LETTERS TO NATURE
`
`Clonal expansion of p53 mutant
`cells is associated with
`brain tumour progression
`David Sldransky*, Tom Mikkelsent,
`Karl Schwechhelmer:j:, Mark L. Rosenblum§,
`Web Cavaneet & Bert Vogelstein*ll
`
`" The Department of Oncology, The Johns Hopkins University School of
`Medicine, Baltimore, Maryland 21231, USA
`t Ludwig Institute for Cancer Research, Montreal, Province of Quebec,
`H3A 1A1, Canada
`; Abteilung Neuropatologl Pathologlscher lnstltut,
`Albert Ludwigs Universitat, Frieberg, Germany
`§ Brain Tumor Research Center, Department of Neurological Surgery,
`University of California Medical Center, San Francisco,
`California 94143, USA
`
`TUMOUR progression is a fundamental feature of the biology of
`cancer'. Cancers do not arise de novo in their final form, but begin
`as small, Indolent growths, which gradually acquire characteristics
`associated with malignancy. In the brain, for example, low-grade
`tumours (astrocytomas) e~olve into fa.'ltcr growing more dysplastic
`and invasive high-grade tumours (gliublas!omas)b. To define the
`genetic events underlying brain tumour progression, we analysed
`the p53 gene in ten primary brain tumour pairs. Seven pairs
`consisted of tumours that were high grade both at presentation
`and recurrence (group A) and three pairs consisted of low-grade
`tumours that had progressed to higher grade tumours (group B).
`In group A pairs, four of the recurrent tumours contained a p53
`gene mutation; In three of them, the same mutation was found In
`the primary tumour. In group 8 pairs, progression to high grade
`was associated with a pS3 gene mutation. A subpopulation of cells
`were present in the low-grade tumours that contained the same
`pS3 gene mutation predominant in the cells of the recurrent
`tumours that had progressed to glioblastoma. Thus, the histologi(cid:173)
`cal progression of brain tumours was associated with a clonal
`expansion of cells that had previously acquired a mutation In the
`p53 gene, endowing them with a selective growth advantage. These
`experimental observations strongly support Nowell's clonal
`evolution model of tumour progression4
`•
`Brain tumours, like most other tumour types, are associated
`with several genetic changes. Among these, loss or mitotio re(cid:173)
`7
`combination of chromosome 17p is common 5-
`• Furthermore,
`10
`in several glioblastomasK, as in many other tumour typcs9•
`,
`loss of one I7p allele correlates with mutation of p53 in the
`remaining allele. To evaluate the relationship between p53 and
`astrocytoma progression, tumour pairs were obtained from
`patients with brain tumours that had been surgically removed.
`Tumours were carefully dissected to ensure optimal removal of
`contaminating normal tissue. A portion of the p53 gene, includ(cid:173)
`ing exons 5-8, was amplified by the polymerase chain reaction
`(PCR) and subcloned into a phagemid vector; pooled clones
`were sequenced as previously described 11
`• Four group A tumours
`contained p53 mutations at recurrence and, in three, the identical
`p53 mutation was seen at initial presentation (Table 1 ). All were
`missense mutations resulting in nonconservative amino-acid
`changes. From the ratio of wild-type to mutant p53 alleles in
`the sequencing gels, it was clear that all group A tumours with
`p53 gene mutations had lost the wild-type allele.
`All three of the advanced tumours from group B (Fig. 1) had
`a p53 gene mutation. Again, all mutations were missense muta(cid:173)
`tions resulting in nonconservative amino-acid changes (Table
`1). Two of the three high-grade tumours (from patients 88 and
`89) had lost the wild-type allele. No evidence of these mutations
`was observed in the less advanced tumours of the pairs in the
`sequencing assays. But were these mutations present in a small
`
`II To whom correspondence shoulli be addressed
`
`846
`
`fraction of cells in the initial tumour, in a proportion not
`detectable by sequencing? We used a sensitive plaque assay to
`identify a potential cell minority containing p53 mutations
`among a majority of cells with normal p53 genes. The p53 gene
`was amplified through PCR as described above and the PCR
`products subcloned into a phage vector. Specific oligonucleo(cid:173)
`tides that recognize a mutant base pair in the p53 gene were
`then used to probe filter lifts of the phage plaques.
`Specific oligonucleotides were synthesized that would detect
`the mutations seen in the tumours of patients B8 and 89 and
`used to probe the cloned PCR products of the low grade tumours .
`A small number of positive plaques were seen in the low-grade
`tumours (Fig. 2). Positive plaques were picked and the phage
`DNA sequenced to confirm the presence of the mutation. Blots
`were then stripped and hybridized with a probe detecting all
`p53 clones (mutant plus wild type) to calculate the percentage
`of mutant p53 genes in the low-grade tumours. This showed
`that 8% and 21% of the tumour cells from patients 88 and 89,
`respectively, contained a mutant p53 gene. The high-grade
`tumours from both patients consisted of cells in which over 95%
`contained mutant p53 genes as assessed by the same assay.
`Histological sections were examined and all tumours were found
`to be contaminated by <10% noma! cells (Fig. 1).
`The low-grade component of the tumour from patient 810
`contained a p53 mutation at codon 253 and a light mutant band
`at codon 273 when pooled clones were sequenced, whereas the
`high-grade component was found to contain mutant bands of
`equal intensity at codons 253 and 273 (Table 1 ). After subcloning
`
`FIG. 1 Micrographs of tumours from patients in group B. The tumour from
`patient 10 had two distinct patterns (a and b). Most of the neoplasm was
`moderately cellular with a dense feltwork of glial processes. The nuclei of
`the glioma cells in a were mildly to moderately pleomorphic. Mitotic figures
`were rare and no vascular endothelial proliferation was identified. This region
`was classified as a moderately anaplastic astrocytoma. Sharply demarcated
`from this region and Invading the leptomeninges was a more cellular
`component of the neoplasm with larger nuclei, nuclear pleomorphism and
`multiple mitotic figures (b, small arrows). The glial feltwork was less exten(cid:173)
`sive and vascular endothelial proliferation (large arrow) was evident. This
`latter region was classified as glioblastoma multiforme. Both regions were
`glial fibrillary acid protein (GFAP) immunopositive, an indicator of their glial
`origin. The tumours in c and e also had features of low-grade glial tumours,
`whereas those in d and f had features of glioblastoma multiforme. a, c.
`and e, Initial low-grade tumours from patients B8, B9, and B10, respectively;
`b, d. and f. high-grade tumours from the same patients.
`
`NATURE · VOL 355 · 27 FEBRUARY 1992
`
`«:> 1992 Nature Publishing Group
`
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`

`

`Patient
`
`Initial
`tumour grade
`
`Initial tumour mutation•
`
`Time to
`progression
`(months)
`
`Progressed
`tumour
`grade
`
`TABLE 1 Mutations In each tumour
`
`Group A
`81
`82
`83
`84
`85
`86
`97
`Group B
`B8
`89
`810
`
`IV
`IV
`IV
`IV
`IV
`IV
`IV
`
`I
`II
`
`Codon 173 GTG-+ ATG, Val-+ Met
`Codon 244 GGC -+ AGC, Gly--> Ser
`Codon 248 CGG .... TGG, Arg-+ Trp
`None
`None
`None
`None
`
`None
`None
`Codon 253 ACC-> ATC, Thr--> Leu
`
`7
`13
`9
`11
`5
`9
`7
`
`15
`28
`0
`
`IV
`IV
`IV
`IV
`IV
`IV
`IV
`
`IV
`IV
`IV
`
`LETTERS TO NATURE
`
`Progressed tumour mutation
`
`Codon 173 GTG-+ATG, Vai-+Met
`Codon 244 GGC--> AGC, Gly .... Ser
`Codon 248 CGG-+ TGG, Arg .... Trp
`Codon 281 GAG-+ GAA, Asp-> Glu
`None
`None
`None
`
`Codon 27 3 CGT-> TGT, Arg .... Cys
`Codon 270 Tn-> Ctt, Phe .... Leu
`Codon 253 ACC-> A TC, Thr -+ Leu
`Codon 273 CGT-> TGT, Arg-+ Cys
`
`Group A tumours consisted of glioblastoma at both presentation and recurrence, whereas group B tumours were low-grade (I and 1\) at presentation and
`high-grade (Ill and IV) at recurrence. DNA was isolated from tumours and exons 5-8 of the p53 gene were amplified by the PCR, subcloned Into lambda
`Zap, and sequenced as described previously11 . The tumour from patient 810 contained two components, histologically and mechanically separable, from
`the same tumour.
`* As assessed by sequencing (see text).
`
`amplified DNA, individual clones were sequenced and these
`two mutations were found to reside on separate alleles. Using
`the oligonucleotide-specific hybridization method described,
`
`Low grade
`
`Tumour
`
`High grade 0 Coni 0 Coni
`
`FIG. 2 Clonal expansion of p53 mutant cells. Over 95% of the clones obtained
`by PCR from low-grade and high-grade tumours in patients 98 (top) and 99
`(bottom) hybridized to an oligonucleotide probe detecting all p53 clones
`(Tot). An oligonucleotide probe specific for mutant p53 at codon 273
`hybridized to only a small number of plaques (8%) In the \ow-grade tumour
`from patient 98 (top left), but hybridized to the majority of plaques (99%)
`in the high-grade tumour. A mutant-specific oligonucleotide probe for the
`high-grade tumour in 99 also hybridized to 21 o/o of clones in the low-grade
`tumour (bottom left) and 95% in the high-grade tumour. Neither of the
`mutant-specific probes hybridized to control plaques (far right) from another
`glioblastoma which contained a p53 mutation at a different codon.
`METHODS. Tumour DNAs were amplified using PCR, ligated to lambda Zap
`arms (Stratagene), packaged, plated at a density of 200-500 plaques per
`plate, and the plaque DNAs transferred to nylon membranes as described11
`.
`Mutant-specific o\igomers corresponding to the mutant sequences in the
`high-grade tumours were synthesized to recognize a mutation at codon 273
`(5' -TIGAGGTGTGTGTTIGTG-3') for tumours of patient 98 and 910; a mutation
`at codon 270 (5' -GGAACAGCCTIGAGGTGC-3') for tumours in patient 89; and
`a mutation at codon 253 (5'-CATCCTCATCATCATCAC-3') for the tumour in
`patient 910. As a control, the filters were rehybridized to an oligonucleotide
`probe for the wild-type sequence at codons 243 to 248 (5'-ATGGGCGGCAT(cid:173)
`GAACCGG-3'), which would identify all p53 clones (mutant or wild type, Tot).
`The oligomers were labelled with [32P]ATP and T4 kinase to a specific activity
`of 5 x 108 d.p.m. Hybridization with o\igomers was at 45 oc for 1 h. After
`hybridization, the membranes were rinsed in 3 xSSC (450 mM NaCI, 18 mM
`sodium citrate, 1 mM Tris, pH 7.2), 0.1% SDS at room temperature for 5 min
`and then washed at 2-5 •c below the calculated melting temperature. The
`blots were then briefly dried and exposed to film . For the one-allele tumours
`from patients 88 and 89, the percentage of mutant cells was calculated by
`counting the mutant clones and dividing this number by the total number
`of clones that contained p53 sequences. we assumed that the subset of
`cells in the \ow-grade tumours which contained a p53 gene mutation also
`had a concomitant deletion of the wild-type allele. as was found in the
`high-grade tumours of these patients.
`
`NATURE · VOL 355 · 27 FEBRUARY 1992
`
`phage plaques from low- and high-grade components were
`probed with an oligonucleotide specific ror the codon-253 muta(cid:173)
`tion, an oligonucleotide specific for the 273 mutation, and a
`normal oligonucleotide detecting all p53 clones (mutant and
`wild type). This analysis revealed that 60% of the cells in the
`low-grade component contained one allele which was wild type
`and one allele that was mutant at codon 253, whereas both
`alleles were mutant (one at codon 253 and one at codon 273)
`in 40% of the cells. Virtually all of the cells in the high-grade
`component contained
`two mutant alleles. Thus progres(cid:173)
`sion appeared to be associated with mutation of the second
`allele at codon 273 in a cell with a pre-existing mutation in the
`first allele.
`Nowell postulated that a cell acquiring a genetic change might
`acquire a selective growth advantage•. Clonal expansion of this
`cell, driven b.,Y succes ive mutation , would lead to tumour
`progression•·•-- Although the occurrence of evcral mutations
`1
`1
`has been observed in human tumours 13
`, evidence for clonal
`·
`'
`expansion of a subpopulation of cells with a specific endogenous
`mutation has until now not been available. The data presented
`here suggest that mutation of p53 leads to a selective growth
`advantage in vivo that seems to be a critical step in transforma(cid:173)
`tion from low-grade to high-grade tumours. This provides a
`direct experimental demonstration of clonal expansion in a
`human tumour: a rare cell carrying a specific change in a critical
`gene became the dominant cell type as the tumour progressed.
`This concept will probably be useful in understanding the role
`of various oncogenes and suppressor gene mutations in the
`pathway to malignancy.
`0
`
`Received 8 November; accepted 12 December 1991
`1 Foulds, L Neap/liStie Development Vols 1 and 2 (Academic, London, 1969 and 1975)
`2 SCherer, H J A mer. J. Cancer 40, 159-198 (1940).
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`(Williams and Wilkins, Baltimore, 1977)
`4 . Nowell, p_ Science 194, 23-28 (1976).
`5. James, C. D ot ol. Cancer Res. 48, 5546- 5551 (1988)
`6 Blgner. 5 H era/. Cancer Res 48, 405-411 (1988).
`7 James, C. D eta/. Proc. naln. Acad. Set. U.S.A 86, 2858-2862 (1989).
`8_ Nigro, J. M et a/. Nature 342, 705-708 (1989)
`9 Baker, S. J. et at. Science 244, 21/- 221 (1989)
`10. Hollstein, M , Sidransky, 0 , Vogelstein, B. & Harris. C. C Science 263, 49-53 (19911.
`11. Sidransl<y, D. er at. Science 2112, 706- 709 (19911
`12 Kerbel R. S. Adv. Cancer Res 56, 87-132 (19901.
`13 Bishop. J. M Cell 64, 235-248 (1991).
`14. Fearon. E. R & Vogel stein, B Cell 81, 759-767 (1990)
`
`ACKNOWLEDGEMENTS We thari< S J Armond for photomicrographs and pathological description of
`the tumour In patient 810. J T Vogel stein for reading, and T. Gwiazda for preparation of the manuscript.
`Supporled by the Preuss Foundation and the NIH.
`
`847
`
`cC 1992 Nature Publishing Group
`
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