Nils Sörensen

Postoperative neoadjuvant chemotherapy before radiotherapy as compared to immediate radiotherapy followed by maintenance chemotherapy in the treatment of medulloblastoma in childhood: results of the german prospective randomized trial hit ’91

Purpose: The German Society of Pediatric Hematology and Oncology (GPOH) conducted a randomized, prospective, multicenter trial (HIT ’91) in order to improve the survival of children with medulloblastoma by using postoperative neoadjuvant chemotherapy before radiation therapy as opposed to maintenance chemotherapy after immediate postoperative radiotherapy. Methods and Materials: Between 1991 and 1997, 158 patients were enrolled and 137 patients randomized. Seventy-two patients were allocated to receive neoadjuvant chemotherapy before radiotherapy (arm I, investigational). Chemotherapy consisted of ifosfamide, etoposide, intravenous high-dose methotrexate, cisplatin, and cytarabine given in two cycles. In arm II (standard arm), 65 patients were assigned to receive immediate postoperative radiotherapy, with concomitant vincristine followed by 8 cycles of maintenance chemotherapy consisting of cisplatin, CCNU, and vincristine (“Philadelphia protocol”). All patients received radiotherapy to the craniospinal axis (35.2 Gy total dose, 1.6 Gy fractionated dose / 5 times per week followed by a boost to posterior fossa with 20 Gy, 2.0 Gy fractionated dose). Results: During chemotherapy Grade III/IV infections were predominant in arm I (40%). Peripheral neuropathy and ototoxicity were prevailing in arm II (37% and 34%, respectively). Dose modification was necessary in particular in arm II (63%). During radiotherapy acute toxicity was mild in the majority of patients and equally distributed in both arms. Myelosuppression led to a mean prolongation of treatment time of 11.5 days in arm I and 7.5 days in arm II, and interruptions in 35% of patients in arm I. Quality control of radiotherapy revealed correct treatment in more than 88% for dose prescription, more than 88% for coverage of target volume, and 98% for field matching. At a median follow-up of 30 months (range 1.4–62 months), the Kaplan-Meier estimates for relapse-free survival at 3 years for all randomized patients were 0.70 ± 0.08; for patients with residual disease: 0.72 ±0.06; without residual disease: 0.68 ± 0.09; M0: 0.72 ± 0.04; M1: 0.65 ± 0.12; and M2/3: 0.30 ± 0.15. For all randomized patients without M2/3 disease: 0.65± 0.05 (arm I) and 0.78 ± 0.06 (arm II) (p < 0.03); patients between 3 and 5.9 years: 0.60 ± 0.13 and 0.64 ± 0.14, respectively, but patients between 6 and 18 years: 0.62 ± 0.09 and 0.84 ± 0.08, respectively (p < 0.03). In a univariate analysis the only negative prognostic factors were M2/3 disease (p < 0.002) and an age of less than 8 years (p < 0.03). Conclusions: Maintenance chemotherapy would seem to be more effective in low-risk medulloblastoma, especially in patients older than 6 years of age. Neoadjuvant chemotherapy was accompanied by increased myelotoxicity of the subsequent radiotherapy, causing a higher rate of interruptions and an extended overall treatment time. Delayed and/or protracted radiotherapy may therefore have a negative impact on outcome. M2/3 disease was associated with a poor survival in both arms, suggesting the need for a more intensive treatment. Young age and M2/3 stage were negative prognostic factors in medulloblastoma, but residual or M1 disease was not, suggesting a new stratification system for risk subgroups. High quality of radiotherapy may be a major contributing factor for the overall outcome.

Somatic mutations of WNT/wingless signaling pathway components in primitive neuroectodermal tumors

Primitive neuroectodermal tumors (PNETs) represent the most frequent malignant brain tumors in childhood. The majority of these neoplasms occur in the cerebellum and are classified as medulloblastomas (MB). Most PNETs develop sporadically; however, their incidence is highly elevated in patients carrying germline APC gene mutations. The APC gene encodes a central component of the WNT/wingless developmental signaling pathway. It regulates the levels of cytoplasmic β‐catenin protein that plays a central role in neural development and cell proliferation. We analyzed 87 sporadic PNETs and 10 PNET cell lines for mutations of the APC gene and β‐catenin (CTNNB1) gene using single strand conformational polymorphism (SSCP) and sequencing analysis. We examined the mutation cluster region of APC (codons 1255–1641) for germline variants and somatic mutations. The medulloblastoma cell line MHH‐MED‐2 carried a Glu1317Gln missense germline variant and a sporadic MB sample showed a somatic Pro1319Leu substitution. Mutational analysis of exon 3 of CTNNB1 uncovered 4 PNETs (4.8%) with somatic missense mutations. These mutations caused amino acid substitutions in 3 of 80 medulloblastomas (Ser33Phe, Ser33Cys and Ser37Cys) and 1 of 4 supratentorial PNETs (Gly34Val). All mutations affected GSK‐3β phosphorylation sites of the degradation targeting box of β‐catenin and resulted in nuclear β‐catenin protein accumulation. Deletions of CTNNB1 were not detected by PCR amplification with primers spanning exons 1–5. Our data indicate that inappropriate activation of the WNT/wingless signaling pathway by mutations of its components may contribute to the pathogenesis of a subset of PNETs.

Mutations of the Wnt antagonist AXIN2 (Conductin) result in TCF-dependent transcription in medulloblastomas

Medulloblastomas (MBs) represent the most common malignant brain tumors in children. Most MBs develop sporadically in the cerebellum, but their incidence is highly elevated in patients with familial adenomatous polyposis coli. These patients carry germline mutations in the APC tumor suppressor gene. APC is part of a multiprotein complex involved in the Wnt signaling pathway that controls the stability of β‐catenin, the central effector in this cascade. Previous genetic studies in MBs have identified mutations in genes coding for β‐catenin and its partners, APC and AXIN1, which cause activation of Wnt signaling. The pathway is negatively controlled by the tumor suppressor AXIN2 (Conductin), a scaffold protein of this signaling complex. To investigate whether alterations in AXIN2 may also be involved in the pathogenesis of sporadic MBs, we performed a mutational screening of the AXIN2 gene in 116 MB biopsy samples and 11 MB cell lines using single‐strand conformation polymorphism and sequencing analysis. One MB displayed a somatic, tumor‐specific 2 bp insertion in exon 5, leading to carboxy‐terminal truncation of the AXIN2 protein. This tumor biopsy showed nuclear accumulation of β‐catenin protein, indicating an activation of Wnt signaling. In 2 further MB biopsies, mutations were identified in exon 5 (Glu408Lys) and exon 8 (Ser738Phe) of the AXIN2 gene, which are due to predicted germline mutations and rare polymorphisms. mRNA expression analysis in 22 MBs revealed reduced expression of AXIN2 mRNA compared to 8 fetal cerebellar tissues. Promoter hypermethylation could be ruled out as a major cause for transcriptional silencing by bisulfite sequencing. To study the functional role of AXIN2 in MBs, wild‐type AXIN2 was overexpressed in MB cell lines in which the Wnt signaling pathway was activated by Wnt‐3a. In this assay, AXIN2 inhibited Wnt signaling demonstrated in luciferase reporter assays. In contrast, overexpression of mutated AXIN2 with a deleted C‐terminal DIX‐domain resulted in an activation of the Wnt signaling pathway. These findings indicate that mutations of AXIN2 can lead to an oncogenic activation of the Wnt pathway in MBs.

Epilepsy in shunt‐treated hydrocephalus

Although epilepsy is commonly associated with shunt‐treated hydrocephalus, its relation to the shunting procedure and the criteria identifying postoperative epilepsy remain controversial. Of 283 patients shunted at Würzburg University Hospital over a 24‐year period (1970 to 1994), 182 were followed up for a minimum of 1 year after shunt insertion and entered the study. The data were analyzed retrospectively in 1995 and 1996. Epilepsy was analyzed in relation to the etiology of hydrocephalus, functional status, time and site of shunt insertion, onset of seizures and seizure type, EEG changes, sex, shunt systems, and shunt revisions. Of the 182 patients studied, 37 (20%) developed epilepsy. The incidence of epilepsy varied according to the etiology of hydrocephalus: posthemorrhagic (5%), postinfectious (4%), connatal/miscellaneous/unknown (3%), myelomeningocele (2%), tumor/arachnoidal cyst/aqueduct stenosis (0%). Early shunting and poor functional status was associated with a higher risk for epilepsy. Epilepsy was not influenced by sex, shunt systems, or number of shunt revisions. Twenty‐two (12%) of 182 patients developed epilepsy (generalized N=13, focal N=9) after intracranial shunting. Focal EEG abnormalities (N=16) were located mainly at the anatomical site of the shunt (N=14), but only three patients (2%) presented with focal seizures contralateral and focal EEG abnormalities ipsilateral to the site of the shunt. The presence of epilepsy was determined by the etiology of hydrocephalus rather than by surgical intervention. The incidence of postoperative epilepsy (12%) was low. Onset of epilepsy, clinical presentation of seizures, and EEG changes did not appear to be valid criteria for identifying shunt‐related epilepsy. Thus, epilepsy as a complication of intracranial shunting might be overestimated in the literature.

Analysis of the max-binding protein MNT in human medulloblastomas†

Medulloblastomas (MBs) are the most frequent malignant brain tumors in children. The molecular pathogenesis of these tumors is still poorly understood. Microsatellite and restriction‐fragment‐length polymorphism studies have revealed allelic loss of genetic material on the short arm of chromosome 17 in the region 17p13 in approximately 50% of MBs, suggesting the presence of a tumor‐suppressor gene in this region. A candidate for this putative tumor‐suppressor is the MNT gene, located at 17p13.3 and encoding a Max‐interacting nuclear protein with transcriptional‐repressor activity. In this study, we analyzed MNT mRNA and protein expression in 44 MB samples, including 32 primary tumors, 3 recurrent tumors and 9 MB cell lines. Allelic loss at 17p13.3 was found in 49% of informative cases. RT‐PCR showed MNT mRNA expression in all cases analyzed. Endogenous Mnt protein with an apparent molecular weight of 72 to 74 kDa was detected in lysates from MB cell lines. The presence and functional integrity of Mnt in MBs were tested in electrophoretic mobility‐shift assays. These experiments demonstrated that Mnt interacts with Max, and that this heterodimer binds DNA specifically, suggesting a functional bHLHZip domain of MB‐derived Mnt. In support, single‐strand conformation‐polymorphism (SSCP) analyses revealed no mutation in the bHLHZip region. Deletion of the Mnt Sin3 interaction domain was shown to convert Mnt from an inhibitor of myc/ras‐co‐transformation into a molecule capable of cooperating with Ras in transformation. This region therefore was screened for mutation by SSCP: again, no alterations were found. These findings indicate that the MNT gene located at 17p13.3 is not likely to be involved in the molecular pathogenesis of MBs. Int. J. Cancer 82:810–816, 1999.

SGNE1/7B2 is epigenetically altered and transcriptionally downregulated in human medulloblastomas.

In a genome-wide screen using differential methylation hybridization (DMH), we have identified a CpG island within the 5′ region and untranslated first exon of the secretory granule neuroendocrine protein 1 gene (SGNE1/7B2) that showed hypermethylation in medulloblastomas compared to fetal cerebellum. Bisulfite sequencing and combined bisulfite restriction assay were performed to confirm the methylation status of this CpG island in primary medulloblastomas and medulloblastoma cell lines. Hypermethylation was detected in 16/23 (70%) biopsies and 7/8 (87%) medulloblastoma cell lines, but not in non-neoplastic fetal (n=8) cerebellum. Expression of SGNE1 was investigated by semi-quantitative competitive reverse transcription–polymerase chain reaction and found to be significantly downregulated or absent in all, but one primary medulloblastomas and all cell lines compared to fetal cerebellum. After treatment of medulloblastoma cell lines with 5-aza-2′-deoxycytidine, transcription of SGNE1 was restored. No mutation was found in the coding region of SGNE1 by single-strand conformation polymorphism analysis. Reintroduction of SGNE1 into the medulloblastoma cell line D283Med led to a significant growth suppression and reduced colony formation. In summary, we have identified SGNE1 as a novel epigenetically silenced gene in medulloblastomas. Its frequent inactivation, as well as its inhibitory effect on tumor cell proliferation and focus formation strongly argues for a significant role in medulloblastoma development.

No evidence for mutations or altered expression of the Suppressor of Fused gene (SUFU) in primitive neuroectodermal tumours

The sonic hedgehog (Shh) and the Wnt signalling pathways are involved in the development of medulloblastomas (MBs), the most frequent malignant brain tumours in children. Components of these two developmental and cancer‐associated pathways, including (Patched) PTCH, SMOH, adenomatous polyposis coli (APC), β‐catenin and AXIN1 show somatic mutations in sporadic MBs. In this study we analysed SUFU (human suppressor of fused), which acts as a negative regulator of both the Shh and Wnt signalling pathways and therefore represents a putative tumour suppressor gene, to find out if it is also involved in the pathogenesis of sporadic MBs. We screened 145 primitive neuroectodermal tumours (PNETs) including 90 classic MBs, 42 of the desmoplastic variant and two medullomyoblastomas as well as 11 MB cell lines for mutations using single‐strand conformational polymorphism (SSCP) and sequencing analysis. 18% of the MBs exhibited allelic losses on chromosome 10q. In contrast to a previous report, in which truncating mutations of SUFU have been identified in 9% of MBs, we were not able to identify somatic mutations of SUFU in our large tumour panel. We uncovered single nucleotide polymorphisms (SNPs) in exon 4, 8, 11 and in intron 2 in the SUFU gene. Expression analysis by competitive reverse transciption‐polymerase chain reaction (RT‐PCR) revealed no difference in SUFU mRNA levels of both MB subtypes and normal foetal or adult cerebellar tissues. Our results indicate that genetic alterations of the SUFU gene, do not contribute significantly to the molecular pathogenesis of MBs.

Human medulloblastomas lack point mutations and homozygous deletions of the hSNF5/INI1 tumour suppressor gene

Medulloblastomas (MBs) are malignant primitive neuroectodermal tumours (PNETs) of the cerebellum occurring predominantly in childhood. The association of monosomy of chromosome 22 with MB is controversial. Atypical teratoid/rhabdoid tumours (AT/RTs) of the brain share clinical and histological features with MBs and supratentorial PNETs (sPNETs). In particular, AT/RTs can be misdiagnosed as MBs and sPNETs because AT/RTs frequently contain areas of primitive neuroepithelial cells similar to PNETs. Recently, mutations of the tumour suppressor gene hSNF5/INI1, located on 22q11.23, have been described in AT/RTs, MBs and sPNETs, with conflicting data on the prevalence of hSNF5/INI1 mutations in the latter entities. Therefore, we screened MBs for point mutations and homozygous deletions of the hSNF5/INI1 tumour suppressor gene. In 90 MBs, no mutations of the hSNF5/INI1 gene were identified. Thus, our study virtually rules out hSNF5/INI1 as a tumour suppressor gene involved in the pathogenesis of medulloblastoma.

Frequent intragenic polymorphism in the 3' untranslated region of the lissencephaly gene 1 (LIS-1).

Candidate gene for: Miller-Dieker lissencephaly syndrome (MDS) and isolated lissencephaly (ILS). Lissencephaly is a neuronal migration disorder characterized by a severe paucity of cortical gyri. Ninety-two percent of the patients with MDS and 38% of the patients with classical ILS have small or subpicroscopic deletions on chromosome band 17~13.3, which includes the recently characterized lissencephaly gene 1 (LIS-1) (Dobyns et al. 1993, Reiner et al. 1993).