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Regular Articles |
From the International Agency for Research on Cancer,*
Lyon, France; and the Departments of
Pathology
and
Neurosurgery,
University Hospital,
Zürich, Switzerland
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Loss of heterozygosity (LOH) on chromosome 10 (LOH#10) is the most frequent genetic alteration in glioblastomas and occurs in approximately 80% of cases.6-14 It is less frequent (40%) in anaplastic astrocytomas6-10,12-14 and absent or rare in low-grade astrocytomas.6,8-10,12-14 Most glioblastomas appear to have lost an entire copy of chromosome 10.6-12 In the remaining cases, three commonly deleted loci have been identified, suggestive of the presence of several tumor suppressor genes. These regions include 10p14-pter, observed in about 80% of cases,8,9,12-15 10q23–24, in about 80% of cases,6,7,10-14,16 and 10q25-qter, in about 90% of cases.6,7,10-14
The PTEN tumor suppressor gene identified on 10q23.317,18 has been found to be mutated in glioblastomas at frequencies of about 20%.11,16,19-23 We recently reported that PTEN mutations are common (32%) in primary glioblastomas but rare (4%) in secondary glioblastomas,24 which corroborates the observation of a significant reciprocal correlation between p53 and PTEN mutations in glioblastomas.23 Because more than 50% of glioblastomas with p53 mutations have been reported to show LOH#10,25,26 these neoplasms are likely to exhibit LOH#10 at loci other than PTEN.
The objective of this study was to identify tumor suppressor loci on chromosome 10 that are involved in the genetic pathway leading to secondary glioblastomas. We analyzed LOH#10 using polymorphic microsatellite markers in DNA from microdissected areas showing histologically an abrupt transition from low-grade or anaplastic astrocytoma to glioblastoma, which suggests the emergence of a new tumor clone. Instead of using normal DNA as a control, microsatellite analysis was carried out for each glioblastoma focus in comparison with genomic DNA from the respective, less malignant precursor lesion, ie, low-grade or anaplastic astrocytoma. We also analyzed LOH on chromosome 19, which may contain one or more putative tumor suppressor loci involved in astrocytoma progression.27-32
The results of this study provide evidence that acquisition of genetic alteration during progression from low-grade or anaplastic astrocytoma to a highly anaplastic glioblastoma phenotype, with marked proliferative activity and lack of glial fibrillary acidic protein (GFAP) expression, is associated with loss of a putative tumor suppressor gene on 10q25-qter.
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Materials and Methods |
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Five secondary glioblastoma cases were selected that histologically showed an abrupt transition from low-grade or anaplastic astrocytoma to glioblastoma. These biopsies were from the Departments of Neurosurgery at University Hospital, Zürich, Switzerland (Cases 1–4) and Tübingen, Germany (Case 5). Tumors were fixed in formalin, embedded in paraffin, and classified according to the WHO grading system.33 Under light microscopic observation, the regions showing different malignancy grades (Grades II-IV) were marked and scraped off into an Eppemdorf tube. Genomic DNA was extracted from each area as described previously.34
Case 1
This 28-year-old female patient was diagnosed with a left frontal
anaplastic astrocytoma (WHO Grade III), which was surgically resected.
One and a half years later, the tumor recurred at the same site and was
diagnosed as glioblastoma (WHO Grade IV). In the second biopsy, an
abrupt transition from anaplastic astrocytoma to glioblastoma was
observed histologically. After partial resection of the glioblastoma,
the patient received whole brain radiation therapy with a boost on
tumor bed and margin. Nine months later, the patient showed massive
cervical lymphadenopathy. Histological examination of a supraclavicular
lymph node revealed the presence of a glioblastoma metastasis. The
patient underwent cervical irradiation and chemotherapy but died 1 year
later. Areas of anaplastic astrocytoma, glioblastoma, and adjacent
normal brain tissue from the second biopsy, as well as a lymph node
metastasis, were microdissected for LOH analysis. (Figure 1
, #1).
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This 49-year-old female patient presented with generalized seizures. A right temporo-occipital tumor was subtotally removed and histologically diagnosed as low-grade fibrillary astrocytoma (WHO Grade II). Two and a half years later, the tumor recurred as a cystic lesion with ventricular infiltration and was diagnosed as glioblastoma (WHO Grade IV). Subsequently the patient received cranial radiation therapy but died 3 months later. The second biopsy contained areas with low-grade astrocytoma features and others that fulfilled the histological criteria for the diagnosis of glioblastoma. Both areas and adjacent normal brain tissue were microdissected and analyzed.
Case 3
This 48-year-old male patient presented with persisting headache
and partial complex seizures. A left parieto-occipital tumor was
extirpated and histologically diagnosed as low-grade fibrillary
astrocytoma (WHO Grade II). Four years later, the tumor recurred as
glioblastoma. The patient did not receive radiation therapy and died 4
months after the second operation. Two different areas of glioblastoma
foci (IV1 and IV2) were microdissected from two tumor blocks taken at
the second biopsy. Histologically, the IV1 area consisted of
polymorphic glioblastoma cells, whereas the IV2 area showed a more
homogeneous pattern (Figure 1
, #3). Three different areas of low-grade
astrocytoma, one adjacent and the others farther away from the area
with glioblastoma histology (IV1), were microdissected.
Case 4
Presenting with a history of generalized seizures, this 40-year-old male patient underwent surgery for a right frontal tumor, which was macroscopically well delineated. This tumor showed histologically the features of low-grade astrocytoma with a gemistocytic component (WHO Grade II). Two years later, the tumor recurred as an invasive lesion that infiltrated the ventricular walls and the corpus callosum. The tumor was resected and diagnosed as glioblastoma. Subsequently the patient received radiation therapy. One year later, he displayed low back and leg pain due to spinal dissemination of the glioblastoma. He died of extensive tumor dissemination after 3 months. Areas of low-grade astrocytoma and glioblastoma identified in the second biopsy were microdissected for LOH analysis.
Case 5
This 69-year-old female patient presented with severe headache and
left hemiparesis that had commenced 3 weeks before admission. A right
frontal cystic tumor was totally resected. The patient subsequently
received radiotherapy. The biopsy showed a focally abrupt transition
from low-grade astrocytoma to glioblastoma. The glioblastoma focus
showed brisk mitotic activity and lacked GFAP expression (Figure 1
, #5, center). One year later the tumor recurred at the same site with
invasion of the basal ganglia and deep white matter. The patient
underwent a second operation and received a second cycle of
radiotherapy but showed no clinical improvement. Treatment was
continued at another hospital and further clinical follow-up data are
not available. For LOH analysis, areas of glioblastoma (IV1) and
low-grade astrocytoma (II) and another more distant area of
glioblastoma (IV2) in the first biopsy were microdissected.
Analyses of LOH on Chromosomes 10 and 19 Using Microsatellite Markers
LOH on chromosomes 10 and 19 was studied by polymerase chain reaction (PCR)-based microsatellite analysis. Microsatellite loci on each chromosome were selected to fully cover reported common deletions on 10p14-pter,8,9,12-15 10q23–24,6,7,10-14 10q25-qter,6,7,10-14 19p13.2-pter,30 and 19q13.2–13.4.27-29,31,32 Thirty-seven and nine microsatellite markers were used for analyses on chromosomes 10 and 19, respectively. All microsatellite markers were purchased from Research Genetics (Huntsville, AL). They were dinucleotide repeats except for D10S1435, D10S527, and D19S246 (tetranucleotide repeats). The size range and heterozygosity of each marker was obtained from the Genome Database (http://gdbwww.gdb.org/). Genetic maps and distances of chromosomes 10 and 19 were obtained from the enhanced location databases at ftp://cedar.genetics.soton.ac.uk/pub/chrom 10/gmap and ftp://cedar.genetics.soton.ac.uk/pub/chrom 19/gmap, respectively.35
The allelic losses for each primer set were determined by comparing the
electrophoretic patterns of PCR product of glioblastomas and their
respective precursor lesions (low-grade or anaplastic astrocytoma
areas). PCR was performed according to the instructions of Research
Genetics with minor modification. Briefly, 1 µl of DNA solution was
subjected to PCR with 2 µl of 5x PCR buffer, 200 µmol/L of each
dNTP, 6 pmol each of forward and reverse primer, 0.5 µCi of
[
-33P]-dCTP (ICN Biomedicals, specific
activity 3000 Ci/mmol), 0.225 units of Taq polymerase
(Sigma, St. Louis, MO) and 1.5 mmol/L of MgCl2 in
a final volume of 10 µl. After an initial cycle of 95°C for 2
minutes, 35 cycles of 94°C for 45 seconds, 57°C for 45 seconds, and
72°C for 1 minute were followed by a final extension 72°C for 7
minutes in a Genius DNA Thermal Cycler (Techne, Cambridge, UK). PCR
products were mixed with an equivalent volume of the denaturing
solution containing 95% formamide, 20 mmol/L EDTA, 0.05% xylene
cyanol, and 0.05% bromophenol blue. Immediately after heating at
95°C for 5 minutes, 4 µl of the mixture was loaded onto a 6 or 7%
polyacrylamide/7 mol/L urea sequencing gel. Gels were run at 70W for
3–5 hours, depending on the length of the products, dried at 80°C,
and autoradiographed for 48–96 hours. The signal intensity of
each allele on the X-ray film was measured by densitometry (Bio-Rad
model GS-670). LOH was assumed when the signal intensity of the allele
in the glioblastoma focus was less than 50% of that in the reference
DNA (normal brain, low-grade, or anaplastic astrocytoma).
PCR-Single-Strand Conformation Polymorphism (SSCP) Analysis and Direct DNA Sequencing for p53 and PTEN Mutations
Prescreening for mutations was carried out by PCR-SSCP analysis34 of exons 5–8 of the p53 gene for all cases and of exons 1–9 of the PTEN gene for cases 1–4.36 PTEN mutations were not analyzed in Case 5 because of the limited amount of tumor tissue available. Samples that showed mobility shifts in the SSCP gels were further analyzed by direct DNA sequencing as previously described.34
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Using 37 microsatellite markers, we examined a total of 285
polymorphic loci on chromosome 10 and obtained 178 informative results
(62%). Loss of an entire copy of chromosome 10 was not found in any of
the glioblastomas analyzed. Among 8 glioblastoma foci analyzed in
biopsies from 5 patients, 7 showed partial LOH#10 at 10q25-qter distal
to D10S597, covering the DMBT137
and
FGFR2 loci38
(Figure 2)
. Six of the 8 glioblastoma foci showed
LOH at one or two flanking markers for PTEN, ie, D10S215 and
D10S541.17
LOH on 10p was found in only 2 glioblastoma
foci from one patient (Figure 2
, Case 5).
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. In Case 2, an area of peritumoral normal brain was also microdissected and analyzed. The allelic patterns of normal brain tissue were identical to those of the low-grade astrocytoma.
In Case 3, one glioblastoma focus (IV1) showed LOH on 10q25-qter,
whereas another glioblastoma focus (IV2) did not show LOH with any of
the microsatellite markers used (Figures 2 and 3)
. Three areas of low-grade astrocytoma
analyzed in Case 3 all showed the same allelic patterns.
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Using 9 microsatellite markers, a total of 68 polymorphic loci were examined, covering chromosomal regions 19p13.2-pter and 19q13.2–13.4 and yielding 49 informative results (72%). Except for one glioblastoma focus (Case 3, IV1), there was no apparent LOH in any of the regions analyzed.
Mutations in the p53 and PTEN Genes
PCR-SSCP followed by direct DNA sequencing revealed two p53 missense mutations and one deletion. In all cases, the mutations were already detected in the first biopsy. The mutations had the following locations: codon 275 (TGT->TAT, Cys->Tyr, glioblastoma focus and metastasis of Case 1), codon 278 (CCT->ACT, Pro->Thr, Case 2) and codon 275–279 (15-bp deletion, Case 4). The mutation data of Cases 2 and 4 have previously been published.34 PCR-SSCP analysis for PTEN mutations (exons 1–9) revealed no mobility shift in any of the tumor foci analyzed.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, #5, center). Such foci resemble the focus-in-focus lesion or
carcinoma-in-adenoma phenomenon described in experimental
hepatocarcinogenesis42-44
and human liver
tumors45
and are likely to be caused by the acquisition of
an additional genetic alteration. We observed five such cases and used
microsatellite analysis to determine whether this sudden change of
morphology is due to LOH on chromosomes 10 and 19. Rather than
comparing the allelic pattern with that of normal DNA, we aligned the
microsatellite patterns of the glioblastoma foci with those of the
adjacent, less malignant precursor lesion, ie, low-grade or anaplastic
astrocytoma. The results obtained provide initial evidence that acquisition of the glioblastoma phenotype during astrocytoma progression is associated with loss of a putative tumor suppressor gene on 10q25-qter, covering the DMBT137 and FGFR2 loci.38 The DMBT1 gene was originally identified from a homozygously deleted region in a medulloblastoma cell line and showed homology to the scavenger receptor cysteine-rich superfamily.37 Although allelic losses and homozygous deletions at DMBT1 were detected in 59% and 23% of glioblastomas, respectively,37 no mutation has been reported so far. FGFR2 encodes fibroblast growth factor receptors and is considered a potential tumor suppressor gene because decreased expression of its two isoforms, FGFR2c (BEK) and FGFR2b (KGF-R),46 have been demonstrated in glioblastomas47 and transitional cell carcinomas of the bladder.48 However, FGFR2 mutations have not yet been identified in human neoplasms.
Loss of an entire copy of chromosome 10 has been observed in a majority of glioblastomas.6-12 However, none of the glioblastoma foci analyzed in this study showed loss of an entire copy of chromosome 10. The possibility exists that partial deletion on chromosome 10 is typical for secondary glioblastomas progressing from low-grade or anaplastic astrocytoma, whereas large deletions and loss of the entire chromosome are prevalent in primary (de novo) glioblastomas.5 Alternatively, newly emerged glioblastoma foci may ultimately become the prevailing tumor cell fraction and may, in the process, develop larger chromosomal deletions due to increased genetic instability.
LOH at the PTEN locus has been found in up to 80% of
glioblastomas,6,7,10-13,16
whereas PTEN
mutations have been detected in less than 25% of
cases.11,16,19-23
In the present study, none of the 6
glioblastoma foci analyzed contained a PTEN mutation,
although 6 of 8 glioblastoma foci showed LOH at one or two of the
flanking markers of PTEN (Figure 2)
. This supports the
observation that mutational loss of PTEN function is rarely
involved in the genetic pathway to secondary
glioblastoma.24
However, other mechanisms of
PTEN inactivation, eg, promoter hypermethylation or
homozygous PTEN deletion, cannot be ruled out.
PTEN promoter hypermethylation has not been found in human
tumors including prostate cancer, bladder cancer, and renal cell
carcinomas,49,50
but data on brain tumors are not
available. Homozygous PTEN deletion appears to be rare in
glioblastomas.16,19,21,24
Deletion on 19q13.2–13.4 has been found in a variety of gliomas,
including low-grade astrocytomas (<20%), anaplastic
astrocytomas (~40%), and glioblastomas
(~30%),27,28,31,32
as well as in oligodendrogliomas
(~70%) and mixed oligo-astrocytomas
(~70%).27,28,31,32,51
There is limited
evidence for the occurrence in gliomas of LOH at a second locus on
19p13.2-pter.30
In this study, LOH on 19q13.2–13.4 was
observed in only 1 of 8 glioblastoma foci analyzed (Case 3, IV1, Figure 2
), suggesting that 19q13.2–13.4 and 19p13.2-pter loci are not
typically involved in the acquisition of the glioblastoma phenotype
during progression from low-grade or anaplastic astrocytoma. It remains
to be shown whether LOH on chromosome 19 occurs at a later stage than
LOH on chromosome 10 in secondary glioblastomas, or whether LOH on
chromosome 19 is more typically associated with the evolution of
primary (de novo) glioblastomas.
Little is known about the effect of radiation therapy on genetic alterations in glioblastomas. Hulsebos et al52 showed that no additional LOH at p16 and Rb loci was found after irradiation. In many other cases, additional genetic alterations were detected in the absence of radiotherapy.52 The present study is largely noninformative because, except in Case 3, all patients received radiotherapy only after the histological diagnosis of glioblastoma. The lymph node metastasis of Case 1 was resected 9 months after cranial irradiation and showed the same LOH pattern on chromosomes 10 and 19 as that of the parent glioblastoma.
Glioblastomas are histologically and biologically heterogeneous, but it
is largely unknown whether this is due to a polyclonal development of
glioblastomas from less malignant, monoclonal precursor lesions or to
genetic instability after the acquisition of the glioblastoma
phenotype. In two cases of this study (Cases 3 and 5), we analyzed two
glioblastoma foci separately. In Case 3, one glioblastoma area showed
LOH at 14 markers on 10q and 19q, but the other glioblastoma area
retained heterozygosity at all of these loci, suggesting that
progression from low-grade astrocytoma to glioblastoma can be
polyclonal. In the biopsy of Case 5, one glioblastoma area showed LOH
on 10p13, 10q23, and 10q25-qter, whereas another glioblastoma area
showed an additional deletion on 10p14. This could signify that both
glioblastoma foci originated from the same tumor clone, but that an
additional deletion occurred in only one area (Figure 2)
. Similar
examples of genetic heterogeneity have been demonstrated on chromosome
8 during prostate cancer progression.53
In conclusion, the present study on secondary glioblastomas with a histologically sudden transition from low-grade or anaplastic astrocytoma indicate that the acquisition of the glioblastoma phenotype is typically associated with LOH at 10q25-qter but not on 10p, 10q23, or 19.
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Acknowledgements |
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Footnotes |
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Supported by a grant from the Foundation for Promotion of Cancer Research, Japan.
Accepted for publication April 13, 1999.
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