Oncogene amplification in medulloblastoma: analysis of a case by comparative genomic hybridization and fluorescence in situ hybridization

Pathology (1999) 31, pp. 337–344

ONCOGENE AMPLIFICATION IN MEDULLOBLASTOMA: ANALYSIS OF A CASE BY COMPARATIVE GENOMIC HYBRIDIZATION AND FLUORESCENCE IN SITU HYBRIDIZATION V E N ITA J AY *, JE R E M Y S Q U IR E †, JA N E B AYA N I†, A H M E D M. A L K H AN I ‡, JA M E S T. R U T K A ‡ A N D M A RIA Z IE L EN S K A ¶ Divisions of Pathology*, Neurosurgery‡ and Molecular Diagnostics¶ , The Hospital for Sick Children-University of Toronto and Ontario Cancer Institute†, Toronto, Ontario, Canada

Summary We describe amplification of the MYCC oncogene in a medulloblastoma with aggressive clinical behavior. The patient was a six year old boy who underwent gross total surgical excision of a cerebellar tumor. Despite chemotherapy and total neuraxis radiation, the clinical course was one of relentless progression, with extensive subarachnoid spread and death within eight months of presentation. The pathological features were consistent with the recently described, “large cell variant” of medulloblastoma. Tumor cells exhibited large vesicular nuclei, prominent nucleoli and strong immunoreactivity for synaptophysin. Polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH) assay revealed no evidence of MYCN amplification or 1p deletion in the tumor. FISH analysis revealed evidence of MYCC amplification in the 20- to 30-fold range. Comparative genomic hybridization (CGH) revealed regions of gains and amplification in three locations, with gains of chromosome 7, amplification of 8q24 (corresponding to the MYCC locus) and gains of the long arm of chromosome 17 (suggestive of isochromosome 17q). While conventional karyotypic analysis was not successful in the present case, CGH provided invaluable information about gene amplification and losses/ gains of chromosomes and chromosomal regions. Thus, CGH is a powerful technique applicable to frozen or paraffinembedded material which helps to ascertain the presence of gene amplification even without prior knowledge of the gene to be tested. Key words: comparative genomic hybridization, fluorescence in situ hybridization, Medulloblastoma, MYCC, MYCN, oncogene Abbreviations: CGH, comparative genomic hybridization; PNET, primative neuroectodermal tumor. Accepted 1 June 1999

INTRODUCTION In 1992, Giangaspero et al. 1 described in four infants a distinct variant of medulloblastoma associated with highly aggressive clinical behavior and proposed the designation of “large cell medulloblastoma”. In contrast to the classic medulloblastoma (primitive neuroectodermal tumor(PNET) of the cerebellum) which exhibits a relatively monotonous population of tumor cells with hyperchromatic oval or carrotshaped nuclei and scanty cytoplasm, these tumors were

associated with large vesicular nuclei and prominent nucleoli and immunohistochemical evidence of neuronal differentiation. All four cases in their report were characterized by early leptomeningeal dissemination and an aggressive clinical course despite radiation and chemotherapy. In the present report, we describe an aggressive tumor with features of “large cell medulloblastoma” in a six year old boy. Our patient died of extensive craniospinal subarachnoid tumor dissemination within eight months of initial presentation. Oncogene amplification is relatively uncommon in medulloblastoma, with a greater frequency in medulloblastoma cell lines. Both MYCN and MYCC amplification are reported in this setting. In one case in the series of large cell medulloblastomas reported by Giangaspero et al.,1 there was evidence of MYCC amplification by Southern blot analysis. In the present case of large cell medulloblastoma, we performed differential polymerase chain reaction (PCR), fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH) to evaluate chromosomal gains or losses and oncogene amplification in the tumor.

CASE REPORT A 6 yr old previously healthy boy presented with a 6 d history of progressive headache, nausea and vomiting associated with double vision. The family history was noncontributory. Physical examination revealed a fully awake and orientated child with normal vital signs. The patient had marked bilateral papilledema and mild ataxic gait. The remainder of the physical examination was unremarkable. A CT scan of the head demonstrated a midline hyperdense lesion in the posterior fossa, occupying the roof of the 4th ventricle, which was compressed resulting in marked obstructive hydrocephalus. The mass enhanced homogeneously with intravenous contrast administration (Fig. 1). The patient underwent a posterior fossa craniotomy the next morning and gross total excision of the tumor arising from the cerebellar vermis. Intraoperative frozen section report indicated a medulloblastoma and the operative course was uncomplicated. An early postoperative CT scan revealed no evidence of enhancing lesions (Fig. 2), however MRI of the spine detected enhancing drop metastases in the subarachnoid space. The postoperative period was complicated by CSF leakage from the surgical wound. This was managed initially with a lumbar drain, but later, a ventriculoperitoneal shunt was inserted. A few weeks later, the patient suffered a grand mal seizure and was started on phenytoin. The patient received whole neuroaxis radiation and was started on ICE (iphosphamide, carboplatinum, and etoposide) chemotherapy. During this

ISSN 0031–3025 printed/ISSN 1465–3931 online/99/040337–8 © 1999 Royal College of Pathologists of Australasia

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Fig. 1 Axial plain (left) and contrast-enhanced (right) CT of the head showing hyperdense mass lesion in the posterior fossa which enhances homogeneously with contrast.

Fig. 2 Immediate postoperative axial, contrast-enhanced CT scan showing complete resection of the posterior fossa mass lesion.

Fig. 3 Gadolinium-enhanced axial MRI scan of the brain taken approximately 10 mo after the patient’s initial surgery and showing diffuse leptomeningeal disease with enhancement of the entire subarachnoid space.

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Fig. 4 Gadolinium-enhanced sagittal (left) and axial (right) spinal MRI showing extensive spread of the tumor which has disseminated and filled the entire subarachnoid space.

period, he was functioning reasonably well, but was admitted to the hospital a few times for thrombocytopenia, fever, neutropenia and poor seizure control. Eight months postoperatively, he started to deteriorate neurologically. The MRI scan of the head revealed widespread leptomeningeal enhancement (Fig. 3). At this time, the MRI of the spine also revealed heavy coating of the subarachnoid space with enhancing tumor (Fig. 4). The patient developed respiratory failure and died within 2 wks. Postmortem examination was limited to examination of the brain and spinal cord.

MATERIALS AND METHODS The surgically excised tumor tissue (2 3 1.5 3 1 cm) was submitted for histological, immunohistochemical, ultrastructural and molecular analysis. After confirmation of tumor by frozen section, a tumor sample was submitted for PCR, FISH and CGH analysis. A tumor sample was also submitted for cytogenetic analysis, which was not successful.

Histology, immunohistochemistry and electron microscopy Tissue was processed for conventional histology with formalin fixation and paraffin embedding, and immunohistochemical staining was performed by the avidin–biotin complex or peroxidase antiperoxidase techniques using the following antibodies: glial fibrillary acidic protein (GFAP, polyclonal, 1:200, DAKO), synaptophysin (monoclonal, 1:5, Behring), phosphorylated neurofilaments (low, mid and high molecular weight subsets, monoclonal, 1:100, Sternberger), nonphosphorylated neurofilaments (monoclonal, 1:50, Sanbio), epithelial membrane antigen (EMA, monoclonal, 1:10, DAKO), low molecular weight cytokeratin (prediluted, monoclonal, Becton-Dickinson), vimentin (monoclonal, 1:300, Sigma), neuron-specific enolase (NSE, polyclonal, 1:250, DAKO), and MIB-1 (monoclonal, 1:50, Immunotech).

For EM, tissue was fixed in the universal fixative (equal parts of 4% formaldehyde and 1% glutaraldehyde) and post-fixed in 1% osmium tetroxide, dehydrated in graded alcohols and propylene oxide and embedded in epon. One-micrometer thick sections were stained with toluidine blue. Ultrathin sections were stained with uranyl acetate and lead citrate and examined under a Philips 400 TEM.

DNA extraction and differential PCR for analysis of MYCN amplification DNA was extracted from tumor specimens and control tissues by overnight digestion at 37°C with proteinase K in TE buffer and 10% sodium dodecyl sulfate followed by phenol-chloroform extraction and ethanol precipitation according to standard methods.2 Five hundred nanograms of target DNA, either MYCN amplification standards or undigested tumor DNA, were used in each determination according to previously described methods. 3 The MYCN primers MB4 (5046) and MB5 (5474) were used at 60 and 65.7 ng/reaction, respectively. These primers, from exon 3 of the MYCN gene, resulted in a product of 428 bp. Primers from exon 3 of the cystic fibrosis gene were used in each reaction as an internal control, and resulted in a product of 170 bp. All samples were tested in duplicate, and gels were stained with 0.5 m g/ml of ethidium bromide. Determination of copy number was obtained by comparing the ratio of the MYCN product to the cystic fibrosis product in the tumor with that of the MYCN standards, as described previously.3

Fluorescence in situ hybridization (FISH) analysis FISH analysis was used according to the previously described method.4 A cosmid genomic DNA probe derived from the MYCN and MYCC genes (generously provided by Dr A. T. Look and colleagues of St. Jude’s Children’s Hospital, Memphis, TN, USA) was labeled with digoxigenin dUTP by nick translation. For all tumors in which a 1p deletion was

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investigated, 200 ng of the cosmid genomic probe AD9 (generously provided by Dr Timothy W. Houseal and Dr Gregory M. Landes of Genzyme Genetics) from chromosome 1p36.2–.3 was used, and was labeled with biotin-14 dUTP. The probe D1Z5, specific for the centromere of chromosome 1, was already labeled with digoxigenin as supplied (Oncor Inc., Gaithersburg, MD, USA). Typically, probes were placed on the slide under a coverslip ringed with rubber solution. Hybridization was done for 16 to 20 h at 37°C. Washing consisted of 3 3 5 min washes at 50% formamide and 2 3 SSC, followed by 3 3 5 min washes in 2 3 SSC at 45°C. The slides were then soaked in 3% bovine serum albumin–0.1% Tween 20 in 43 SSC for 15 min at 37°C prior to incubating with mouse antidigoxigenin antibody (Boehringer Mannheim, Germany) diluted 1:50 for 30 min at 37°C in fluorescent isothiocyanate conjugated avidin (Oncor), and washed 3 times for 5 min each in phosphate-buffered detergent (Oncor) at 45°C. The signal was amplified by adding mouse antidigoxigenin (Boehringer Mannheim) diluted at 1:50 in antiavidin (Oncor) for 15 min at 37°C, and washing 3 times for 5 min each in phosphate-buffered detergent at 45°C. Next, 3% BSA–0.1% Tween 20 in 43 SSC was added and allowed to incubate for 15 min at 37°C, after which rhodamine-conjugated antidigoxigenin (Boehringer Mannheim) in fluorescent isothiocyanateconjugated avidin (Oncor), diluted 1:5 for 15 min at 37°C, was added. This was washed off with phosphate-buffered detergent, using 33 5 min washes at 45°C, and the preparations were counterstained and mounted with a 2:3 mixture of 49,6-diamidin-2-phenylindol-dihydrochloride in antifade (Oncor). A minimum of 200 interphase nuclei were scored for FISH analysis of MYCN and MYCC copy number and 1p deletion. Quality checks on all probes used for FISH established that hybridization efficiencies of > 90% were obtained with control specimens prior to use of the probes in this study. Representative areas of the microscope slides were scanned systematically to ensure complete assessment of FISH results. Negative controls (normal diploid lymphocytes) and a positive control (IMR32)5 were run with every assay. Photomicrographs were obtained with a Nikon Microphot-FXA epifluorescence microscope equipped with triple-band fluorescent isothiocynate/Texas red/DAPI or with double-band fluorescent isothiocyanate/Texas red filters (Omega Optical Inc., Brattleboro, VT, USA). The image was captured by a cooled CCD camera (Photometrix, Tucson, AZ, USA) and overlaid electronically using Gene Join Software (courtesy of Tim Rand and David Ward, Yale University, New Haven, CT, USA).

Comparative genomic hybridization (CGH) CGH was used to identify the locations of all regions of copy number changes in the tumor. Metaphase spreads from normal human lymphocytes were prepared using standard protocols.6 The slides were aged for 2 to 3 d prior to denaturation at 70°C by 70% formamide in 23 SSC, followed by dehydration in an ethanol series. The slides were treated with proteinase K at a concentration of 0.1 m g/ml in 20 mM Tris, pH 7.5, 2 mM CaCl2 prior to hybridization. The CGH procedure was similar to published standard protocols.6 ,7 Tumor and normal spleen DNA were labeled with digoxigenin and biotin, respectively, by nick-translation to produce labeled fragments of 500 to 2000 bp in size. Equal amounts (200 ng each) of normal and tumor DNA were mixed in the presence of Cot-1 DNA (5 m g) and denatured in a mixture of 70% formamide in 23 SSC (Hybrisol VII, Oncor) at 75°C for 5 min. The DNA was allowed to reanneal at 37°C for 1 h prior to hybridization to the denatured metaphase slides. Hybridization under sealed coverslips was carried out for 60 to 72 h at 37°C in a humid environment. Washing was performed at 45°C including 33 5 min washes in 50% formamide, 23 SSC and 3 washes in 23 SSC. To prevent nonspecific antibody binding, the slides were soaked in a mixture of 3% BSA in 43 SSC and TNB (0.1 M Tris–HCl, pH 7.5, 0.15 M NaCl and 0.5% Boehringer blocking agent (Boehringer Mannheim) for 30 min. The fluorescence was detected by 5 m g/ml avidin–fluorescein isothiocyanate (FITC) (Oncor) and 2 m g/ml rhodamine-conjugated sheep anti-DIG antibody (Boehringer Mannheim). This step was followed by 33 5 min washes in 0.1% Tween 20, 43 SSC at 45°C. The slides were mounted in 20 mM Tris–HCl, pH 8.0, 90% glycerol containing 2.3% of the antifade – 1, 4, diazabicyclo-(2.2.2) octane (Oncor). Twenty images were captured using a Nikon Labophot-2 microscope equipped with an automatic filter

Pathology (1999), 31, November wheel and 83,000 filter set (Chroma, Brattleboro VT, USA) with single band pass exciter filters for UV/FITC (490 nm), DAPI (360 nm) and rhodamine (570 nm) and were analyzed using the ISIS CGH software version 2.0 (MetaSystems, Heidelberg, Germany). With this software the ratio of rhodamine:FITC signal is expressed as a red:green ratio with a deviation from a 1:1 ratio indicating gain or loss of chromosomal material. In keeping with established conventions for identifying regions of chromosomal loss and gain using CGH, a ratio of 0.8 was used to assign losses and 1.20 for gains.7 – 1 0 If the profiles exceeded a gain ratio of 1.5, the region was considered to be highly amplified and indicated as a thicker vertical line. A 99% confidence limit was selected in the designation of copy number changes.

RESULTS Light microscopy and electron microscopy Histological examination of the surgical specimen revealed a tumor infiltrating cerebellar folia and extending up to the pia and focally breaking into the subarachnoid space. There was focal desmoplasia related to the leptomeningeal spread of the tumor. The tumor cells were large with prominent nucleoli (Fig. 5), significant nuclear pleomorphism, abundant mitoses and extensive necrosis. The cells displayed scanty cytoplasm and there was no evidence of Homer Wright rosettes or any other architectural pattern. Immunostaining for MIB1 revealed a labeling index of 56%, indicating a high proliferative potential. The tumor cells were strongly immunopositive for synaptophysin. There was also positivity for NSE, scattered positivity for GFAP and no staining for neurofilaments, vimentin, EMA and cytokeratin. EM confirmed neuronal differentiation with neuritic cell processes filled with microtubules and neurosecretory granules. Tumor cells showed marked variability in nuclear size and shape and prominent nucleoli. There were no rhabdoid features in this neoplasm. Postmortem examination of the central nervous system revealed three metastatic subdural tumor nodules measuring between 0.5 mm and 1.5 cm. There was extensive leptomeningeal opacification due to tumor infiltration, which obscured the basal cisterns and formed a thick coating along the cranial nerves and blood vessels. The brain weighed

Fig. 5 Surgical specimen: high-power light micrograph demonstrates the features of a large cell medulloblastoma. The tumor exhibits large nuclei with prominent nucleoli and scanty cytoplasm. (H & E, original magnification, 31,000).

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firmed by FISH analysis, 20 to 30 fold (Fig. 7a) and gain of the long arm of chromosome 17 suggesting the presence of i(17q).

Fig. 6 Autopsy specimen: low-power light micrograph shows extensive leptomeningeal dissemination of tumor. The tumor fills and expands the subarachnoid space and extends into the Virchow Robin space along the cerebral vasculature. (H & E, original magnification, 3100).

1494 g (normal for age, 1263 g). A right ventriculoperitoneal shunt was found to be patent at autopsy. There was diffuse gyral flattening. A cuff of tumor tissue surrounded and compressed the medulla. Coronal sections of the brain revealed extensive subarachnoid and intraventricular tumor spread, with expanded Virchow Robin spaces plugged with tumor. The mode of metastasis was primarily leptomeningeal and no discrete intraparenchymal metastasis was seen even in the posterior fossa. Extensive subarachnoid tumor deposits were seen along the entire length of the spinal cord. Microscopy confirmed extensive leptomeningeal and intraventricular tumor spread with involvement of the Virchow Robin spaces (Fig. 6). There was extensive necrosis and high mitotic activity in the tumor. Differential PCR assay for MYCN There was no evidence of MYCN amplification by differential PCR in the tumor sample. FISH analysis for MYCN and MYCC amplification and 1p deletion FISH analysis revealed no evidence of MYCN amplification or 1p deletion. Of 200 nuclei examined in the tumor sample, 95% of nuclei with signals showed two signals/ interphase nucleus for the MYCN probe, while 98% of nuclei with signals showed two p58 signals/interphase nucleus. The positive control exhibited discrete domains of yellow/green fluorescence consistent with homogeneously staining regions with the MYCN probe and also had one signal with the p58 probe in the majority of nuclei examined. Background hybridization was not apparent. FISH analysis revealed evidence of MYCC amplification. Ninety-four per cent of nuclei with signals showed 20 to 30 signals/interphase nucleus for the MYCC probe (Fig. 7a). CGH analysis Regions of amplification were detected in three locations (Fig. 7b). There was gain of chromosome 7, 8q24 amplification corresponding to MYCC gene locus (con-

DISCUSSION Giangaspero et al.1 proposed the designation of “large cell” medulloblastoma as a distinctive variant of medulloblastoma associated with large vesicular nuclei and prominent nucleoli. The tumor morphology in our patient is akin to that observed by Giangaspero et al. in four infants,1 although our patient was six years of age at the time of presentation. Similar to the cases reported by Giangaspero et al., our case was strongly synaptophyin positive and revealed evidence of neuronal differentiation by EM including neuritic processes and dense core granules. While malignant rhabdoid tumor should be considered in the differential diagnosis of large cell medulloblastoma,11 there were no rhabdoid features in the present tumor, which showed neuronal differentiation by immunohistochemistry and EM. As in the cases described by Giangaspero et al.,1 our patient developed widespread craniospinal dissemination. The extremely high MIB1 labeling index of 56% also confirmed the proliferative potential of the tumor. Spinal drop metastases were observed early in the clinical course and documented by MRI of spine shortly after the surgical resection. The tumor remained unresponsive to chemotherapy and total neuraxis irradiation and the patient died of disseminated subarachnoid and intraventricular tumor spread within eight months of initial presentation. At autopsy, there was extensive coating of the leptomeninges of the brain and spinal cord with tumor extension along Virchow–Robin spaces. The mode of tumor dissemination was via the cerebrospinal pathways rather than a discrete intraparenchymal tumor recurrence. We examined the tumor sample by PCR, FISH, and CGH for the presence of chromosomal gains and losses and oncogene amplification. Conventional cytogenetic analysis was also attempted, but was not successful. While cytogenetic analysis provides invaluable information on karyotypic abnormalities, brain tumor cytogenetics is often frustrating as tumors often fail to grow in culture. In the present case, although karyotype analysis was unsuccessful, application of newer tools such as CGH provided important information about genomic losses or gains and oncogene amplification. CGH methodology is a convenient and rapid way to screen for chromosomal changes and provides accurate profiles of the overall genomic alteration that may be of benefit in understanding the molecular basis of tumor development. The ability to survey the whole genome in a single hybridization experiment provides an enormous advantage over conventional cytogenetic analysis, particularly for solid tumors where it is often difficult to obtain sufficient G-banded metaphases of good quality for detailed analysis. The frequency of oncogene amplification is greater in medulloblastoma cell lines than in primary tumor samples.1 2 – 19 Karyotypic evidence of oncogene amplification is provided by the identification of double minute chromosomes. Double minute chromosomes were described in 10 to 20% of medulloblastomas by Bigner et al.2 0 In

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Fig. 7 (A) MYCC analysis by FISH: close-up of nuclei viewed to detect FITC for amplified MYCC. (B) Ideogram summarizing the results of CGH analysis of large cell medulloblastoma: gains are represented by vertical lines to the right of schematic chromosomes. CGH diagram shows increased copy number of chromosome 7, amplification corresponding to MYCC gene locus on chromosome 8 and gain of long arm of chromosome 17 corresponding to i(17q).

medulloblastomas, amplification of MYCC and less commonly of MYCN is described. Oncogene amplification has traditionally been determined quantitatively by Southern blotting, a time consuming technique which often requires more DNA than is available from small tumor biopsies. Recently, the application of the novel technique, differential PCR, has provided a useful alternative to Southern blotting for evaluation of MYCN copy number.2 1 Using this technique, we have previously reported MYCN amplification in a case of medulloblastoma with prominent neuronal differentiation.21 In the present case of large cell medulloblastoma, there was no evidence of MYCN amplification by differential PCR assay. The technique of FISH has the extraordinary advantage over conventional cytogenetic analysis in its applicability to interphase cells. FISH provides information about

translocations, gene amplifications and other chromosomal alterations. 2 2 In the present study, we applied the FISH technique to study abnormalities reported in medulloblastoma: amplification of the oncogenes MYCN or MYCC and deletions of chromosome 1. FISH analysis for MYCN complimented the results of the differential PCR assay and confirmed the absence of MYCN amplification in our case. There was also no evidence of 1 p deletion, which is a common abnormality in tumors such as the neuroblastoma. While techniques such as karyotype analysis or FISH require interphase nuclei or metaphase spreads to determine the genetic composition of a tumor, CGH, in contrast, uses DNA extracted from the tumor sample. CGH measures the relative DNA sequence copy number of the tumor without the need for cultures, by co-hybridizing with differentially labeled tumor DNA and reference normal DNA to normal

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metaphase chromosomes. 7,2 3 Recognition of previously unknown abnormalities is thus possible by CGH, which is based on competitive binding of tumor and control DNA to normal metaphase chromosomes. Gains or losses of whole chromosomes or segments of chromosomes are visualized as intensity differences in the fluorescence, which can be quantified. Thus CGH is applicable to both frozen and paraffin-embedded material and is a genome wide test that assesses changes in the entire genome in a single step, without prior knowledge of which gene or chromosome is involved. In a CGH study of 27 cases of medulloblastoma, Reardon et al.24 found a number of genomic abnormalities including, nonrandom losses in regions on chromosomes 10q, 11, 16q, 17p and 8p, gains of chromosomes 17q and 7 and amplifications of chromosome bands 5p15.3 and 11q22.3. In a study of 18 primitive neuroectodermal tumors (PNETs) Schutz ¨ et al.2 5 found loss of 17p, gain of 17q, amplification of 2p24 corresponding to MYCN and amplification of 8q24 corresponding to MYCC. We previously reported 2p24 amplification in another medulloblastoma by CGH, corresponding to the MYCN locus.26 While there was no evidence of MYCN amplification or 1p deletion in the present case, CGH revealed gains of chromosome 7, 8q24 and the long arm of chromosome 17. In contrast to malignant gliomas where the most prevalent change is gain or loss of whole chromosomes, the dominant findings in medulloblastomas are deletions and unbalanced translocations.2 7– 30 Gains of whole chromosomes with deletions or unbalanced translocations result in partial trisomies. Deletions of extra copies of chromosome 1, resulting in trisomy of 1p or 1q, are reported in medulloblastoma. 2 0 Thus, trisomy of 1q and monosomy for 17 p are common in these tumors. Isochromosome 17q has been recognized as an important abnormality in the CNS PNETs, most of which have been cerebellar medulloblastomas.2 0,2 7– 29 ,3 1 In our patient, CGH revealed a gain of the long arm of chromosome 17, suggesting the presence of i(17q) in the tumor. In the study of Reardon et al.,24 chromosomal gain most commonly involved 17q and 7, both of which were observed in our case. The frequency of oncogene amplification in medulloblastoma cell lines may be greater than 80%, with MYCC being the gene most frequently amplified.12 Bigner et al.12 reported MYCC amplification in three cell lines and four xenografts derived from four primary medulloblastomas with double minutes, and suggested that the MYCC gene provides a growth advantage to the tumor cells in vitro and in nude mice. Friedman et al. found a 20-fold amplification of MYCC in a medulloblastoma-derived cell line.14 In a large series of 27 medulloblastomas, Badiali and collegues found a single case which had a large number of double minutes and revealed 27-fold MYCC amplification.1 3 This tumor had an unusual rapidly aggressive course with extensive CSF unresponsive to chemotherapy. Tomlinson et al.18 reported MYCN amplification as well as a rearranged MYCC gene in an aggressive medulloblastoma in a 27 year old man. In the report of Schutz ¨ et al.,2 5 high copy number amplifications found in chromosomal regions in which MYCN and MYCC were localized, suggesting a “pathogenetic” role of these genes in PNETs. These authors found that none of the three patients with high copy number amplifications of MYCN responded to therapy. While other

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studies of primary PNETs have not detected high levels of MYCN or MYCC amplification, Schu¨ tz et al.,2 5 suggested, based on CGH analysis, that amplification of MYCC genes (which was seen in 17% of their cases) may be higher in medulloblastoma than expected from previous data. In the original report of large cell medulloblastoma by Giangaspero et al.,1 one case revealed a 27-fold MYCC amplification. In our patient, CGH revealed amplification of 8q24, corresponding to the MYCC locus. Amplification of MYCC was also reaffirmed by Southern blot analysis in our case and estimated to be in the 20-fold range. In summary, we describe a case of large cell medulloblastoma with MYCC amplification, gain of chromosome 7 and i(17q). The tumor pursued an aggressive course with widespread meningeal dissemination, and was unresponsive to radiation and chemotherapy. Even though conventional cytogenetics was unsuccessful, the technique of CGH allowed detection of chromosomal gains and amplifications in this tumor. CGH allows detection of deletions, gains and amplifications on a genome wide scale in a single hybridization. In the future, analysis of a greater number of cases will permit recognition of previously unknown genetic aberrations in medulloblastoma and its aggressive variants. Address for correspondence: V. J., Division of Pathology, The Hospital for Sick Children, 555 University Avenue, Toronto M5G 1X8, Ontario, Canada. E-mail: [email protected]

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