Medulloblastoma arises in the cerebellum and is the most common malignant brain tumour of childhood, however its molecular basis is not well understood. To assess the role of aberrant epigenetic events in medulloblastoma and identify critical genes in its development, we profiled the promoter methylation status of 11 candidate tumour-suppressor genes (TSGs; p14ARF, p15INK4b, p16INK4a, CASP8, HIC1, EDNRB, TIMP3, TP73, TSLC1, RIZ1 and RASSF1A) in medulloblastoma cell lines, primary tumours and the normal cerebellum. Gene-specific TSG methylation was a significant feature of both medulloblastomas and the cerebellum. Extensive hypermethylation of RASSF1A was detected frequently in medulloblastomas but not in the normal cerebellum (41/44 primary tumours versus 0/5 normal cerebella). In contrast, complete methylation of HIC1 and CASP8 in a subset of primary tumours (17/44 and 14/39) occurred against a consistent background of partial methylation in the normal cerebellum. These data therefore indicate that extensive methylation of RASSF1A, HIC1 and CASP8 are tumour-specific events in medulloblastoma. Moreover, methylation of these genes in medulloblastoma cell lines was associated with their epigenetic transcriptional silencing and methylation-dependent re-expression following treatment with the DNA methyltransferase inhibitor, 5-aza-2′-deoxycytidine. The remaining genes studied showed either low frequency methylation (p14ARF, p16INK4a, RIZ1; <7% of cases), no evidence of methylation (p15INK4b, TIMP3, TP73, TSLC1), or comparable patterns of methylation in the normal cerebellum (EDNRB), suggesting that their hypermethylation does not play a major role in medulloblastoma. Our data demonstrate that tumour-specific hypermethylation affects only a subset of genes, and does not support the existence of a concordant methylation phenotype in this disease. We conclude that epigenetic TSG inactivation is a significant feature of medulloblastoma, and identify RASSF1A, HIC1 and CASP8 as potentially critical genes in its pathogenesis. Furthermore, methylation observed in the normal cerebellum emphasises the requirement for appropriate control tissues when assessing the tumour-specificity of TSG hypermethylation.

Abbreviations: 5-aza CdR, 5-aza-2′-deoxycytidine; COBRA, combined bisulphite and restriction analysis; MSP, methylation-specific PCR; TSG, tumour-suppressor gene

Introduction

Medulloblastoma, the most common malignant brain tumour of childhood, is a primitive neuro-ectodermal tumour arising in the cerebellum. The aggressive clinical behaviour of the tumour and the cognitive and endocrinological long-term side effects of current therapies make both the development of prognostic indicators for disease stratification and the identification of new therapeutic targets a major goal. Current understanding of the molecular biology of medulloblastoma is limited. Cytogenetic studies have described consistent chromosomal aberrations, however molecular genetic studies have identified specific genetic abnormalities in only a small proportion of tumours (reviewed in refs 1,2).

Hypermethylation of promoter-associated CpG islands leading to transcriptional silencing has emerged as an important mechanism of epigenetic inactivation of tumour suppressor genes (TSGs) in cancer development (reviewed in ref. 3). Using a genome-scanning approach, aberrant patterns of CpG island methylation have been described in medulloblastoma (4,5), although few gene-specific events have been identified to date. Recently, we have demonstrated bi-allelic epigenetic inactivation by promoter methylation of the tumour suppressor gene RASSF1A (ras-association domain family protein 1, isoform A) in medulloblastoma (6), further highlighting the potential significance of epigenetic TSG inactivation in the development of this tumour.

To examine more widely the prevalence and role of aberrant promoter methylation events in medulloblastoma and to identify further tumour-specific events in its pathogenesis, we have determined the methylation status of the promoter-associated CpG islands of a series of known or candidate TSGs in primary medulloblastoma tumours and the normal cerebellum. In addition to RASSF1A, three groups of candidate genes were selected for analysis: (i) genes which have been reported previously to show evidence of methylation in medulloblastoma [CASP8 (Caspase 8, cysteine-aspartic acid protease 8), HIC1 (hypermethylated in cancer 1) and p16INK4a] (ii) genes which are epigenetically inactivated in other brain tumours [p14ARF p15INK4b, TIMP3 (tissue inhibitor of metalloprotease 3) and TP73] and (iii) genes which exhibit methylation in several different cancer types, suggesting frequent involvement in tumour development [EDNRB (endothelin B receptor), TSLC1 (tumour suppressor gene in human lung cancer) and RIZ1 (retinoblastoma protein interacting zinc finger gene)] This parallel examination of multiple promoter-associated CpG islands has allowed the compilation of an extensive profile of gene-specific methylation events in medulloblastoma. Importantly, the analysis of normal cerebellar tissue has permitted the identification of tumour-specific hypermethylation of a subset of genes, which we show is associated with their transcriptional silencing. This highlights the potential importance of these epigenetic events in medulloblastoma pathogenesis. Finally, we make a preliminary assessment of the clinico-pathological significance of tumour-specific methylation in medulloblastoma.

Material and methods

 Cell lines and patient material

Eleven medulloblastoma cell lines (DAOY, D283 Med, MHH-MED-1, MHH-MED-8A, D341 Med, D384 Med, D425 Med, D458 Med, UW402 and UW228) were studied. D425 Med and D458 Med were derived from the primary and a metastatic tumour from a single patient, respectively; UW228-2 and UW228-3 were derived from the same tumour but exhibit phenotypic differences (21), all other cell lines were derived independently. All cells were grown under recommended culture conditions, and cell line identity was confirmed prior to use by karyotyping (data not shown). Cell line DNA was extracted using the Qiagen DNeasy kit (Qiagen, Crawley, UK).

A cohort of 44 primary medulloblastomas were analyzed, representative of all the major histopathological subtypes (25 classic, seven large cell/anaplastic and 12 nodular/desmoplastic tumours), and adult and paediatric patients (10 infants <3 years, 30 children 3–16 years and four adults >16 years). Clinical and pathological data were centrally reviewed for this study. The presence of metastases was detected on a pre-operative MRI scan. DNA was extracted from frozen tissues using standard methods and from formalin-fixed, paraffin-embedded tissue using a Nucleon hard tissue kit (Amersham Biosciences, Little Chalfont, UK). Normal cerebellar control DNA consisted of post-mortem material from two infants (new born and 25 months) and three adults (60, 67 and 68 years) who had died of non-neoplastic conditions. Normal peripheral blood DNA consisted of a pool of 20 newborn cord blood DNAs. Local Ethical Committee and Institutional Review Board approval has been obtained for the collection, storage and biological study of all material.

Analysis of promoter methylation status

Bisulphite treatment of DNA was carried out using a CpG genome DNA modification kit (Serologicals, Livingston, UK) according to the manufacturer’s instructions. The methylation status of the CpG islands of p14ARF, p15INK4b, p16INK4a, CASP8, HIC1, EDNRB, TIMP3 and RIZ1 was determined by methylation-specific PCR (MSP) (22) using previously published primer sets (13,16,19,22 - B24#B24–25). The methylation status of the CpG islands of RASSF1A, TP73 and TSLC1 was determined by direct sequencing of the reverse strand and estimation of the relative peak heights of PCR products generated from bisulphite treated DNA using previously published primers (14,17,26). The unmethylated control for MSP consisted of pooled normal neonatal cord blood DNA (see above), the methylated control was the same sample methylated in vitro by SssI methylase (New England Biolabs, Hitchin, UK).

Thirty nanograms of bisulphite treated DNA was used per reaction, the PCR reactions were carried out using previously published conditions. MSP PCR products were separated on a 2.5% agarose gel in 1x TBE [0.09 M Tris–Borate, 0.002 M EDTA (pH 9)]. Products analysed by sequencing were directly sequenced with a CEQ DTCS kit (Beckman Coulter, High Wycombe, UK) using the antisense primer to obtain the reverse sequence. Sequenced products were analyzed on a CEQ 2000XL DNA analysis system (Beckman Coulter), and the methylation status at each CpG residue determined by assessment of the relative peak intensities.

COBRA (combined bisulphite and restriction analysis) of the CASP8 promoter was performed by overnight digestion of a PCR product generated using a non-differential primer set CASP8SQ2 (9) with Taq1 (MBI Fermentas, Hanover, MD) at 65°C, which cuts once within the 455 bp PCR product generating products of 396 and 59 bp. The 455 bp uncut product and the 396 bp digested product were separated on a 4% Nusieve 3:1 agarose gel in 1x TBE.

5-Aza-2′-deoxycytidine (5-aza CdR) treatment and RT–PCR

Four cell lines (D425 Med, MED-8A, DAOY and D283 Med) were grown in the presence or absence of the demethylating agent 5-Aza CdR (5 µM) for 4 days. Medium was renewed daily. HeLa cells were also grown as a positive control as these expressed all four transcripts. RNA was extracted from 107 cells using an RNeasy kit (Qiagen). RNA concentration was determined by spectrophotometry, and agarose gel electrophoresis was used to confirm consistent RNA integrity and quantity from each cell line. One microgram of total RNA was used to synthesize cDNA using a reverse transcription system (Promega, Southampton, UK). cDNA was synthesized using random primers and oligo-d(T) primers in separate reactions, followed by pooling. Equivalent amounts of this cDNA was used for PCR amplification of the RASSF1A, CASP8, HIC1 and ß-actin transcripts. The primers for detecting RASSF1A expression were as described previously (26) from exons 2aß and exon4, generating a 242 bp product. CASP8 primers were a previously published set from exons 1 and 3 generating a 379 bp product (9). HIC1 primers were designed for exon 1B 5′-GCGCGGCGGGGGCTGAGAC and exon 2 5′-GCCCTTGGTGCGCTGGTTGTTGAG, product size 244 bp, to amplify the major transcript (28). ß-Actin, a housekeeping gene, was used as a control for RNA integrity using primers BA67 and BA68 (29). Annealing temperatures for RASSF1A, CASP8, HIC1 and ß-actin were 60, 57, 66 and 60°C respectively. ß-Actin PCRs were amplified for 25 cycles, all other reactions were amplified for 35 cycles. PCR products were electrophoresed on a 2.5% agarose gel in 1x TBE.

Results:

Methylation status of CpG islands in medulloblastoma cell lines

In order to highlight genes that are potentially methylated in medulloblastoma tumours, the methylation status of the 11 candidate genes (RASSF1A, CASP8, HIC1, p16INK4a, p14ARF, p15INK4b, TIMP3, TP73, EDNRB, TSLC1 and RIZ1) was initially investigated in a panel of 11 medulloblastoma cell lines by MSP and bisulphite sequencing, using previously validated primer sets and conditions (13,14,16,17,19,22- B23#B23 - B24#B2426). A summary of these results is presented in Table I. All medulloblastoma cell lines showed evidence of methylation of multiple genes, with between two and six of the 11 CpG islands examined methylated in each cell line.

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Oncology Research,¹ Discovery Technologies, Novartis Institutes for BioMedical Research, Cambridge, Massachusetts 02139²

Received 29 November 2006/ Returned for modification 1 February 2007/ Accepted 11 April 2007

Bmi-1 and Mel-18 are structural homologues that belong to the Polycomb group of transcriptional regulators and are believed to stably maintain repression of gene expression by altering the state of chromatin at specific promoters. While a number of clinical and experimental observations have implicated Bmi-1 in human tumorigenesis, the role of Mel-18 in cancer cell growth has not been investigated. We report here that short hairpin RNA-mediated knockdown of either Bmi-1 or Mel-18 in human medulloblastoma DAOY cells results in the inhibition of proliferation, loss of clonogenic survival, anchorage-independent growth, and suppression of tumor formation in nude mice. Furthermore, overexpression of both Bmi-1 and Mel-18 significantly increases the clonogenic survival of Rat1 fibroblasts. In contrast, stable downregulation of Bmi-1 or Mel-18 alone does not affect the growth of normal human WI38 fibroblasts. Proteomics-based characterization of Bmi-1 and Mel-18 protein complexes isolated from cancer cells revealed substantial similarities in their respective compositions. Finally, gene expression analysis identified a number of cancer-relevant pathways that may be controlled by Bmi-1 and Mel-18 and also showed that these Polycomb proteins regulate a set of common gene targets. Taken together, these results suggest that Bmi-1 and Mel-18 may have overlapping functions in cancer cell growth.

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P53 mutations are relatively uncommon in medulloblastoma, but abnormalities in this cell cycle pathway have been associated with anaplasia and worse clinical outcomes. We correlated p53 protein expression with pathological subtype and clinical outcome in 75 embryonal brain tumors. The presence of JC virus, which results in p53 protein accumulation, was also examined.

Methods

p53 protein levels were evaluated semi-quantitatively in 64 medulloblastomas, 3 atypical teratoid rhabdoid tumors (ATRT), and 8 supratentorial primitive neuroectodermal tumors (sPNET) using immunohistochemistry. JC viral sequences were analyzed in DNA extracted from 33 frozen medulloblastoma and PNET samples using quantitative polymerase chain reaction.

Results

p53 expression was detected in 18% of non-anaplastic medulloblastomas, 45% of anaplastic medulloblastomas, 67% of ATRT, and 88% of sPNET. The increased p53 immunoreactivity in anaplastic medulloblastoma, ATRT, and sPNET was statistically significant. Log rank analysis of clinical outcome revealed significantly shorter survival in patients with p53 immunopositive embryonal tumors. No JC virus was identified in the embryonal brain tumor samples, while an endogenous human retrovirus (ERV-3) was readily detected.

Conclusion

Immunoreactivity for p53 protein is more common in anaplastic medulloblastomas, ATRT and sPNET than in non-anaplastic tumors, and is associated with worse clinical outcomes. However, JC virus infection is not responsible for increased levels of p53 protein.

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A team of investigators led by St. Jude Children’s Research Hospital has announced that improvements in the treatment of the childhood brain cancer medulloblastoma have significantly increased the rate of survival of children with this disease.

The treatment increased the overall five-year survival for 86 children with average-risk medulloblastoma from the current rate of 70 percent to 85 percent; and raised the rate of survival among the 48 high-risk patients from 55 percent to 70 percent. Patients are considered to be at average risk of treatment failure if their cancer has not spread following initial surgery to remove the tumor, or if the remaining tumor is very small. Patients are considered at high risk of failure if their tumor has spread following surgery or if the remaining tumors are larger than those of low-risk patients.

A report on these results appears in the September 7 issue of Lancet Oncology.

The investigators were able to achieve the improved survival rates while reducing the amount of radiation and length of chemotherapy following surgery in average-risk patients from levels used in standard treatments, according to Amar Gajjar, M.D., co-chair of the St. Jude Department of Oncology and director of its Neuro-Oncology Division. The chemotherapy for average-risk patients was completed in 16 weeks, versus the standard treatment, which lasts 48 weeks. The new treatment not only reduced radiation to the brain and spinal cord, but also reduced the level of two chemotherapy drugs by 75 percent and 50 percent, respectively. This reduction in radiation and chemotherapy holds promise for lessening the long-term, troublesome side effects on intellectual development of young patients treated for this cancer, Gajjar said.

In addition to improving overall five-year survival of average-risk patients to 85 percent, this group had an 83 percent rate of event-free survival and a rate of five-year, event-free survival of 70 percent for high-risk patients. Event-free survival means that a child did not have medical complications or relapse that required further treatment.

Moreover, the improved treatment achieved a survival rate of 66 percent as compared to 30-40 percent among children whose cancer had spread.

The results of the current clinical trial, SJMB96, are especially significant because they represent a dramatic change from the 45 percent survival rate achieved two decades ago using just surgery and irradiation, according to Gajjar. The subsequent addition of chemotherapy before or after radiotherapy improved that survival rate to 65 percent for children aged 3 years or older who had medulloblastoma.

“We attribute our very promising results to the early use of high-dose radiotherapy after surgery - rather than waiting until after chemotherapy - in combination with short-term, intense chemotherapy,” Gajjar said. “Shorter-term, intense chemotherapy is an especially important component of treatment that contributes to the improved survival of high-risk patients.”

The researchers also showed that genetic differences exist in medulloblastoma tissues among children. These differences could be used to differentiate between children whose tumors are very aggressive and require novel experimental treatment and those children whose tumors are less aggressive and who could benefit from further reduction in treatment.

“These additional studies suggest that we can further reduce the long-term effects of treatment among some children by further reducing the treatment intensity,” Gajjar said. The researchers previously reported that medulloblastoma consists of several distinct subgroups of this cancer that can be identified according to specific genetic abnormalities.

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The author has no significant financial interest or other relationship with the manufacturers of any products or providers of any service mentioned in this article.

JAMES M. OLSON, MD, PhD

Sri Gururangan and Henry Friedman present a thoughtful review of advances in pediatric neurooncology. Coupled with the recent review of pediatric brain tumor biology written by Richard Gilbertson, these articles highlight the value that the pediatric neuro-oncology community places on translating signal transduction modifiers into clinical practice.[1] The remainder of this commentary focuses on the challenges and opportunities associated with developing more effective and less toxic therapies for children with brain tumors.

Over the past 30 years, the cure rate for children with all types of malignant cancer has improved from less than 15% to more than 75%. Improvement for children with malignant brain cancer has been more limited. Gururangan and Friedman point out the limitations of surgical and radiotherapeutic approaches for tumors surrounded by precious, developing brain and the limitations of chemotherapy caused by the blood-brain barrier and intrinsic resistance mechanisms. Additional obstacles include insufficient biologic material and appropriate models for studying the diseases as well as the relatively small number of patients available for participation in clinical trials.

Biologic Materials and Models

In the past, the relatively low incidence of pediatric brain tumors limited the quality of data generated in single-institution studies. The Cooperative Human Tissue Network (CHTN) addressed this problem bydeveloping a national tumor bank that has steadily accrued specimens and provided them to researchers (wwwchtn. ims.nci.nih.gov). Unfortunately, the size of specimens provided to CHTN is typically < 50 mg, which severely limits the number of studies that can be conducted. Given that the average pediatric brain tumor weighs approximately 13 g at diagnosis, this represents only 0.3% of surgical material. In a small number of centers, surgeons and pathologists now provide gram quantities of tumor material for research, demonstrating the feasibility and safety of providing adequate samples.

There are no cell lines or mouse models for most types of pediatric brain cancer. A small number of medulloblastoma cell lines and xenograft models exist, but laboratory subculturing selects for cells that differ markedly from patient material. A genetically precise medulloblastoma model was developed in Matt Scott’s laboratory by targeted disruption of the patched gene, a negative regulator of the sonic hedgehog pathway.[2] Although this model has been helpful in understanding medulloblastoma biology, tumors typically arise in only 10% to 15% of animals. This limits the utility of this model for drug testing. Some investigators have generated tumors more rapidly and with higher frequency by crossing the mice onto a p53-deficient background or irradiating young mice. These approaches have been criticized as artificial because p53 mutations are rare in medulloblastoma, and patients have rarely received irradiation prior to diagnosis. A new genetically precise mouse model that activates the hedgehog pathway through constitutively active smoothened (a protein) has recently been reported. These mice have a 48% medulloblastoma incidence with a median age of onset at 25.7 weeks.[3]

The National Cancer Institute BrainTumor Progress Review Group established the following priorities for pediatric brain tumor research: (1) identify the signaling pathways involved, their relationship to developmental neurobiology, and their implications for new therapy, and (2) use knowledge of tumor phenotype and genetic alterations to generate genetically precise animal models that can be used to evaluate and prioritize potential new therapies. Laboratories are actively engaged in these pursuits.

Accelerating Clinical Trials

One of the greatest challenges facing investigators in the field of pediatric neuro-oncology is the relative paucity of patients for clinical trials. In the upcoming Children’s Oncology Group (COG) study of high-risk medulloblastoma/primitive neuroectodermal tumor (PNET), it will take 5 years to accrue 300 patients. At that rate, it will take over 30 years just to test the classes of compounds that already show promise in preclinical studies. For less common brain tumors, the challenge is amplified. As survival rates improve, even larger cohorts will be needed for appropriate statistical power if clinical trial design fails to evolve. The challenge is to identify new clinical trial end points to rapidly identify treatments that are failing so that other drugs can be tested. In doing so, we will be able to test more agents in individual patients, particularly in phase II trials.

The National Institutes of Health (NIH) roadmap for research may help accelerate clinical trials (nihroadmap. nih.gov). The roadmap establishes molecular imaging and nanomedicine as NIH research priorities. Molecular imaging refers to emerging techniques that enable noninvasive imaging of cell death, enzyme activity, protein interactions, or other molecular events, which have traditionally been measured only in laboratory studies. Forexample, magnetic resonance imaging contrast agents that bind to cancer cells undergoing cell death may be useful for determining whether an experimental therapy is effective in a matter of days, rather than the current standard of measuring tumor volume every few months. Applying a clinical trial end point that takes days rather than months will enable us to rapidly stop using ineffective agents and optimize effective drug combinations in individual patients.

Nanomedicine-the use of medical therapies, diagnostics, or response indicators that are nanometers in size- likewise has the potential to accelerate clinical investigation. Ideas range fromnanoparticles that deliver a “payload” of chemotherapy to tumor cells to nanoarrays that detect genetic mutations in minute quantities of tissue.

Molecular pathology-the determination of whether a drug target is present, absent, or present and mutated by polymerase chain reaction, immunocytochemistry, or other techniques- may also accelerate clinical trials by identifying subsets of patients who are most likely to benefit from a candidate therapy. Eliminating likely nonresponders based on molecular profiles of their tumor samples sharply reduces the number of patients required to statistically detect a drug response.

The transition from intensifying chemotherapy to targeting vulnerable signal transduction pathways has been facilitated by the extraordinary infrastructure of the Pediatric Brain Tumor Consortium and the COG. Investigatorsaffiliated with these organizations are actively developing genetically precise animal models, identifying vulnerable signal transduction pathways, and recognizing molecular signatures that are pertinent to drug efficacy. These organizations are poised to embrace new clinical trial end points and designs that enable advances even for uncommon cancers.

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In very young children with surgically treated medulloblastoma with metastases, lengthy remissions can be obtained with intensive chemotherapy without radiation therapy, according to a report in the March 10th issue of The New England Journal of Medicine. Investigators also found that cognitive function during follow-up is not affected as drastically as it is when radiation therapy is applied.

The prognosis for young children with medulloblastoma is poor and radiotherapy-induced cognitive deficits are common, lead researcher Dr. Stefan Rutkowski, a pediatric oncologist at the University of Wurzburg in Germany, and his associates note.

In their study, they resected the tumors of 43 patients younger than 3 years old, and administered three 2-month cycles of chemotherapy (cyclophosphamide, methotrexate, vincristine, carboplatin and etoposide).

In addition, an intraventricular subcutaneous reservoir was implanted in the anterior horn of a lateral ventricle, “with the aim of prolonging cytotoxic methotrexate levels in the cerebrospinal fluid by giving repeated small single doses,” the investigators report.

Complete resection of the tumor was accomplished in 17 patients, while residual tumor remained in 14 and macroscopic metastases were evident in 12 patients. Residual disease was further treated with surgery, chemotherapy or radiation therapy.

Altogether, the 5-year progression-free and overall survival rates were 58% and 66%, the authors report.

The 5-year progression free survival rates were 82%, 50% and 33% for those with complete resection, residual tumor, and metastases, respectively, and overall survival rates were 93%, 56% and 38%.

Risk of relapse or death was significantly lower for children with desmoplastic medulloblastoma, no evidence of metastasis, and those older than 2 years. There were no signs of methotrexate-induced neurotoxicity.

Among 14 patients who underwent detailed neuropsychological testing at a median of 4.8 years, IQ was lower than the general population of the same age. Among those not receiving any radiation therapy, IQ was higher than documented in other studies of patients who underwent radiotherapy.

“Results are especially promising for patients without initial metastases, in whom radiotherapy should be reserved for salvage strategies at relapse,” Dr. Rutkowski’s group indicates.

This report “represents a substantial step forward in the treatment” of medulloblastoma, Dr. Lisa M. DeAngelis, at Memorial Sloan-Kettering Cancer Center in New York, notes. She agrees that “radiotherapy may be withheld in the very young without compromising disease control.”

(Source: N Engl J Med 2005;352:978-986,1036-1038: Reuters Health: Oncolink: March 2005.)

Related Articles:

—      Improved treatment raises medulloblastoma survival rate

—      Technique could speed new treatments for medulloblastoma

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David M. Berman,^125* Sunil S. Karhadkar,^125* Andrew R. Hallahan,⁶⁷ Joel I. Pritchard,⁷ Charles G. Eberhart,² D. Neil Watkins,⁴ James K. Chen,¹⁵ Michael K. Cooper,¹³⁵ Jussi Taipale,¹⁵ James M. Olson,⁶⁷ Philip A. Beachy¹⁵

Constitutive Hedgehog (Hh) pathway activity is associated with initiation of neoplasia, but its role in the continued growth of established tumors is unclear. Here, we investigate the therapeutic efficacy of the Hh pathway antagonist cyclopamine in preclinical models of medulloblastoma, the most common malignant brain tumor in children. Cyclopamine treatment of murine medulloblastoma cells blocked proliferation in vitro and induced changes in gene expression consistent with initiation of neuronal differentiation and loss of neuronal stem cell-like character. This compound also caused regression of murine tumor allografts in vivo and induced rapid death of cells from freshly resected human medulloblastomas, but not from other brain tumors, thus establishing a specific role for Hh pathway activity in medulloblastoma growth.

Departments of ¹ Molecular Biology and Genetics, ² Pathology, ³ Neurology, ⁴ Oncology, and ⁵ Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. ⁶ Clinical Research Division, Fred Hutchinson Cancer Research Center, and ⁷ Division of Pediatric Oncology, University of Washington/Children’s Hospital, Seattle WA 98105, USA.
^*   These authors contributed equally to this work.

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Department of Radiation Oncology, Cancer Hospital (Institute), Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China and ² Department of Radiation Oncology, St Jude Children’s Research Hospital, Memphis, TN, USA

For reprints and all correspondence: Yueping Liu, Department of Radiation Oncology, Cancer Hospital (Institute), Chinese Academy of Medical Sciences, PO Box 2258, Beijing 100021, PR China. E-mail: wanyx@public.bta.net.cn

Received October 20, 2004; accepted January 2, 2005

Background: Although the optimal treatment mode for medulloblastoma is frequently discussed, results based on large series of cases, especially those treated in Asia, have rarely been reported. Our purpose was to evaluate the efficacy of postoperative radiation therapy, and to identify prognostic factors, in a relatively large cohort of patients with limited-stage medulloblastoma treated at a single institute in China.

Methods: Between January 1996 and April 2001, 69 patients with Chang stage M0/M1 medulloblastoma were referred to our hospital for radiation therapy after total or subtotal resection of the primary tumor. All patients received 30 Gy to the craniospinal axis followed by a 20–25 Gy boost to the posterior fossa (median fraction, 1.8 Gy).

Results: Sixty-four patients were followed for a median period of 38.5 months. The rates of 3-year and 5-year overall survival were 68.8% and 55.7%, respectively; corresponding disease-free survival were 57.8% and 51.4%, respectively. Patients who had received radiation treatment within 25 days after resection had a greater probability of 3-year survival (81.5% versus 59.5%; P = 0.11) and 3-year disease-free survival (74.1% versus 46.0%; P = 0.03) than patients who began radiation treatment later. No relationship was found between survival and age, sex or tumor size.

Conclusions: This regimen was comparatively ineffective in preventing recurrence of postoperative medulloblastoma; however, we found that the interval between surgery and radiation is a significant prognostic factor for disease-free survival.

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Aaron was born on the afternoon of 9^th January at Waitakere Hospital in West Auckland.  Aaron was our second born (proud parents being Sue and Murray) and we already had a bubbly 2 year old daughter Karina who was thrilled to have a new brother.  Murray and myself were just thankful he was healthy.

Aaron had the usual childhood illnesses, and usual knocks and falls for a boy, even a few scars to show off.   His first five and a half years were spent in Auckland.  Aaron thoroughly enjoyed mixing with other children, and enjoyed most of his pre-school time playing outside, his favourite place was in the sand pit.  As a toddler he was very happy, loved hugs (which he never outgrew), was friendly and confident.  In 1994 we moved to the Far North.  Aaron always loved the beach and by the end of most summers he would be so brown!!  He loved catching waves with the boggy board and snorkelling.  He joined Cubs and later Scouts with his sister and dad who was a leader, this encouraged him to grow in confidence and enjoy the outdoors. Other things he enjoyed were reading, magic cards (a complicated card game with endless rules), computer games and movies (especially science fiction and horror).  He also had a good sense of humour, always loved a good joke.  He was tolerant, kind and caring, a sensitive and thoughtful young person.

In June 2001 when Aaron was 12 he complained of headaches, saying he had had a few lately but this particular headache wouldn’t go away.  I took him to see our family doctor who said it was a build up of fluid in his nasal/ear passages and his glands were slightly inflamed, gave him antibiotics. Two days later I noticed Aaron was a bit wobbly on his feet and he still had the headache.  The next day he felt dizzy, had double vision, the right side of his face felt numb, and later he vomited and we noticed his speech was slurred.  He stopped taking the antibiotics straight away and we went back to the doctor.  He still insisted it was fluid build up etc, keep taking the antibiotics, come back if you feel the need.

I wasn’t happy or satisfied with the diagnosis and decided to get a second opinion.  This doctor really gave Aaron a thorough examination, he made a phone call to a paediatrician and arranged for us to go Whangarei Hospital the next day.  At the time, Murray was in the middle of the Pacific on a yacht and on his way to Vanuatu so it was just the three of us, mum, Karina and Aaron.  We arrived at the hospital and the doctor examinations and questions started (there would be many in the next few days) and Aaron had a CT scan. The doctors informed us Aaron’s gag reflex wasn’t normal, there was a slight droop (noticeable when he smiled) and numbness on the right side of his face and he had less power to the limbs on his left side.  The scan showed something on the brain, but they couldn’t, or wouldn’t say anymore and the specialists at Starship would do an MRI.  I knew then this was serious but tried to keep positive and hopeful.  I’ll always remember Aaron’s words after they told us … “I don’t won’t to die”.

We arrived at Starship that evening.  After extensive examinations they put Aaron on dexamethasone to help reduce swelling.  He had to undergo a MRI scan that required him to lie still for an hour.    It was amazing the change within a couple of days after being on the dex.  All the symptoms improved rapidly, headaches, dizziness, and speech.  Only problem was it gave him a constant and huge appetite for a couple of weeks.

Murray arrived within the next day after making a hurried flight back from Vanuatu, and we heard the worst news any parent could hear, our son had an inoperable brain tumour, a pontine brainstem glioma. We were told there are 4-5 children a year in New Zealand diagnosed with this tumour.  They said surgery was not possible because of where the tumour was situated and offered radiotherapy that would slow the growth, there was nothing else they could do for him, and Aaron could have anything from 4 months to 2 years to live.  We were all in shock, totally numb, our world was turned upside down, literally.  I kept asking myself, why Aaron, what has he done to deserve this.  We didn’t realise it fully then, but our lives had changed forever.

The next week we moved into Ronald McDonald House and started the process of preparing for radiotherapy which consisted of visiting an oncologist and getting a mask made up so the radiotherapy could be aimed directly at the tumour.  The next week he started his treatment and this continued for 6 weeks, same time everyday, Monday to Friday, we would walk up to Oncology for his 18 seconds of treatment.   After two weeks, we decided it was best if Murray and Karina went back home, to go back to work and school.  Life has to go on and we had to pay our bills somehow.  Most weekends they would come down and visit and once Aaron and I flew home for the weekend.  The side effects from the radiotherapy were difficult, he lost some hair and became very tired.  The side effects of the dex were becoming very apparent also and after some weeks on this drug he gained weight and tiny red spots appeared on his face (which are all very devastating for a 12 year old).

From the time Aaron was diagnosed we were investigating and trying alternative treatments, Aaron went along with most of these but in the months to come he eventually drew the line when it came to diet!  Murray and I could not accept that nothing else medically could be done for Aaron and so continued looking overseas for possible treatments, in this day and age there must be something out there.  This led us to Australia, America, Brazil and Germany.

On the 22^nd August 2001 after 6 weeks of radiotherapy and being away from home for 8 weeks, we arrived back, it was so fantastic to be home again.  We regularly visited a paediatrician in Kaitaia to monitor Aaron’s medication, he was in contact with a palliative care nurse and oncologists at Starship and Auckland Hospital.  Aaron was still very tired so he wasn’t able to go back to school, yet he was determined to stay fit and healthy as much as he could.   Some days he would go for a bike ride and workout in the gym.  One day he went shooting at a friend’s farm and went swimming with dad (they were the only brave ones as it was September and the water was freezing).  A favourite pastime during his days at home was playing Monopoly, he loved to beat everyone, even Grandad who was an accountant and visiting us at the time!  He also had times when he couldn’t do much other than sit in front of the TV watching his favourite movies or programs.  Some of his favourite pastimes were becoming increasingly difficult for him to do, he had double vision sometimes and he didn’t have the same control of his fine motor skills.  He loved playing with Warhammer, which involved tiny models being painstakingly joined and painted.  He didn’t attempt this any more.

One of Aaron’s wishes was to see the snow so through a trust set up for children like Aaron we were able to spend a week in Queenstown in September, all four of us!!  We stayed at the Sunshine Lodge and enjoyed jet boating, paragliding, visiting the Bluff, skiing/snowboarding and of course having snow fights and making snowmen.  We ate out lots, and had some great family times.  During the week we were away Aaron finished his dex but unfortunately his headaches started coming back.  He put on a brave face and enjoyed the holiday as much as he could. 

After returning from our holiday in Queenstown we returned to Auckland for consultations with oncologists and it was decided Aaron should go back on dex and have another MRI.  Thank goodness the MRI wasn’t as long as the first.  I still can’t believe how brave Aaron was through all of this, he didn’t complain much.  The news was devastating, the tumour had grown.  No further radiotherapy could be done as the brain can only take so much and he’d had his maximum dose, they couldn’t offer any other treatment, other than dex.  We all took the news very badly, Karina had to walk out part way through the consultation and Aaron went very quiet.  The week that followed I heard him crying some nights and I went to his side. We had been attending our local church and found great comfort and support there, Aaron especially, he started to develop a faith in God.  He had matured in many ways.

In October Aaron made the decision to return to school.  He was determined to get back to his usual activities and to see his friends, to make the most of what time he had left.  He was very nervous on the first day back, wondering what kids would say or ask him.  His appearance had changed dramatically since being on the dex.  His year 7/8 class were then practising for the cross-country and Aaron insisted on joining in.  He didn’t want to feel any different from his friends and be given any special treatment so he would run the length of the beach with his classmates.  At home he was still working out but he had times when all he wanted to do was sleep, rest or meditate.  These times became more frequent.

We had a friend in the medical profession who lived in Germany and he tracked down a neurosurgeon who said it was possible to operate with very good chances of removing the tumour and good chances of recovering from the operation.   We decided to take the risk and go for it.  Aaron was now finding it difficult to walk, his left foot dragged and he would often trip.  His still had double vision and he often felt dizzy.   We made the necessary arrangements to fly to Germany and left in early November.  The surgeon removed 70% of the tumour with little damage.  In early December we returned determined to have a normal Christmas, as close as possible anyway.  By this time Aaron was unable to walk, numbness returned to the right side of his face, swallowing was difficult, the use of his left hand was limited and worst of all, he lost his ability to speak. 

Over the next two months we had lots of visitors especially from family who all lived a long distance away, grandparents, aunts, uncles and cousins came for miles to see him and support us any way possible.

We had the use of a swimming pool that had an easy entry. Three or four times a week we would take the 10 minute trip to cool off and do some physiotherapy with Aaron.  He loved the freedom of the water but eventually this trip became too tiring for him.  Aaron’s symptoms grew worse, eating a meal sometimes took hours, his blood pressure was high, just sitting and holding his head up became difficult and tiring for him.  He watched TV, he read his favourite computer magazines and people read to him.  He did see the Lord of the Rings at our local cinema and we started to read “The Hobbit” to him.  He was given The Lord of the Rings book for his 13^th birthday but he insisted we read The Hobbit first.

In January the Scout Association awarded Aaron the Cornwell Scout Badge, an award given to Scouts who have shown extraordinary perseverance and courage through suffering or illness.  This award is rarely given in New Zealand.  It had been arranged for Aaron to officially receive this award on scout night but sadly this was not to be.

On Monday the 11^th February 2002 Aaron woke us early, we knew his time had come, we could see it in his eyes.  He died at 7.20 that morning, God came down and took his hand, and He took Aaron to Heaven, relieving him of all his suffering. Aaron stayed with us at home until his farewell, we often sat in his room talking, praying and crying, sharing memories and happy times.

The day we celebrated his life the weather was spectacular, the church overlooks a huge bay and the sun shone brilliantly, the water was sparkling blue and there wasn’t a cloud in the sky.  Both the weather and seeing so many family, friends, school students and staff, Scouts and people from the community, elevated our spirits.

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Medulloblastoma is a primitive neuronal tumour, usually arising in childhood. It is characterized by its propensity for spread via the cerebrospinal fluid (CSF). Treatment is by surgical resection followed by craniospinal radiotherapy (CSRT) and adjuvant chemotherapy. CSRT is one of the most complex radiotherapy (RT) techniques planned in most oncology departments [1]. We report a case of medulloblastoma which highlights the need for better resources for RT departments in the UK in order to accommodate patients requiring complex planning within a short time frame, such as required for CSRT.

A 10-year-old boy presented with headaches and vomiting. CT and MR scans revealed a cerebellar tumour. He had a radiologically confirmed complete macroscopic resection of the tumour and histology confirmed medulloblastoma.

He had a normal MR scan of the supratentorial area and spine and no evidence of tumour cells on cytospin of CSF from lumbar puncture. He was planned to receive CSRT and a boost to the posterior fossa with weekly vincristine during RT, followed by eight courses of chemotherapy with cisplatin, vincristine and CCNU given 6-weekly.

During preparation for CSRT planning, he developed more headaches. A CT scan 33 days after surgery suggested recurrence. When he was about to commence CSRT his symptoms deteriorated. An MR scan performed 21 days after the CT demonstrated definite evidence of tumour progression within the surgical bed.

Following his recurrence he had a further complete resection and commenced CSRT 34.2 Gy in 19 fractions followed by 21.6 Gy to the posterior fossa, giving a total dose of 55.8 Gy to the posterior fossa. 6 weeks after the completion of RT he commenced chemotherapy. However, when he was due to receive his third course of chemotherapy he became unwell with nausea, vomiting and back pain. MR scan at that stage revealed extensive leptomeningeal relapse. He was given palliative treatment and died 8 months after initial presentation.

Tumour volumes on the sequential CT and MRI data sets were measured using the Volume Viewer package on an Advantage Workstation (GE Medical Systems, Erlangen, Germany). Image slices were loaded into the package and the tumour volume manually segmented by drawing a region of interest around the tumour in each slice. The software’s volume algorithm was used to calculate the volume of the segmented tumour.

The two volume measurements were 3.88 cm³, and 3 weeks later 32.55 cm³. The volume doubling time (T_d) was 6.84 days, [Using the formula: T_d=t₂ x log 2/(log V_B–log V_A), where T_d=volume doubling time, V_A=Initial tumour volume and V_B=Tumour volume after t₂ days].

It is generally recommended that children with medulloblastoma should be treated by “immediate” RT. This should commence as soon as feasible after surgery. However, in the European PNET-3 study [2] which recruited patients between 1992 and 2000, where 58.1% of patients were from the UK or Ireland, only 13.5% of patients could commence RT within 28 days, and for 31.5% of patients the surgery to RT interval was greater than 42 days. Extended waiting times are still experienced in many RT departments in the UK. A recently published national audit [3] has reported that a greater proportion of patients waited an unacceptably long time for RT in 2003 compared with 1998.

There is only limited information available on the doubling time for patients with medulloblastoma. Since patients are managed by surgical resection, there is generally no opportunity to observe the rate of tumour growth on sequential scans. However, sequential scans are occasionally available, allowing an estimate of T_d.

In a series reported from the University of California, San Francisco [4], 26 patients had BUdR (bromo-deoxyuridine) labelling index performed to estimate the potential doubling time (T_pot). This was relatively short, 25–82 h. In three cases sequential CT scans were available in order to estimate the actual volume doubling time (T_d). In these three cases the T_d values were 20.7 days (T_pot=56 h), 24.4 days (T_pot=82 h) and 23.8 days (T_pot=25 h). The significantly lower values for T_pot compared with T_d reflect a high cell loss factor for medulloblastoma. In an earlier series, the mean T_d estimated from sequential CT scans for 3 medulloblastomas was 19.8 days [5].

In the case reported here the volume doubling time was estimated from scans using two different modalities, i.e. initially CT and subsequently MR, and the introduction of some inaccuracy in the estimation cannot be excluded. However the T_d is significantly shorter than that reported in the other series. Because of the paucity of information on the growth rate of medulloblastomas it is not clear what range of T_d these tumours may exhibit. It is likely that this doubling time represents the shorter end of the range. To concur with this the clinical course of his disease was aggressive, with tumour progression during adjuvant chemotherapy.

RT departments in the UK need to be better resourced in order to cope with the sudden need to accommodate a child with medulloblastoma requiring timely treatment with complex planning. In the European PNET-4 randomized study of conventionally fractionated compared with hyperfractionated RT for children with medulloblastoma, recently opened in the UK, the requirement is for RT to start preferably with 4 weeks and no later than 40 days after surgery.

In the case reported here the volume doubling time was significantly shorter than previously reported cases, and this was associated with a very poor clinical outcome, and for this patient tumour doubling time was probably at the shorter end of the range. However this case underlines the need for better resources for UK radiotherapy departments in order to provide the flexibility to accommodate patients requiring complex planning within a short time frame, such as required for CSRT.

S Bacon, J Clinkard and R E Taylor

Departments of ¹ Medical Physics and ² Clinical Oncology, Cookridge Hospital, Leeds, LS16 6QB, UK

Received for publication June 13, 2005. Accepted for publication June 15, 2005.

References

—      Taylor RE. UKCCSG Radiotherapy and Brain Tumour groups. Medulloblastoma/PNET and craniospinal radiotherapy (CSRT). Report of a workshop held in Leeds 30.6.99. Clin Oncol 2001;13:58–64.

—      Taylor RE, Lucraft H, Bailey CC, Robinson KJ, Weston CL, Ellison D, et al. Impact of radiotherapy parameters on outcome in the International Society of Paediatric Oncology (SIOP)/United Kingdom Children’s Cancer Study Group (UKCCSG) PNET-3 study of pre-radiotherapy chemotherapy for M0-1. Int J Radiat Oncol Biol Phys 2004;58:1184–93.[Medline]

—      Ash D, Barrett A, Hicks A, Squire C. Re-audit of radiotherapy waiting times 2003. Clin Oncol 2004;16:387–94.[CrossRef]

—      Ito S, Hoshino T, Prados MD, Edwards SB. Cell kinetics of medulloblastomas. Cancer 1992;70:671–8.[CrossRef][Medline]

—      Yamashita T, Kuwubara T. Estimation of rate of growth of malignant brain tumors by computed tomography scanning. Surg Neurol 1983;20:464–70.[Medline]

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