations, was shorter = than=20 that for patients whose primaries showed balanced profiles (43 and=20 128 months, respectively, P =3D 0.18). Similar = results were=20 obtained for PFS. The estimated median PFS for patients, whose tumors = initially=20 showed genomic aberrations, was 18 months, whereas the median PFS = for=20 patients whose primaries showed balanced profiles, was 58 months=20 (P =3D 0.09).=20 <DIV class=3DFigure><A name=3DFig3></A><IMG=20 alt=3DMediaObjects/401_2008_422_Fig3_HTML.jpg=20 src=3D"http://www.springerlink.com/content/tt4661418w621n24/MediaObjects/= 401_2008_422_Fig3_HTML.jpg"></DIV> <DIV class=3DCapt>Fig. 3 Acquired = cytogenetic=20 aberrations in progressed MB detected by FISH analysis. a Case = 26,=20 primary tumor: balanced profile for chromosomal loci 17p13 (red), = 17q21=20 (green), and 6q23 (blue) with two signals for each probe. = b=20 Local progression of this tumor after 96 months: appearance of = 17q21 and 6q=20 gains (three and more signals). c Case 25, primary tumor: = balanced=20 profile for chromosomal loci 2p24/MYCN (green) and = 2p11-q11=20 (red) with two signals for each probe. d Local progression = of this=20 tumor after 91 months: appearance of MYCN oncogene = amplification=20 (numerous green signals and signal clusters) </DIV> <HR> </DIV></DIV> Analysis of samples from recurrent tumors disclosed an = unchanged=20 cytogenetic profile for the investigated loci in 19 cases (68%). Among = these,=20 six tumors were initially balanced, and 13 samples already showed = chromosomal=20 aberrations in their primaries. In the latter 13 cases, we found no = differences=20 in the extent of cytogenetic abnormalities when primary and recurrent = specimens=20 were compared. Seven recurrent tumors with initially balanced profile = showed=20 appearance of acquired cytogenetic abnormalities involving various loci = of=20 interest. Among them, five tumors showed acquired abnormalities of 17q=20 accompanied by gain of 6q in two cases. Two other tumors with initially = balanced=20 profile disclosed appearance of MYCN oncogene amplification. = Besides=20 this, analysis of two intracranial metastases, which developed from = primaries=20 with concomitant MYCN amplification and i(17q), revealed that = tumor cells=20 bearing oncogene amplification were not detected in secondary lesions, = whereas=20 cells with i(17q) were retained. </DIV></DIV> <DIV class=3D""><A name=3DSec8></A> <HR> <DIV class=3Dheading2>Discussion</DIV> The concept of anaplastic MB has been expanded for = clinical purposes=20 hypothesizing that even a lower degree of anaplasia and/or focal = anaplasia may=20 affect patient outcome. Most studies on the role of gradual anaplasia = revealed=20 that even focal anaplasia had a significant negative effect on = progression-free=20 and overall survival [<CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR3">3</A></CITE>=96<CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR7">7</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR10">10</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR13">13</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR14">14</A></CITE>].=20 However, some other studies showed no association between the degree of=20 anaplasia and MB prognosis [<CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR12">12</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR16">16</A></CITE>].=20 The current concept of the molecular pathogenesis of MB = suggests=20 that these tumors have the ability to undergo stepwise progression as it = has=20 been demonstrated for other tumors, including neuroectodermal neoplasms=20 [<CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR3">3</A></CITE>].=20 The hypothetic model of MB pathogenesis includes primary =93initiation = events=94=20 (i.e. Hedgehog, NOTCH, and WNT activation) and subsequent =93progression = events=94=20 (i.e. chromosome 17 aberrations, MYC/MYCN amplifications, and = others).=20 Nevertheless, this has been a hypothesis only, because conclusive = evidence of=20 temporal molecular MB progression has not been presented yet. Only one = study=20 reported on CGH analyses of a single pair of primary MB and its = recurrence=20 [<CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR6">6</A></CITE>].=20 In that case, an increased number of overall chromosomal aberrations was = found=20 in the recurrent tumor, one of the acquired lesions being a deletion of = 17p.=20 In the current study investigating 28 pairs of primary MB = and their=20 recurrences, we found an increase in anaplastic features by = histopathological=20 assessment in approximately 40% of tumors. Notably, the time to = progression was=20 longer for tumors with increasing degree of anaplasia in the recurrence, = suggesting that a certain interval is required for gradual accumulation = of such=20 changes within the histological appearance. To test the likelihood of = molecular=20 progression in MB, we analyzed five chromosomal loci that have been = reported to=20 be crucial for MB biology and prognosis [<CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR2">2</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR6">6</A></CITE>=96<CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR9">9</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR11">11</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR13">13</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR15">15</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR17">17</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR18">18</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR23">23</A></CITE>=96<CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR25">25</A></CITE>].=20 We found that patients bearing primary tumors with characteristic = chromosomal=20 aberrations such as MYC or MYCN oncogene amplification, = 17q gain,=20 or 6q gain had a shorter median PFS and OS, suggesting that tumor cells = with=20 these chromosomal abnormalities represent cytological sources for = selective=20 growth advantage underlying further clinical progression per se. These = findings=20 are exactly in line with our results obtained from larger series of MB = [<CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR19">19</A></CITE>].=20 MYCN amplification is a hallmark of tumors with = poor clinical=20 outcome [<CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR13">13</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR19">19</A></CITE>].=20 Intriguingly, two MB showed unexpected loss of cells bearing MYCN = amplification within their intracranial metastatic deposits. Metastatic = lesions=20 have been widely thought to arise from a small selected fraction of = cells in the=20 primary tumor, which share molecular alterations associated with loss of = cell=20 adhesion and increased cell motility. Perhaps, MYCN amplification = has no=20 effects on the capability of MB cells to disseminate throughout the CNS. = However, this alteration seems to induce locally aggressive behavior, as = we=20 found acquired MYCN amplification in two local MB recurrences = that were=20 removed 5 and 7.5 years after the primary operation, respectively. = To our=20 knowledge, this is the first report on postponed oncogene amplifications = in MB=20 progression. Interestingly, both these tumors showed local tumor = progression,=20 but no signs of metastatic disease (Table <A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#Tab2">2</A>).=20 Undoubtedly, the analysis of two cases only allows for careful general=20 conclusions. Nevertheless, these findings appear to be intriguing and = further=20 analysis of genetic alterations associated with MB dissemination is = warranted.=20 Five of 13 tumors (38%), whose primaries disclosed = balanced profiles=20 for all investigated loci, showed acquired anomalies of chromosome 17,=20 accompanied by gains of 6q in two tumors. Notably, cases with isolated = gain of=20 17q (without concomitant deletion of 17p) prevailed in this group (four = out of=20 five), whereas i(17q) comprised the vast majority of abnormalities = detected in=20 primary tumors (13 out of 15 tumors). These findings strongly support = the notion=20 that i(17q) may represent a unique primary chromosomal aberration in a = subset of=20 MB [<CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR3">3</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR15">15</A></CITE>,=20 <CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR18">18</A></CITE>].=20 On the contrary, isolated gain of chromosome 17q appears to be a = centrally=20 important aberration for the development of metastatic disease. This is = in line=20 with our recent demonstration of isolated gain of 17q being associated = with=20 unfavorable prognosis, whereas isolated 17p deletion is not [<CITE><A=20 href=3D"http://www.springerlink.com/content/tt4661418w621n24/fulltext.htm= l#CR19">19</A></CITE>].=20 In general, all our findings indicate that sequential = chromosomal=20 aberrations of the type seen in other neuroectodermal tumors can occur = in MB as=20 well. Nevertheless, it remains uncertain whether these cytogenetic = abnormalities=20 are already intrinsically present in a small (and accordingly = =93invisible=94)=20 fraction of primary tumor cells with their sequential clonal expansion = after=20 treatment or accumulated over time in a stepwise cytogenetic fashion. = In conclusion, we found that MB can undergo temporal = molecular=20 progression with accumulation of =93prognostically unfavorable=94 = cytogenetic=20 changes, including even acquisition of oncogene amplifications. Thus, = treatment=20 failures after standard combined treatment of MB patients may, at least = in part,=20 be caused by acquired and steady progressing changes in tumor biology,=20 especially in tumors showing late recurrence. Particularly, when = considering=20 targeted therapy approaches for relapsed patients, secondary biopsy = should be=20 performed to assess the biologic properties of the recurrent tumor, = which may=20 differ substantially from the primary tumor. </DIV> <DIV class=3DAcknowledgments><SPAN=20 class=3DAcknowledgmentsHeading>Acknowledgments <SPAN=20 class=3D"">This study was supported by a grant from the Deutsche=20 Kinderkrebsstiftung to S. Pfister. <DIV class=3DFormalPara> <DIV class=3D"">Open=20 Access 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> <HR> <H2><A name=3DBib1></A>References </H2> <TABLE> <TBODY class=3DCitation> <TR vAlign=3Dtop> <TD>1.</TD> <TD><A name=3DCR1></A>Albright A, Wisoff J, Zeltzer P, Boyett J, = Rorke L,=20 Stanley P (1996) Effects of medulloblastoma resections on outcome = in=20 children: a report from the Children=92s Cancer Group. = Neurosurgery=20 38(2):265=96271. doi:<A=20 = href=3D"http://dx.doi.org/10.1097/00006123-199602000-00007">10.1097/00006= 123-199602000-00007</A>=20 <A=20 = href=3D"http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=3DRetrieve&= db=3DPubMed&dopt=3DAbstract&list_uids=3D8869053"=20 target=3D_blank><IMG height=3D20 alt=3DPubMed=20 = src=3D"http://www.springerlink.com/content/tt4661418w621n24/pubmed_link.g= if"=20 width=3D65 border=3D0></A> <A=20 href=3D"http://dx.doi.org/10.1097/00006123-199602000-00007"=20 target=3D_blank><IMG height=3D20 alt=3DCrossRef=20 = src=3D"http://www.springerlink.com/content/tt4661418w621n24/crossref_link= .gif"=20 width=3D65 border=3D0></A> <A=20 = href=3D"http://chemport.cas.org/cgi-bin/sdcgi?APP=3Dftslink&action=3D= reflink&origin=3Dspringer&version=3D1.0&coi=3D1%3ASTN%3A280%3= AByiD383mvFc%253D&md5=3D11aadfe149c021ae8e99115dda759d04"=20 target=3D_blank><IMG height=3D20 alt=3DChemPort=20 = src=3D"http://www.springerlink.com/content/tt4661418w621n24/chemport_link= .gif"=20 width=3D65 border=3D0></A> </TD></TR> <TR> <TD> </TD></TR> <TR vAlign=3Dtop> <TD>2.</TD> <TD><A name=3DCR2></A>Clifford S, Lusher M, Lindsey J et al (2006)=20 Wnt/wingless pathway activation and chromosome 6 loss characterise = a=20 distinct molecular sub-group of medulloblastomas associated with a = favourable prognosis. 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