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Clinical Cancer Research 13, 2651, May 1, 2007. doi: 10.1158/1078-0432.CCR-06-1779
© 2007 American Association for Cancer Research
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Cancer Therapy: Clinical

Prognostic Relevance of Clinical and Biological Risk Factors in Childhood Medulloblastoma: Results of Patients Treated in the Prospective Multicenter Trial HIT'91

Stefan Rutkowski1, André von Bueren4, Katja von Hoff1, Wolfgang Hartmann5, Tarek Shalaby4, Frank Deinlein1, Monika Warmuth-Metz2, Niels Soerensen3, Angela Emser7, Udo Bode6, Uwe Mittler8, Christian Urban9, Martin Benesch9, Rolf D. Kortmann10, Paul G. Schlegel1, Joachim Kuehl1, Torsten Pietsch5 and Michael Grotzer4

Authors' Affiliations: 1 University Children's Hospital of Wuerzburg; Departments of 2 Neuroradiology and 3 Pediatric Neurosurgery, University of Wuerzburg, Wuerzburg, Germany; 4 Department of Pediatric Oncology, University of Zurich, Zurich, Switzerland; 5 Department of Neuropathology, University of Bonn; 6 University Children's Hospital of Bonn, Bonn, Germany; 7 Institute of Medical Biostatistics, Epidemiology and Informatics, University of Mainz, Mainz, Germany; 8 University Children's Hospital of Magdeburg, Magdeburg, Germany; 9 University Children's Hospital of Graz, Graz, Austria; and 10 Department of Radiation Oncology, University of Leipzig, Leipzig, Germany

Requests for reprints: Stefan Rutkowski, Children's University Hospital, Josef-Schneider-Strasse 2, D-97080 Wuerzburg, Germany. Phone: 49-931-201-27728; Fax: 49-931-201-27722; E-mail: Rutkowski_S{at}klinik.uni-wuerzburg.de.


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: To identify better risk stratification systems in childhood medulloblastoma based on clinical factors and analysis of routinely processed formalin-fixed tumor material.

Experimental Design: Formalin-fixed paraffin-embedded tumor samples from well-documented patients treated within the prospective randomized multicenter trial HIT'91 were analyzed for DNA amplification of c-myc and N-myc (n = 133) and mRNA expression of c-myc and trkC (n = 104; compared with human cerebellum) using validated methods of quantitative PCR and reverse transcription-PCR. Results were related to clinical data and outcome.

Results: TrkC and c-myc mRNA expression were identified as independent prognostic factors by multivariate analysis. Three risk groups were identified. (a) Favorable risk group: all 8 patients (2 metastatic) with high trkC (>1x human cerebellum) and low c-myc mRNA expression (≤1x human cerebellum) remained relapse-free [7-year event-free survival (EFS), 100%]. (b) Poor risk group: 10 of 15 patients with metastatic disease and high c-myc and low trkC mRNA expression relapsed (7-year EFS, 33%). (c) Intermediate risk group: the 7-year EFS of the remaining 78 patients was 65%. Among 47 M0 stage patients, all 10 patients with high trkC mRNA expression remained relapse-free compared with 15 events in 37 patients with low trkC mRNA expression levels (7-year EFS, 100% versus 62%; P = 0.056).

Conclusions: Whereas the collection of fresh-frozen tumor samples remains a major challenge in large clinical trials, routinely processed paraffin-embedded tissue samples can be used to quantitate the prognostic biological markers trkC and c-myc. On prospective validation of cutoff levels, this may lead to improved stratification of treatment for children with medulloblastoma.

Medulloblastoma, the most frequent malignant brain tumor of childhood, is an invasive embryonal tumor of the cerebellum with an inherent tendency to metastasize via the subarachnoidal space (1). Up to 60% to 80% of children can be successfully treated using the current therapeutic strategies, including neurosurgical resection, radiotherapy, and chemotherapy, but significant tumor- and treatment-related side effects have to be considered (2–4). In the last decades, stratification of patients to risk groups has been based on age, staging, and presence or absence of significant postoperative residual tumor (5, 6). Between 20% to 40% of patients meeting the current clinical standard risk criteria (nonmetastatic disease, no significant postoperative residual tumor) will experience tumor relapse but cannot be identified at diagnosis as patients with an elevated risk of tumor relapse. Additionally, a potential subset of low-risk patients, who may be cured with less intensive than standard treatment strategies, cannot be identified by clinical criteria alone. Molecular markers, such as N-myc, c-myc, trkC, and ERBB2, and gene expression profiles have been investigated: amplification and mRNA expression of the proto-oncogene c-myc have been associated with an unfavorable prognosis (7–10). In contrast, the neurotrophin-3 receptor trkC (11–13) has been shown to correlate with a favorable prognosis in children with medulloblastoma. The large cell anaplastic medulloblastoma variant has been related to an adverse outcome (14), and desmoplasia was identified as a favorable risk factor in young children (15). Consequently, new stratification criteria combining clinical, histologic, and molecular risk factors have been proposed, aiming to improve risk-adapted treatment recommendations in future (16–20). Most of the recent studies analyzing candidate biological prognostic factors in childhood medulloblastoma are limited by relatively small patient numbers. Therefore, the confidence about which biological variables are most predictive is limited. Accordingly, researchers have proposed large prospective biological studies for validation both in the U.S. and in Europe. However, central collection of fresh-frozen tumor material in large multicenter clinical studies is extremely challenging. For many institutions to supply fresh-frozen tissue samples for molecular analyses, the logistics will be complex and the cost will be high. Routine diagnostics of brain tumors in clinical practice are based on analysis of histology on formalin-fixed and paraffin-embedded (FFPE) tissues. Recent technological improvements, including the introduction of highly sensitive real-time reverse transcription-PCR procedures, now allow for rapid and specific quantification of even small amounts of mRNA, making this assay amenable to studies where the RNA is moderately or even highly degraded. We have optimized RNA isolation from FFPE archive medulloblastoma samples and found that trkC and c-myc mRNA measurements significantly correlated with those obtained from matching fresh-frozen tissues frozen tissues (n = 20; trkC, r = 0.786; P = 0.0002; c-myc, r = 0.948; P < 0.0001, Pearson correlation; ref. 21).

Here, we present an analysis of clinical, histopathologic, and molecular risk factors in a large series of 133 medulloblastoma children >3 years at diagnosis, which were well-documented study patients treated according to the prospective randomized multicenter trial HIT'91 (22).


   Materials and Methods
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 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Patients and therapy. Tumor material was available from 133 children between 3 and 18 years of age (median, 7.6 years) with medulloblastoma, registered between August 1991 and December 1997 by 29 centers in Germany, Austria, and Switzerland to the prospective randomized multicenter trial HIT'91. After surgery, patients were randomized to receive either craniospinal radiotherapy followed by maintenance chemotherapy (‘maintenance arm’) or neoadjuvant chemotherapy and craniospinal radiotherapy (‘sandwich arm’) as described (22). Briefly, neoadjuvant ‘sandwich’ chemotherapy consisted of procarbazine followed by two cycles of ifosfamide/etoposide, high-dose methotrexate, and cisplatin/cytarabine, and poor responders received additional eight cycles of carboplatin/1-(2-chloroethyl)-3-cyclohexyl-L-nitrosourea/vincristine after radiotherapy. Maintenance chemotherapy consisted of eight courses of cisplatin, 1-(2-chloroethyl)-3-cyclohexyl-L-nitrosourea, and vincristine. Radiotherapy consisted of 35.2 Gy to the neuraxis, a boost of 20 Gy to the posterior fossa, and an additional boost to macroscopic metastases if present. Potential biological prognostic factors were analyzed blind to clinical information. The study HIT'91 was approved by the ethics committee of the University of Wuerzburg. Informed consent was obtained from legal representatives of all patients before registration.

Histopathology and M staging. The histologic diagnosis of medulloblastoma was confirmed by central review in 131 of 133 patients according to the WHO classification of brain tumors (1), and histologic subtypes (classic, desmoplastic, and large-cell/anaplastic) were reevaluated in the year 2005 by an experienced neuropathologist (T.P.). In two patients, medulloblastoma was diagnosed by the local pathologist only. Postoperative residual tumor, defined as a measurable lesion on early postoperative imaging (magnetic resonance imaging or computed tomography), was confirmed in 38 of 133 children. M staging was done according to the Chang criteria (M1, microscopic dissemination into the cerebrospinal fluid; M2, macroscopic intracranial metastases; M3, macroscopic spinal metastases; and M4, extraneural metastases; ref. 23).

DNA extraction and semiquantitative PCR for c-myc and N-myc DNA amplification. FFPE tumor samples were available from 133 patients. DNA was extracted with the QIAamp DNA mini kit (Qiagen). To quantify c-myc and N-myc gene copy numbers, exogenous DNA standard competitors were generated with internal deletions by in vitro mutagenesis as described before (24). Primer sequences, specific PCR conditions, and the fragment sizes were as follows: c-myc, 5'-TCTGGATCACCTTCTGCTGG-3' and 5'-AGGATAGTCCTTCCGAG TGG-3', target 126 bp, standard 108 bp, and annealing temperature 61°C; N-myc, 5'-TAAACGTTGGTGACGGAAGG-3' and 5'-TACAGAAATGTTCCCCAGGG-3', target 167 bp, standard 150 bp, and annealing temperature 54°C; and APRT (control gene), 5'-CAGGGAACACATTCCTTTGC-3' and 5'-TGGGAAAGCTGTTTACTGC-3', target 135 bp, standard 121 bp, and annealing temperature 54°C. To ensure coverage of the equimolar range of the competitors and the corresponding target DNAs, we did titration experiments with serial dilutions of the competitors in a pool of tumor and normal tissue DNAs. Optimal titration was defined as the point of equal signal intensity of exogenous competitor and target. Subsequent PCR amplification of all products was carried out with a fluorescently labeled reverse primer. Products were analyzed in 4.5% denaturating acrylamide gels on a DNA Sequencer (ABI 377) using Genescan software (ABI). Gene copy numbers were calculated as follows: [(TARGETsample/TARGETstandard) / (APRTsample/APRTstandard)]. Gene amplification was defined as higher than mean plus 2-fold SD of the whole set of samples. APRT was chosen as it is located in the chromosomal region 16q24.3, which is not a region of frequent losses or gains in medulloblastomas.

RNA isolation and quantitative reverse transcription-PCR for c-myc and trkC mRNA expression. Isolation of total RNA from FFPE tumor tissue and real-time quantitative reverse transcription-PCR for analyses of c-myc and trkC mRNA have been done from 104 samples with sufficient tumor material available as described previously (21). The Optimum FFPE for paraffin block RNA isolation kit (Ambion Diagnostics) was used to isolate RNA from FFPE tumor tissue. Briefly, paraffin from 1 x 20 to 2 x 20 µm slices of FFPE medulloblastoma samples was removed by washing each sample with xylene for 30 min. Samples were collected by centrifugation, washed with ethanol, and allowed to air dry at room temperature following final centrifugation. Samples were then resuspended in 10 µL (60 units/µL) Proteinase K and 100 µL digestion buffer and incubated for 3 h at 37°C followed by 12 h at room temperature. After complete digestion, RNA was obtained according to the manufacturer's protocols.

After treatment with 1 µL RNase-free DNase I (2 units/µL), RNAs were quantitated by spectrophotometer, and A260/280 nm ratios were calculated for quality assurance. Afterwards, the samples were stored at –80°C. cDNA synthesis was done as described (21).

Kinetic real-time PCR quantification of c-myc and trkC mRNA was done using the ABI Prism 7700 Sequence Detection System (Applied Biosystems) as described (21). The amount of c-myc and trkC, normalized to the endogenous control 18S rRNA, was related to the commercially available calibrator human cerebellum (Becton Dickinson).

Statistical analysis. Associations of clinical variables and molecular variables (categorical variables) were analyzed by {chi}2 tests. Functions for overall survival (OS) and event-free survival (EFS) were estimated using the method of Kaplan and Meier, and the log-rank test was used for comparison. EFs was defined as time from the date of diagnosis to the date of first progression, to death of any cause, or to the date of the last contact (the earliest event was counted). OS was defined as time from the date of diagnosis to death or last contact. The median follow-up of surviving patients was 7.5 years (range, 3.8-12 years). Eighteen patients without cerebrospinal fluid analyses were not included in analyses comparing patients with localized and metastatic disease. Groups of continuous nonparametric data were compared using the Mann-Whitney U test. All univariable analyses were done on a local significance level of 5% (not adjusted for multiple comparison). Multivariable Cox regression was applied to analyze the prognostic value of following potential explanatory prognostic factors with respect to EFS: c-myc DNA amplification, N-myc DNA amplification, c-myc mRNA expression (continuous; >1 versus ≤1), and trkC mRNA expression (continuous; >1 versus ≤1), therapy (maintenance-arm and sandwich-arm), extent of initial resection (complete versus incomplete), staging (metastatic and localized), sex, histologic subtype (desmoplastic, anaplastic, and classic), and age at diagnosis (continuous; <7.6 versus ≥7.6 years). A Cox regression model was built using a stepwise variable selection procedure (P value of likelihood ratio test ≤0.05 as inclusion criterion and P ≥ 0.10 as exclusion criterion) described by Collett (25). The multivariable Cox regression analysis is regarded as explorative. Statistical analyses were done in the year 2006 using SPSS version 12.0 (SPSS, Inc.).


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Staging and histology. Among the 133 patients with material for analyses of DNA amplifications and mRNA expression levels, 64 patients had localized disease at diagnosis, 29 patients had disseminated tumor cells in cerebrospinal fluid (M1 stage according to Chang ref. 23), and 22 patients had macroscopic metastases at diagnosis (M2/M3 stage). In 18 patients without macroscopic metastases, cerebrospinal fluid for analysis of microscopic tumor cell dissemination was not available (M0/M1 stage). Of 53 events, 49 were due to tumor progression or relapse, 1 patient died due to toxicity, and 3 patients succumbed to a secondary malignancy. Patient characteristics and univariable analyses of clinical variables are summarized in Table 1 .


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  		Table 1. Patient characteristics and clinical variables

 
DNA amplification of c-myc and N-myc. c-myc DNA amplification was detected in 5 of 116 evaluable samples (17 not evaluable), and 3 of them had a tumor relapse (1 M0, 1 M1, and 1 M2/3, no significant association to the presence of metastases; P = 0.680, {chi}2 test). Amplification of N-myc DNA was observed in 6 of 100 samples (33 not evaluable), and 1 patient relapsed (Table 2 ). No patients with N-myc amplification presented with macroscopic metastases at diagnosis (1 M0, 2 M0/1, and 3 M1; P = 0.110, {chi}2 test). The amplification of c-myc or N-myc was not a significant prognostic factor for EFS (P = 0.285 and 0.195).


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  		Table 2. Histologic variables and biological markers

 
Expression of c-myc and trkC mRNA expression. Adequate material to isolate RNA was available from 104 tumors. In 101 of these, the quality of isolated RNA, based on spectrophotometry (A260/280 nm ≥1.0) and CT values for 18S RNA was sufficient to do quantitative reverse transcription-PCR for trkC and c-myc. Expression values of the housekeeping gene 18S RNA did not differ depending on storage time (data not shown). Median levels of c-myc and trkC mRNA expression were 1.2 (range, 0-40,599; 75% quartile, 12.4) and 0.06 (range, 0-148; 75% quartile, 0.8), respectively.

The amount of trkC and c-myc mRNA was related to human cerebellum. Consequently, cutoff values of 1 were chosen to analyze patient groups with high or low expression of trkC and c-myc mRNA according to survival. No difference in Chang stage (M0 versus M1-3) was found in 48 patients with a c-myc expression ≤1 ("low") compared with 53 patients >1 ("high"; P = 0.195, {chi}2 test). Differences in EFS between patients with high and low c-myc expression did not reach statistical significance (7-year EFS, 71% versus 56%; P = 0.19).

Only 5 of 23 patients (10 M0, 2 M0/1, 7 M1, and 4 M2/3) with trkC levels >1 relapsed compared with 35 events in 78 patients with trkC ≤1 (7-year EFS, 83% versus 58%; P = 0.044; Fig. 1A ). Frequencies of metastases (M0 versus M1-3) were not different (P = 0.745, {chi}2 test). All 9 patients with a very high trkC expression >9 remained relapse-free (EFS, 100%; 5 M0, 1 M0/1, 1 M1, and 2 M2/3), whereas 40 of 92 patients with a trkC expression <9 had an event (7-year EFS, 60%; P = 0.023; Fig. 1B).


Figure 1
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  		Fig. 1. EFS according to relative trkC mRNA expression levels compared with normal cerebellum. A, EFS was different between children with relative trkC mRNA expression levels >1 and ≤1 (EFS, 0.83 versus 0.58; P = 0.044). B, all patients with a trkC expression >9 remained relapse-free compared with children with trkC levels ≤9 (7-y EFS, 1.0 versus 0.60; P = 0.023).

 
Ten of 47 patients with M0 stage had trkC levels >1.0, and none of these patients had tumor relapse or progression (1 died from a secondary malignancy) compared with 15 patients with tumor relapse or progression of 37 M0 stage patients with lower trkC levels (7-year EFS, 100% versus 62%; P = 0.055; Fig. 2A ). Fourteen of 24 metastatic patients with c-myc mRNA expression >1 had tumor relapse compared with 6 of 17 patients with lower c-myc expression levels (7-year EFS, 42% versus 71%; P = 0.106; Fig. 2B).


Figure 2
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  		Fig. 2. A, M0 stage patients with trkC levels >1.0 had a trend for a better EFS compared with patients with lower trkC levels (7-y EFS, 1.0 versus 0.62 ± 0.08; P = 0.055). B, patients with metastatic medulloblastoma (M+) and c-myc expression >1 had a trend for an inferior EFS compared with children with lower c-myc levels (7-y EFS, 0.42 versus 0.71; P = 0.106). C, among patients with complete tumor resection (R0), high trkC mRNA expression was related to a better EFS compared with patients with low trkC expression (EFS, 1.0% versus 0.67%; P = 0.026). D, among patients with incomplete tumor resection (R+), patients with low c-myc expression had a better EFS than patients with high c-myc mRNA expression (EFS, 0.77% versus 0.33%; P = 0.039).

 
In patients without postoperative residual tumor, only 1 of 15 patients with high trkC mRNA expression had tumor relapse or progression compared with 20 of 55 patients with low trkC expression (7-year EFS, 100% versus 67%; P = 0.026; Fig. 2C). Among patients with incomplete tumor resection, patients with low c-myc expression fared better than patients with high c-myc mRNA expression (EFS, 77% versus 33%; P = 0.039; Fig. 2D). Subgroup analyses of patients according to treatment arm, completeness of resection, and metastases are summarized in Table 3 .


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  		Table 3. Subgroup analyses about treatment arm and staging

 
Histopathologic subtypes of medulloblastoma. 120 (90%) patients had classic medulloblastoma, 5 (4%) patients were diagnosed as large-cell anaplastic medulloblastoma, and 8 (6%) patients had desmoplastic medulloblastoma, respectively. None of the 5 patients with large-cell anaplastic medulloblastoma (3 M0 and 2 M0/1; 2 patients relapsed) had c-myc DNA amplification, 1 of them had c-myc mRNA expression >1.1 (30.5), and all patients had trkC levels <1. None of 8 patients with desmoplastic medulloblastoma (5 M0, 1 M1, and 2 M2/3; 2 patients relapsed) had c-myc amplification, 4 patients had a c-myc m-RNA expression >1, and 3 patients had trkC levels >9.

Combination of molecular and clinical variables. Three different risk groups resulted from the combined evaluation of clinical staging results and mRNA levels of c-myc and trkC: the best prognostic subgroup consisted of eight patients with high trkC (>1) and low c-myc (<1) mRNA expression. All eight patients remained relapse-free (7-year EFS, 100%) regardless of M stage (1 M1 and 1 M2/3). The most unfavorable subgroup consisted of 15 patients who had metastatic disease and low trkC/high c-myc mRNA expression levels: 10 patients had tumor relapse or progression (7-year EFS, 33%). All other patients (M0 or M0/1 without high trkC/low c-myc, 52 patients; M1/2/3 without high c-myc/low trkC, 22 patients) were assigned to a third group of intermediate risk (30 of 78 patients relapsed; 7-year EFS, 65%; Fig. 3 ). Inclusion of c-myc or N-myc amplification, or histologic subtypes did not further improve the power of this stratification model.


Figure 3
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  		Fig. 3. Survival according to staging and expression of c-myc and trkC. Low risk: 8 patients (2 metastatic) with high trkC (>1) and low c-myc (<1) levels (7-y EFS, 1.0). High risk: 15 patients with metastases and c-myc amplification or low trkC/high c-myc expression (7-y EFS, 0.33). The remaining 78 patients were at intermediate risk (EFS, 0.65).

 
Multivariable analyses. A Cox regression analysis identified trkC mRNA expression as continuous variable as a positive prognostic factor (hazard ratio, 0.86; 95% confidence interval, 0.73-1.01; P = 0.008) and c-myc mRNA expression >1 as a negative prognostic factor for EFS (hazard ratio, 2.33; 95% confidence interval, 1.15-4.71; P = 0.017). In addition, sandwich therapy compared with maintenance therapy (hazard ratio, 3.48; 95% confidence interval, 1.66-7.27; P = 0.001) and metastatic disease (M1/2/3 versus M0: hazard ratio, 1.92; 95% confidence interval, 0.98-3.74; P = 0.056) were independent negative prognostic factors for EFS.


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
The present analysis of clinical, histopathologic, and molecular risk factors of childhood medulloblastoma is based on 133 well-documented pediatric medulloblastoma patients with a long clinical follow-up and represents one of the largest studies on this issue thus far. In terms of medulloblastoma patients and tumor material, it is completely independent of previous studies about trkC and c-myc. Our results perfectly validate the results of previous studies about c-myc and trkC. In addition, we newly show that the current medulloblastoma risk stratification system (based on clinical factors only) can be improved by addition of the biological markers c-myc and trkC. The collection of fresh-frozen tumor samples remains a major challenge in large clinical trials. It is therefore of practical relevance that the analyses of the present study were all made from routinely processed FFPE medulloblastoma material.

Previous studies on c-myc in medulloblastoma, obtained from snap-frozen tumor samples (8, 11, 18, 26), were based on smaller series due to the limited availability of unfixed tumor tissue, costs, and logistics. In contrast, our study shows that meaningful mRNA expression levels of c-myc and trkC can be obtained from FFPE samples in a large patient series. To avoid arbitrary threshold selection, cutoff values of 1 were chosen for analyses of trkC and c-myc mRNA expression, indicating higher or lower mRNA expression levels compared with normal cerebellum.

TrkC expressed as a continuous variable was identified as an independent favorable risk factor by multivariable analysis. In addition, expression of trkC mRNA was the only nonclinical prognostic factor found by univariable analysis in our study. Interestingly, none of the nine patients with a very high trkC expression (>9) had a tumor relapse, regardless of metastatic disease (three patients). By further addition of patients with low c-myc expression to our prediction model, a considerable subgroup (8%) of patients could be identified as a very favorable risk group (7-year EFS, 100%). On the other hand, we identified a high-risk group of 15 patients with metastatic disease, low trkC expression, high c-myc levels, or c-myc amplification (7-year EFS, 33%), a rate that is lower than recent results obtained by intensified ‘high-risk’ regimen (27).

N-myc and c-myc, members of the myc family of proto-oncogenes, are involved in fundamental cellular processes, including proliferation, growth, apoptosis, and differentiation (28, 29). We detected c-myc or N-myc gene amplifications in 5 of 116 (4.3%) and 6 of 100 (6%) tumors, respectively. Similar frequencies have been reported by others (9, 19). Others found c-myc amplification predominantly in metastatic medulloblastoma and no N-myc amplification in metastatic disease (7, 19). In our series, c-myc amplification was not highly associated with metastatic disease. Three of five children with c-myc amplification relapsed. The fact that c-myc amplification was not identified as an independent prognostic factor in contrast to others (18, 19, 26) may be explained in part by patient numbers. We observed a high variability of c-myc mRNA levels (0-40,599), and a c-myc mRNA expression higher than in normal cerebellum (>1) was identified as an independent adverse risk factor in our series. It has been shown previously that c-myc DNA amplification does not correlate with c-myc mRNA expression (8, 30, 31). Mechanisms to activate c-myc other than gene amplification are well recognized in various solid tumors. They include retroviral insertional mutagenesis, chromosomal translocation, somatic mutations, or activation by transcription factors (32).

None of our patients with anaplastic medulloblastoma had an amplification of c-myc, and mRNA levels of c-myc were not higher in this entity compared with patients with classic medulloblastoma. Therefore, the current pathophysiologic concept of a strong association of the proto-oncogene c-myc, anaplasia, and metastatic disease (18) can be supported only in part by our data.

The frequency of anaplastic medulloblastoma varies between different studies and has been described in up to 20% of children with medulloblastoma (18). Here, we observed 4% of large-cell anaplastic medulloblastomas, only counting those cases with severe anaplasia and/or a significant fraction of large tumor cells with typical prominent nucleoli. A double age peak has been postulated for desmoplastic medulloblastoma, with one peak in early childhood and one in adolescence (15). This is in line with the 6% of children with desmoplastic medulloblastoma, which we found in this study, referring to patients from both parts of this age pattern.

In most current trials, standard-risk medulloblastoma is defined by localized disease (M0) and favorable tumor resection. Our subgroup analyses give evidence that high trkC expression may be especially meaningful in patients with a favorable constellation of clinical risk factors (nonmetastatic disease, complete tumor resection; Fig. 2A and C). In contrast, high c-myc expression may be of special relevance in patients with a clinical risk profile (metastatic disease, incomplete tumor resection; Fig. 2B and D). In our series, the treatment arm was identified as an independent risk factor, with children treated by the sandwich strategy having an inferior outcome. Our retrospective subgroup analyses for trkC and c-myc showed a trend for a better outcome in nonmetastatic children treated in the maintenance arm, and all 10 nonmetastatic patients with trkC levels >1 (21% of nonmetastatic patients) remained relapse-free. On prospective validation, this finding may have implications on stratification in future trials. Clearly, a prospective validation of cutoff values has to be interpreted in the context of clinical risk factors and the applied treatment regimen.

In future, favorable risk patients may be stratified to receive less intensive radiotherapy or chemotherapy. Furthermore, high-risk patients may be identified at diagnosis as candidates for more intensified primary treatment regimens.

In conclusion, we have shown that definitions of favorable and unfavorable risk groups in childhood medulloblastoma, also within the subgroup of patients with nonmetastatic disease, can be improved by the determination of trkC and c-myc expression. Notably, the findings showed in this study were obtained by analyzing routinely processed FFPE medulloblastoma samples. Existing archives of medulloblastoma samples from homogeneously treated patients can be therefore further exploited to validate results from recent gene expression studies. On prospective validation of cutoff levels, assessment of trkC and c-myc mRNA expression may be incorporated in clinical trials to improve the risk-dependent stratification in patients with medulloblastoma.


   Acknowledgments
 
We thank Wiebke Treulieb and Julia Becker for excellent data management and the contributing pathologists for the submission of tumor samples: H. Arnholdt, Klinikum Augsburg; M. Bergmann, Klinikum Bremen-Ost; I. Bluemcke, University of Erlangen; W. Brueck, University of Goettingen; H. Kretzschmar, University of Munich; H.H. Goebel, University of Mainz; A. von Deimling, University of Berlin-Virchow; M. Deckert, University of Cologne; W. Feiden, University of Homburg/Saar; H. Frenzel, Klinikum Karlsruhe; V. Hans, Evang. Krankenhaus Bielefeld; Ch. Hagel, University of Hamburg; F. Hofstaedter, University of Regensburg; M. Kiessling, University of Heidelberg; K. Kuchelmeister, University of Gießen; R.H. Laeng, Klinikum Aarau, H-D. Mennel, University of Marburg; R. Meyermann, University of Tuebingen; W, Paulus, University of Muenster; G. Reifenberger, University of Duesseldorf; E. Reusche, Med. Hoshshule Luebeck; W. Roggendorf, University of Wuerzburg; C. Sommer, University of Ulm; A. Stan, Med. Hochschule Hannover; M. Tolnay, Klinikum Basel; B. Volk, University of Freiburg; J. Weis, University of Aachen; H.H. Hugo, University of Kiel; and J.R. Iglesias-Rozas, Katharinenhospital Stuttgart.


   Footnotes
 
Grant support: German Childhood Cancer Foundation-Deutsche Kinderkrebsstiftung and German Cancer Aid-Deutsche Krebshilfe (S. Rutkowski, K. von Hoff, F. Deinlein, P.G. Schlegel, and J. Kuehl), the Swiss National Fonds, the Swiss Research Foundation Child and Cancer (A. von Bueren, T. Shalaby, and M. Grotzer), the German Research Foundation-Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 400, German Ministry for Education and Research-Bundesministerium für Bildung und Forschung, Kompetenznetz Pädiatrische Onkologie (W. Hartmann and T. Pietsch).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: A. von Bueren and K. von Hoff contributed equally to this work. J. Kuehl is deceased.

Received 7/20/06; revised 1/23/07; accepted 2/12/07.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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