Deletion mapping of the medulloblastoma locus on chromosome 17p

GENOMICS

8, %?#-285

(1990)

Deletion Mapping of the Medulloblastoma on Chromosome 17p PHILIP Departments

H. COGEN,*+*,’ LALEH DANESHVAR,*-$ ANDREW AND MICHAEL 5. B. EDWARDS*+*

Locus

K. METZGER,*

of *Neurological Surgery and t Pediatrics, Division of Pediatric Neurosurgery, and the #rain School of Medicine, University of California, San Francisco, California 94143 Received

February

12, 1990;

revised

Tumor Research Center,

April 24, 1990

spread throughout the subarachnoid pathways in the brain and spinal cord develop in 20 to 40% of patients, despite the adjuvant use of radiation therapy and chemotherapy (Packer and Finlay, 1989). Patients with medulloblastoma grouped into “good-risk” and “poor-risk” categories based on the clinical, radiologic, and pathologic features of their tumor when diagnosed have a 75 and 40% survival rate, respectively, at 5 years after therapy (Kopelson et al., 1983; Packer and Finlay, 1989). Currently, treatment for medulloblastoma consists of as complete as possible surgical removal of the primary mass lesion followed by radiation therapy for patients staged in the goodrisk group and the addition of systemic chemotherapy to this treatment regimen for those in the poor-risk group* Recent studies have shown that neoplastic transformation may result from the inactivation in tumor tissue of both copies of a class of genes known as tumor suppressors or recessive oncogenes (Friend et al., 1988; Ponder, 1988). The existence of these genes was first proposed by Knudsen (1971) on the basis of epidemiologic studies of familial and sporadic retinoblastoma. Confirmation of his hypothesis came from molecular biologic techniques, such as RFLP analysis, which showed that specific chromosome 13 alleles present in control tissue were lost or deleted in retinoblastoma tumor specimens from the same individual (Benedict et al., 1983; Cavenee et aZ., 1983; Dryja et al., 1984). The absence of these sequences suggested that there was loss of both copies of a functional tumor suppressor gene. This gene (Rb) subsequently was cloned for retinoblastoma (Friend et al, 1986; Fung et al., 1987; Lee et al., 1987a), and its product was found to be a DNA-binding protein (Lee et al., 1987b). Deletion of chromosome-specific DNA sequences consistent with loss of tumor suppressor gene activity occurs in several other pediatric solid neoplasms, most notably nephroblastoma (Wilms tumor)

Isochromosome 17q has previously been observed consistently in cytogenetic studies of medulloblastoma, the most common posterior fossa neoplasm in children. We performed a restriction fragment length polymorphism (RFLP) investigation of medulloblastoma which showed a loss of chromosome 17p sequences in 45% of these tumors. This finding was predictive of a poor clinical response to treatment. A contiguous panel of markers permitted mapping of the deletion to 17p12-~13.1, the same chromosomal region for which loss of alleles has been shown in tumor specimens from patients with colon cancer, and the same region to which the ~63 gene has been mapped. This suggests that medulloblastoma is associated with a recessive oncogene on chromosome 17p that may be involved in the genesis of several embryologically unrelated neoplasms and that the absence of this gene in tumor tissue has prognostic significance. 0 1990 Academic Press, Inc.

INTRODUCIION

Primary intracranial neoplasms are the most common type of solid cancer in children (Allen, 1985), yet little is known about their etiology at the genetic or molecular level. Childhood brain tumors most often arise in the posterior fossa within the cerebellar vermis or hemispheres (Allen, 1985; Packer and Finlay, 1989). Medulloblastoma, the most common neoplasm in this location, accounts for 25% of all brain tumors in children (Packer and Finlay, 1989). While advances in surgical technique now permit complete resection of the primary tumor mass in most cases, there remains a negative long-term prognosis for many of these children (Packer and Finlay, 1989). Recurrence of the tumor at the same site or metastatic 1 To whom correspondence should be addressed at Department of Neurological Surgery, c/o The Editorial Office, 1360 Ninth Avenue, Suite 210, San Francisco, CA 94122. 279

o&w7543/90$3.00

Copyright 0 1990 All rights of reproduction

by

Academic Press, Inc. in any form reserved.

280

COGEN

ET

AL.

TABLE

1

Chromosome 1’7 RFLP Analysis of Medulloblastoma Probe: Location: Enzyme: Tumor Med-1 Med-2 Med-3 Med-4 Med.5 Med-6 Med-7 Med-8 Med-10 Med-11 Med-12

p144-D6 17p13.3

pYNH37.3 17p13.3

RsaI

BamHI

BamHI

-

12 12 1 12 12 1

12 12 12

1 12 12 12

12 12 2 12 2 2 12 2 12 12 12

1

1 12 1

pHRP5.5 17p13.1

MspI

-

1

pYNZ22.1 17p13.3

Hind111

12 12 12 12 12 12 12 12

pHP53b 17p13.1 Ban11

BglII

&a1

2 1 2 2

2 12 1 2 2 -

12 -

MYH2 17~12

EW503 17~12

pHF12-2 17~12

Hind111

MspI

MspI

RsaI

12 12 1 12 2 1 1 12

12 12 2

-

12 12 12

-

2 12 2 12 2

2 12 12 2 2 12 2

pYNM67 17~11.2

12 12 12 12 12 12 12

Note. Results are shown in the following way: “12”, both alleles are seen in DNA from blood and tumor tissue (heterozygous, no loss); “1” or “2”, both alleles are seen in DNA from blood but one allele is missing in DNA from tumor tissue (hemizygous, loss); “-‘I, only one allele is seen in DNA from blood and tumor tissue (homozygous, not informative); blank entry, study not performed.

(Fearon et al., 1984; Orkin et al., 1984). Convincing evidence for a functional role of recessive oncogenes in tumorigenesis has been shown by the introduction of a human chromosome 11 into a Wilms tumor cell line previously identified as having a deletion of chromosome llp, with subsequent reversal of the neoplastic phenotype (Weissman et al., 1987). Further investigations have revealed chromosome-specific allelic deletions in a number of common adult solid tumors, including colon (Fearon et al., 1987; Law et al., 1988; Baker et al., 1989; Vogelstein et al., 1989), lung (Brauch et al., 1987; Naylor et al., 1987), and breast (Lundberg et al., 1987) cancers, as well as in neoplasms of the adult central nervous system (CNS), including acoustic neuroma (Seizinger et al., 1986, 1987b), meningioma (Seizinger et al., 1987a; Cogen, 1990), and adult glioma (James et al., 1988, 1989). Histologic similarities at the light microscopic level of medulloblastoma, retinoblastoma, and small-cell lung carcinoma (Rorke, 1983) suggest that these tumors might be caused by a common mechanism, i.e., a tumor suppressor gene. Asisochromosome 17q has been shown to be the most consistent cytogenetic anomaly in 30 to 60% of medulloblastomas (Bigner et al., 1988; Griffen et al., 19&3), we performed an RFLP study to see whether there was allelie loss of chromosome 17 sequences in DNA from this tumor. MATERiALS

AND

METHODS

Eleven surgical specimens were obtained from nine patients who underwent resection of a medulloblastoma in the posterior fossa. Qne patient underwent

three separate procedures within the same year because the tumor recurred at the primary site. Homogeneity of the tumor cells and the lack of inclusion of normal cerebellum in the pathologic specimens precluded a need for microdissection of the tissue (Vogelstein et al., 1989). Peripheral blood and, when available, normal brain tissue surrounding the tumor were also obtained from each patient. Protocols for collecting specimens were previously approved by the Committee on Human Research, University of California, San Francisco. Freshly excised tumor tissue that was flash-frozen in liquid nitrogen was used for analysis to avoid artifact from chromosomal rearrangements that may appear after multiple passages in tissue culture (Rorke, 1983). Initial investigations were performed with probes derived from chromosome 17 DNA (Table 1). Although it appeared that loss of DNA would most likely be shown for chromosome 17p, studies were performed with markers that spanned the length of chromosome I7 (vanTuinen et al., 1987,1988). Additional studies were also performed with probes derived from other chromosomal loci (Table 2). Included in the markers selected from chromosomes 1, 5,6,10,11,13,15,18, and 22 were sequences that have previously been shown to be informative in other CNS solid tumors. They included the Rb gene, which is deleted in retinoblastoma (Friend et al., 1986; Fung et al, 1987; Lee et al, 1987a); the markers DlOSl and cyp2c, which have shown losses in adult astrocytomas (James et al., 1988); and the D22Sl and X light chain sequences, which are deleted in acoustic neuroma and meningioma (Seizinger et al., 19,86, 1987a,b; Cogen,

CHROMOSOME

17p DELETION

Table pHPP 17pll.l-qter MspI

12 12 12

ErbAl 17qll-q21

BS3 17q21-q22

PULL11

Ban11

Sac1

12 12

-

12 12 12 12 12 12

12 12 12 12 12 12

12 12 12 12 12

281

IN MEDULLOBLASTOMA

l-Continued

pHMP2K 17q21-q24

RAR 17q21.1

PstI

EeoRI

12 12 12 12 12 12 12 12

12 12 12 12 12 12 12

pE51 17q21-q22 XmnI

12 12 12 12 12 12 12 12 12 12

1990). All of the probes were known before the study to be polymorphic for one or more enzymes. Southern blot technique described earlier was used (Feder et al., 1985). The radioactive signal was allowed to decay before rehybridization of the sameblot with additional probes. Radiolabeling was performed using the random primer technique (Feinberg and Vogelstein, 1983). Positive findings were replicated at least one additional time. The use of probes with a variable number of tandem repeat (VNTR) sequences increased the number of informative results. Dosimetry studies of informative chromosome 17p loci were performed with an LKB Ultroscan XL unit (LKB, Gaithersburg, Maryland). The same filters that were previously hybridized to chromosome 17 probes were rehybridized to sequences from other chromosomal loci, to correct for the differences in signal produced by the amount of DNA loaded in each lane. Ratios were then obtained by comparing the signal from the informative chromosome 17 sequence to that of the standardizing marker for the blood and tumor tissue. These adjusted figures were then used to examine the chromosomal content of the samples from each individual in whom a loss of chromosome 17p DNA was detected. Patients were categorized as good risk or poor risk on the bases’of their age at diagnosis, the size of the tumor, the extent of the surgical resection, and the presence or absence of metastatic tumor deposits in the brain and/or spinal cord (Kopelson et al., 1983). RESULTS

Initial studies (Fig. 1) showed that there was a loss of alleles from DNA sequences that mapped to the

Hind1 12 12 12 12 12 12 12 12 12 12 12

L1.31 17

THH59 17q23-q25.3 PUUII

RsaI

12 12 12 12

12 12 12 12 12

PstI

12 12 12 12 12 12 12 12 12

p9TAU 17

BgZII

XbaI

12

12

12

12 12 12 12 12 12 12 1 12

12 12 12 12 12

distal part of chromosome 17~ in the tumor DNA from four of nine patients (45%). Proximal 17~ sequences and all 17q markers were preserved (Table 1). Further investigations carried out with probes in the region of chromosome 17~11.2-13.3 showed a common location of allelic loss for informative medulloblastoma tumor specimens at chromosome 17p1213.1 (Fig. 2). Loss of DNA sequences has previously been reported in this same location in a large percentage of colon cancers (Fearon et cd., 1987; Law et al., 1988; Baker et al., 1989; Vogelstein et al., 1989). Results of the densitometric analysis of informative chromosome 17~ loci (Table 3) showed that the absence of signal in medulloblastoma tumor tissue was consistent with true deletion of the chromosomal region. Mitotic recombination, reported for chromosome 17~ in adult cerebral astrocytomas (James et cd., 1989), was not observed in any of the medulloblastoma specimens. Allelic loss was also detected with probes from chromosomal region 1Oq in tumor specimens from two individuals, in Med-2 and in Med-3, Med-6, and Med-8. One additional tumor, Med-10, was found to have a loss with a chromosome 6 probe. No loss of DNA was detected with the Rb gene (Fig. 1). These results are consistent with the reports of random chromosomal loss in other types of solid tumors (Vogelstein et al, 1989) and highlight the specificity of the loss of chromosome 17~ DNA in medulloblastoma. The clinical state of the patients at the time of diagnosis and their response to surgery, radiation therapy, and chemotherapy were correlated with loss and/or alteration of chromosome 17p DNA sequences. None of the patients whose tumor specimens

COGEN ET AL.

I I I I I I

I I I

IIIIII

I21

I

IIIIIII

showed loss or alteration of chromosome 17p DNA sequences had a good response to treatment, as demonstrated by local recurrence and/or metastatic spread of the tumor. Four of the five patients whose specimens did not reveal these changes are alive after treatment; the other patient recently died from recurrence of tumor. All of the patients in whom loss and/ or alteration of chromosome 17 DNA was shown following the initiation of their therapy underwent irradiation and chemotherapy in addition to surgical resection based on clinical and radiographic findings, in an attempt to control tumor growth. Despite this treatment, all of these patients, three of whom were classified in the good-risk group at the time of diagnosis, had recurrence of tumor. Ohe of the patients with tumor recurrence was the child from whom Med-3, Med-6, and Med-8 were obtained. He died 1 year after his initial resection, despite receiving maximum doses of radiation therapy and several combinations of chemotherapy. Molecular analysis of these three specimens has shown a large deletion of all of chromosome 17p distal to 17~11.2 as well as a rearrangement of the region of 17~13.1-13.3 as shown by the findings with the probes HRP5.5 and YNZ22.1 (Fig. 2). This finding was observed in the biopsy sample obtained before the institution of irradiation and chemotherapy. Loss and alteration of chromosome 17 DNA were thus found to be better prognostic indicators of outcome than were conventional clinical and radiologic evaluations for this group of patients with medulloblastoma. DISCUSSION The data from this study suggest the presence of a tumor suppressor gene for medulloblastoma on chromosome 17~12-13.1 (Fig. 2). Loss of chromosome 17p sequences has also been shown in colon (Fearon et al., 1987; Law et al., 1988; Baker et al., 1989; Vogelstein et al., 1989) and small-cell lung cancers (Brauch et al., 1987; Naylor et al., 1987), adrenocortical (Yano et al., 1989) and cervical (Atkin and Baker, 1989) carcinomas, and adult cerebral astrocytomas (James et al., 1988,1989). A molecular biologic correlation between medulloblastoma and astrocytoma might be anticipated, as astrocytic differentiation within medulloblastoma has been described (Rorke, 1987); however, none of the tumor specimens in this series demonstrated astrocytic pathology. The discord between the histology and the embryology of neoplasms demonstrating selective loss of chromosome 17p sequences is similar to findings for the Rb gene, which has been shown to be deleted in osteogenic sarcoma (Friend et al., 1986; Fung et al., 1987; Lee et al., 1987a), small-cell lung carcinoma (Harbour et al., 1988), and breast cancer (T’Ang et al., 1988). It was interesting that Rb was not lost or altered in any of the medulloblastomas

CHROMOSOME

17p DELETION

IN

283

MEDULLOBLASTOMA

a Med 3,6,8 (1) BI

(2) Br

Tu

81

Tu

pYNZ22.1IBam

BI

Br

Tu

BI

HI

(3) Br

Tu

El

Tu

MYH2/Hind

b Med5

BI

Br

Tu

BI Br Tu

111

BI

pG14E1.9

Tu

BI

(Rb)/Dra

Br

Tu

I

c Med12

(1)

(2) BI

81

Tu

(2)

(1)

1

Tu

2

2

pHFlP-2/Msp pHPSBb/Ban

1

pYNZ22.11Bam

HI

II

FIG. 1. Allelic losses in medulloblastoma. DNA was extracted from peripheral blood leukocytes (Bl), normal brain adjacent to tumor (Br), and medulloblastoma tumor specimens (Tu) (13, 14). Polymorphic fragments have been described previously (20), and alleles are labeled as “1” or “2”. (a) Med-3, -6, -8, Plates (1) and (2): Loss of chromosome 17 DNA sequences are seen for three tumor specimens obtained from the same patient. His deletion includes all of the region distal to chromosome 17~11.2. Plate (3): Preservation of alleles in this same patient with the retinoblastoma (Rb) gene. (b) Med-5: Loss of alleles with a chromosome 17 DNA probe is seen in this tumor, which has a smaller deletion than those in (a). (c) Med-12: DNA from this tumor shows loss of DNA sequences in the region of chromosome 17p12~13.1, with preservation of more distal markers.

in this study, even though the gene was informative for all of the tumors examined and even though medulloblastoma and retinoblastoma are histologically similar. The loss of chromosome 17p DNA sequences in a variety of histologically dissimilar neoplasms suggests that a single gene involved in tumorigenesis may have a more general mechanism of function. The product of the ~53 gene, located on chromosome 17~13.1, was initially shown to be oncogenic on the basis of transformation capabilities in rodent cell lines (Lamb and Crawford, 1986). However, further investigation revealed that the gene used for these studies was in a mutated form and subsequent work has shown that the gene product has tumor suppressor activity in uitro (Lamb and Crawford, 1986). Recent studies have demonstrated that single amino acid substitutions resulting from mutations in the ~53 gene are present in tumor specimens obtained from colon, lung, and brain tumor in adult patients (Fearon et al., 1987; Law et al., 1988, Baker et al., 1989; Nigro et al., 1989; Takahashi et al., 1989; Vogelstein et al., 1989). While several of these patients demonstrated loss of the p53 gene se-

quence by RFLP analysis, mutations in the ~53 gene were also observed in those individuals in whom the ~53 sequence appeared to be preserved by Southern blot technique (Nigro et al., 1989; Takahashi et aZ., 1989). Data from our study have shown loss of the ~53 sequence in two medulloblastoma specimens; however, many of the patients were uninformative at this

TABLE

3

Densitometry Data for RFLP Analysis of Medulloblastoma Tumor

Marker

Med-3

MYH2

Med-5

MYHP

Med-8

YNZ22.1

Med-12

HF12-2

Tissue

Chr 17 probe/ control probe

Tumor Blood Tumor Blood Tumor Blood Tumor Blood

2.64 4.15 3.40 6.60 1.61 3.25 0.85 1.47

Tumor/ blood 0.64 0.51 0.49 0.58

284

COGEN

locus. Mutation of the p53 gene may therefore be associated with tumor suppressor activity in medulloblastoma. As a base-pair mutation in the p53 gene would not be detectable with the methodology used in this study, we are now analyzing our tumor specimens for mutations in the ~53 gene sequence. Such mutations could explain the failure to detect loss and/or alteration of chromosome 17p DNA in the remaining 65% of our medulloblastoma specimens. Alternatively, contamination of tumor tissue by blood cells may have prevented detection of all of the chromosome 17p losses and/or rearrangements actually present in the medulloblastoma specimens. The consistent picture of highly aggressive medulloblastomas in patients with loss and/or deletion of chromosome 17p DNA sequences has considerable clinical significance. A recent study of colorectal carcinoma showed that patients with the greatest number of losses and alterations of chromosomal loci had the worst outcome (Fearon et al., 1987; Law et al., 1988; Baker et al., 1989; Vogelstein et al., 1989). This finding suggests that allelic variations may represent stages of progression to more aggressive biologic be-

ET

AL.

havior, consistent with the multistep nature that has been proposed for carcinogenesis (Cairns, 1981). The correlation of chromosome 17p loss and alteration with a negative prognosis for medulloblastoma suggests that screening of tumor tissue with specific markers may be an important predictive procedure, particularly when the exact location and sequence of the recessive oncogene have been identified. ACKNOWLEDGMENTS The authors thank the following individuals for providing probes: E. Wright, R. White, Y. Nakamura, D. Barker, B. Vogelstein, T. Takeuchi, N. Spurr, A. Ferguson-Smith, S. Weil, P. Chambon, M. Chao, E. Bakker, C. Buys, C. Schwartz, M. Litt, and R. Neve. We also thank Susan Eastwood for editorial advice. This work was supported in part by grants from the School of Medicine, University of California, San Francisco, and the Morris Stulsaft Foundation. Andrew Metzger is a Howard Hughes Medical Institute Medical Student Research Training Fellow.

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YNH37.3 YNz22.1

Y

13.2 HRP5.5 HP53b

13.1

P

MYHP Ew503

12 :

HF12-2

I

YNMW

@

21.3 q

8.

9.

@

Preserved

l

Lost

0

Noninformative 10.

FIG. 2. Map of chromosome 17 demonstrating the common region of deletion in medulloblastoma. The position of each sequence is a consensus of the most recent available data (42,43). The key for interpretation is shown. Adapted from Baker et al. (3) with permission.

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IN MEDULLOBLASTOMA

42.

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