Departments of ^*Neurosurgery, ^**Biochemistry and ^§Neuropathology,
All India Institute of Medical Sciences, New Delhi 110 029, India

†Corresponding author (Fax, 91-11-6862663; Email, parthoaiims@hotmail.com).

Genetic alterations in the p53 tumour suppressor gene of human chromosome 17 have been frequently found to be associated with various tumours. Glial tumours arise from the supporting cells of the brain. They have a poor outcome and often recur. The present study was undertaken to compare the loss of heterozygosity (LOH) of p53 gene in pairs of primary and recurrent gliomas and to try and correlate it to the degree of malignancy and recurrence interval. LOH of p53 was taken as an indicator of the inactivation of p53 due to genetic changes. Ten patients with recurrent gliomas were studied. Three patients had LOH at primary surgery and two of these had LOH at recurrent surgery. One patient had LOH of p53 only at recurrent surgery but not at primary surgery. No indicative correlation was found between p53 heterozygosity status on one hand and grade of malignancy (primary and recurrent) and recurrence interval on the other.

1. Introduction

Gliomas, the commonest of human brain tumours, are histopathologically divided into four grades: grade I to IV; based on morphological evidences of increasing malignancy. However, at the genetic level the whole process of tumourigenesis and its progression involves an interplay between two classes of genes: oncogenes and tumour suppressor genes. Most human malignancies arise after multiple genetic aberrations and each tumour type has a rather unique combination of these genetic changes (Vogelstein and Kinaler 1993). Molecular genetic studies of the common gliomas implicate the epidermal growth factor receptor (EGFR) and platelet-derived growth factor (PDGF) and its receptors as oncogenes and genes on chromosomes 1p, 9p, 10p, 13q, 17p, 19q and 22q as tumour suppressors.

p53 acts as the guardian of the genome, arresting cell cycle after DNA damage, activating DNA damage repair genes and allowing time for repair before the cell progresses through cell cycle, and if the damage is not repairable or proliferative signals are too strong, to push the cell to apoptosis so that the deleterious damages are not passed to future generations. Some of the literature on glial tumours indicates that p53 inactivation is probably an early event in glial tumourigenesis as it is inactivated in about half of low and high grade tumours and has been supported by similar observations in our laboratory (Chattopadhyay et al 1997). In astrocytomas the loss of heterozygosity (LOH) of 17p was found to be relatively frequent (30–50%) and the remaining p53 allele was often (60–70%) simultaneously mutated (von Deimling et al 1992). In a finding at variance with others, Sidransky et al (1992) identified p53 mutations in recurrent tumours. Many of these tumours had resulted from clonal overgrowth of cell having mutated p53 gene in the primary tumours. However, in our experience (Ghosh et al 1994), inactivation of the p53 gene in glioma is compatible with prolonged survival. In light of all these findings and the important role played by p53 in genome protection, a comparison of the heterozygosity status of p53 gene between primary and the recurrent gliomas was studied. LOH of p53 was taken as an indication of the involvement of p53, as widely used in literature (Fults et al 1992; Frankel et al 1993). There were technical difficulties of sequencing the p53 gene of all the paired primary and recurrent tumours as the quality of retrieved template from unbuffered formalin fixed
and paraffin embedded tumours was not up to the mark for sequencing.

2. Methodology

2.1 Collection of samples

2.2 Isolation of WBC from blood

Ten ml of the collected blood was mixed with 5 volumes of ice-cold RBC Lytic buffer (155 mM Tris HCl, 10 mM KHCO₃, 0·1 mM EDTA), kept at 4°C for 1 h and centrifuged at 6000 g for 10 min to pellet the leucocytes. The pellet was then washed once with 5 ml of tissue Lytic buffer (10 mM Tris, HCl, 10 mM EDTA, 50 mM NaCl, pH 7·5), resuspended in 0·5 ml of the same buffer and stored at – 70°C and later sorted during DNA extraction, based on histopathological report.

2.3 Microdissection of embedded tumour samples

Five 5 mm sections of paraffin embedded tumour tissue –both primary and recurrent – of these patients were cut form the blocks available form the archives of the Neuropathology Department. The third section was stained with haematoxylin and eosin. The tumour areas with more than 90% neoplastic cells were marked on the stained slide by an experienced neuropathologist. The marked areas were traced to the other unstained slides after superimposition and the tissue within the area was scrapped and collected in sterile 1·5 ml microcentrifuge tubes.

2.4 Isolation of DNA from WBC and paraffin embedded microdissected tumour tissue

DNA was isolated form the patients’ leucocytes as per the standard technique (Sambrook et al 1989). DNA from the tumour areas scraped form paraffin embedded sections was isolated by a slightly modified procedure. The scraped sections were briefly treated with 0·5 ml of chloroform to dissolve the paraffin. The chloroform was aspirated off, the sections were allowed to dry briefly and 0·5 ml of modified tissue Lytic buffer (500 mM Tris HCl, 20 mM EDTA, 10 mM NaCl, 0·1% Tween 20 and 500 m g/ml proteinase K, pH 9·2) was added to it and the sample was kept at 40°C overnight on a slow revolving vertical turn-table. Further processing was as per standard technique (Sambrook et al 1989). Working solution of DNA was made by diluting a part of the stock with TE to give a final concentration of 100 ng and 200 ng of DNA/ml for leucocyte and tumour respectively. The difference in working concentration of the DNA from the two sources was to give allowance for the degradation of DNA that occurs during fixation and paraffin embedding of the tumour. DNA was initially extracted from 34 pairs of paraffin embedded primary and recurrent tumours. However, DNA extracted from many of the older samples was not PCR amplifiable, possibly because of degradation during routine hospital processing (usage of unbuffered formalin). The analysis could finally be carried out in tumour DNA from 8 males and 2 females.

2.5 Heterozygosity studies of the p53 locus (17p13·1)

Three sites of normal polymorphism have been described in intron 1 and 6 and exon 4 in p53. As the heterozygosity index of these sites taken separately is not very high, heterozygosity status was studied sequentially in the three loci (in the order of exon 4, intron 1, intron 6; depending on their heterozygosity index) till a site demonstrated loss of heterozygosity or the three polymorphic sites were exhausted, whichever occurred earlier.

2.5a At exon 4: The RFLP in exon 4 was studied by PCR amplification of the region by taking 100–200 mg of template and 20 pmol of each primer (GAT GCT GTC CGC GGA CGA TAT T and CGT GCA AGT CAC AGA CTT GGC) in a total volume of 20 ml and cycling 35 times through 1 min at 94°C, – 1 min at 57°C and – 30 s at 72°C. The PCR product was then extracted once with equal volume of chloroform, precipitated by ethanol and dried. The pellet was resuspended in sterile DDW and digested with 2 U BstUI in its buffer at 50°C overnight and analysed by electrophoresis in a 3% agarose gel and EtBr staining.

If the 2 alleles of the exon 4 region of p53 locus of the DNA sample were polymorphic for the BstUI restriction site, 2 sets of band(s) will be visible: the 259 bp set and a (160 + 99) bp set corresponding to the alleles with and without a BstUI, respectively. Often in agarose gels even at 3% concentration the 99 bp band is not well resolved from the excess primers and primer dimer bands.

2.5b At intron 1: The PNRP site in intron 1 was amplified using 20 pmol of each primer ACT CCA GCC TGG GCA ATA AGA GCT and ACA AAA CAT CCC CTA CCA AAC AGC and 1 µl of working template DNA in a total volume of 20 ml under the following conditions: 35 cycles of denaturation at 94°C for 1 min, annealing at 64°C for 1 min and extension at 72°C for 30 s.

The resulting products were electrophoresed in a 15% non-denaturing polyacrylamide gel electrophoresis (PAGE) in TBE buffer (100 mM Tris Borate, 1 mM EDTA, pH 7·8) at 25°C and 30 mA current. After electrophoresis, the gel was stained in EtBr (1 µg/ml) and observed on a UV transilluminator.

Tumour DNA which has lost 1 allele will amplify only the remaining allele while leukocyte DNA from the same patient, if heterozygous, will show both the bands of different sizes. However, if the patient is homozygous (both the alleles are same), then leukocyte DNA will show amplification of a single band, corresponding to both the alleles, after PCR.

2.5c At intron 6: The RFLP in intron 6 was studied in the same way as for exon 4 RFLP except that the primers used were AGG TCT GGT TTG CAA CTG GG and GAG GTC AAA TAA GCA GCA GG, annealing temperature was 60°C and 2 U of MspI in its buffer was used for digestion at 37°C overnight.

The band sizes for the allele with MspI site is 63 and 44 bp and that without the site is 107 bp. Constitutional heterozygosity and LOH in tumours were decided on the basis of the amplified and digested bands given with leukocyte and tumour DNA.

3. Results

The analysis could finally be carried in 8 males and 2 females. The age ranged from 18 to 35 years. Of these ten tumours, three each were in the frontal and parietal lobes, two in the frontotemporal region and one each in the temporal lobe and in the cerebellum. The recurrences were in the same anatomical location as the primaries. The DNA from the patients’ WBC were used to see the constitutional p53 heterozygosity status of the respective patients before commenting on the heterozygosity status of p53 in the tumours.

The time interval between the initial and second surgery ranged from 27 to 54 months (mean 38·25 months) for the group in which there is a change to a higher grade of malignancy while in the group in which the tumour grade remained unchanged at the first and second surgery, the time interval between the surgeries ranged form 8 to 48 months (mean 23 months).

Seven of these ten patients had a low grade glioma at the first surgery while three had a high grade glioma (table 1). Four of the patients from the low grade group were found to have a higher grade of malignancy at recurrent surgery (cases 1 to 4) while the remaining three (cases 5 to 7) had same grade glioma. Two of seven patients of low grade glioma at initial surgery showed a LOH of p53 allele in both primary and recurrent tumours (figure 1). One of these patients (case 4) demonstrated a high grade GBM while the other (case 7) recurred in the same low grade astrocytoma.

All the three patients with a high grade glioma at initial surgery had the same grade of glioma at the time of recurrence surgery (cases 8 to 10). Of these three patients (figure 1), one (case 10) had LOH of p53 in the primary tumour but at the time of recurrence the tumour cells were found to have retained heterozygosity of p53. However, another case (case 8) which was found to be heterozygous for p53 in the primary tumour, was found to have lost it in the recurrent tumour.

There was some intensity variation in the bands in some cases because of variations in template quality and quantity. Only those cases were considered where qualitatively unambiguous amplified bands were seen.

4. Discussion

Though tumours arise from a single malignant cell, during its growth different cells accumulate diverse genetic changes which are passed on to their progeny. Ultimately the tumour becomes polyclonal, with families of cells having different genetic alterations. Of these clones, some may acquire genetic alterations which may give them
a growth advantage over others, so that they outgrow nd can cause recurrences. Cytogenetic and molecular biological studies of adult gliomas have demonstrated frequent and consistent abnormalities in certain genes and chromosomal regions. These have been used to indicate the existence of several genetic pathways leading to the development of GBM. Although GBM is pathologically heterogeneous, it arises from a single cell whose endpoint in transformation is usually marked by loss of tumour suppresser gene on chromosome 10 (Louis and Gusella 1997; Furnari et al 1996). Loss of 17p, usually associated with mutations of the p53 gene, are seen in both low and high grade astrocytomas (Rasheed and Bigner 1991). LOH of an allele on the short arm of chromosome 17 as an indication of p53 inactivation has been documented in 29–50% of gliomas in various series (Fults et al 1989; von Deimling et al 1992; Furnari et al 1996). In our series, it has been seen in 30% of the gliomas at the first and recurrent surgery. Some workers (Fults et al 1992) report nearly no LOH in low grade astrocytomas and a higher incidence in more malignant while others (Frankel et al 1993) report nearly an equal occurrence of LOH in all grades of gliomas. However, these studies used a polymorphic probe, YNZ22, which maps quite far from p53. To overcome this problem we have studied LOH using intragenic polymorphisms of p53.

In this study, of 3 patients (cases 5 to 7) in whom low grade tumours recurred in the same low grade, 2 (cases 5 and 6) had NLOH of p53 both in primary and recurrent tumours with a recurrence interval of 26 and 13 months respectively. However, in 1 (case 7) patient where LOH of p53 was found in both primary and recurrent tumours, the recurrence interval (24 months) was in the above range. Therefore, LOH of p53 alone may not hasten the recurrence and/or increase the malignancy grade, at least in some cases.

Of 3 patients (cases 8 to 10) where both the primary and recurrent tumours were of the same high grade variety, one (GBM, case 9), which had no loss of heterozygosity (NLOH) of p53 in both the primary and recurrent tumours, recurred after just 9 months. In another (GBM, case 10), the clone of cells, which made up probably most of the primary tumour and had LOH of p53, did not show up in the tumour which recurred after just 8 months (recurrence interval in this case comparable to the previous GBM) indicating that p53 alterations were not critical to the recurrence. Moreover, in a case of recurrent malignant astrocytoma (case 8), which had a recurrence interval of 48 months (one of the highest among all the tumours studied), there was NLOH of p53 in the primary tumour but the recurrent tumour had LOH of p53. This further emphasizes the different pathways in glial tumourigenesis and the discrepancy of histopathological and molecular gradings.

Out of 4 patients (cases 1 to 4) who recurred with a higher grade, 3 (cases 1 to 3) had NLOH of p53 both in the primary and recurrent tumours with recurrence interval being 27, 29 and 54 months, respectively; again suggesting that p53 alterations were not central to the increased histological grading of the recurrence. Only 1 (case 4) had LOH of p53 in both the primary (low grade) and recurrent (high grade) tumour with a recurrence interval (43 months) in the above range. This again validates our previous observation about the dissociation of recurrence, histological grading and p53 status.

Though the number of tumours that could be studied is small, this study gives a preliminary indication that p53 alterations may not be critical for the recurrence of a majority of gliomas. This is also supported by our previous observation (Ghosh et al 1994) that p53 LOH did not have any effect on the survival of a patient with a low grade astrocytoma nor was any effect seen on in vivo and in vitro proliferation index as measured by AgNOR and BrDU staining. We have no evidence to support the findings of Sidransky et al (1992) who had demonstrated p53 alterations in recurrent tumours as well as in regions of their primaries.

Some of the genetic culprits that are involved in astrocytic tumourigenesis and progression are: p53 mutation and LOH, PDGF and/or PDGFR overexpression in the genesis of low-grade astrocytoma; RB mutation and LOH, 9p LOH, 19q LOH in the transition from astrocytoma to anaplastic astrocytoma and GBM; and EGFR amplification, 10p loss, 10q loss in de novo genesis of GBM (Louis and Gusella 1997). As other genes are discovered and complex genetic interactions unravelled, their association with tumour progression can be assessed and, coupled with current histopathology and molecular studies, can be used to determine the prognosis more accurately and help in planning strategies for intervention.

MS received 22 January 1999; accepted 4 August 1999

Corresponding editor: Seyed E Hasnain