Transcript Genetic Alteration of p53 in Brain Astrocytic Tumors

Genetic Alterations of TP53 Gene in
Brain Astrocytic Tumours
Ammira H Al-Shabeeb
Hypothesis
University of Sydney
Brain Astrocytic tumours could be caused by
multiple factors including continuous exposure to a
chemical, radiation or due to genetic factors. On
the molecular level there are certain genetic
mutations can occur. One of the most commonly
mutated genes is the TP53 which was investigated
in this study.
Θ Eighty-three brain tumor biopsies were
collected and used in this study. Thirty were
diagnosed as brain astrocytoma as
confirmed
by
the
histopathological
examination. H&E slides were prepared for
the
paraffin
embedded
blocks
for
histological typing and grading.
Θ Specimens from thirty patients with
astrocytoma
were
selected
to
be
investigated in more details. All had
Astrocytic brain tumors of different grades
and stages. Sex and age matched
individuals were also included as a healthy
controls.
Θ Genomic DNA was isolated from fresh
frozen tissues and formalin-fixed paraffin
embedded tissue blocks.
Θ PCR-SSCP Analysis was used to
determine the frequency of TP53 mutations
within exons 5 -9 in astrocytoma patients.
Θ Results were analysed using statistical
and genetic methods.
Sequences
5' ACC CTG GGC AAC CAG CCC TG 3'
5' TTT ATC TGT TCA CTT GTG CCC 3'
5' CTC CCA GAG ACC CCA GTT GC 3'
5' TCA GAT AGC GAT GTG AGC AG 3'
5' CAG TGT GCA GGG TGG CAA GT 3'
5' GCC ACA GGT CTC CCC AAG GC 3'
5' CCA CCG CTT CTT GTC CTG CT 3'
5' GAC CTG ATT TCC TTA CTG CC 3'
5' GGA GAC CAA GGG TGC AGT TAT 3'
5' GTT AGT TAG CTA CAA CCA GGA GCC 3'
MW
6128
6409
6103
6286
6318
6128
6051
6099
6120
6308
bp
152
167
132
136
Table 1: TP53 Single Strand
Conformational
Polymorphism
SSCP primers sequence , exons
5, 6, 7, 8, and 9 respectively.
Faculty of Medicine
Children’s Hospital at Westmead
Molecular Pathology Department
This study investigated the genetic alterations of TP53 in a group
of patients with brain astrocytoma from Iraq form 2001-2004. A total of 30 patients with WHO
grade I, II, and IV astrocytoma were studied (24 males and 6 females). The mean age was
52 ± 4.55 years. TP53 mutations were detected in 76.7% of astrocytoma. Mutations occur
most frequently at exon 5 (16/23, 69%), followed by exon 7 (15/23, 65.2%), exon 6 (14/23,
60.9%), exon 8 (9/23, 39.1%) and exon 9 (4/23, 17.4%). Multiple mutations among TP53
exons 5-9 in the same examined samples were found in 73.9% of astrocytoma patients. Our
results further showed that there were strong age, sex differences and the number of TP53
mutations (p< 0.01), the old age group of patients 50-85 years have increased rate of TP53
exons mutations compared with other age groups. Male sex significantly different with the
mean number of TP53 mutations than the female sex (3.2 folds). There is association
between TP53 mutations and tumour grade, G IV shows the higher mutation rates (15/15,
100%). In the case of tumour location, 80% of astrocytoma were supratentorial in location
classified as G IV and 20% were G II and infratentorial in location. This indicate that TP53
mutations may serve as a potent prognostic factor for astrocytoma outcome together with
the age and tumour grade.
Variables No
MALE
24
Age
TP53 Mutations
FEMALE
6
Age
TP53 Mutations
Mean ± SE a Median SD b Mini Value Maxi Value 99% CI c Correlation Value
52.8 ± 5.01
2.08 ± 0.29
50
2
32.8 ± 11.6
0.66 ± 0.21
24
1
25
1.47
6
0
28.4
0.51
85
4
6
0
80
1
38.2-66.8
1.25-2.90
0-75.7
0-1.43
Astrocytoma's
Histopathology
Grades
-0.012
GI
G II
** - 0.88
G IV
p < 0.001
p < 0.001
a= SE of Mean b= SD, c = CI,
C
** Highly Significant
Exon 5
Total Number of
TP53 Mutated
Exons
Total Number of TP53
Non-Mutated
Exons
Total Number of
TP53 Exons
O - Value
5
2.8
18
20.8
33
30.5
0
0
52
48.2
42
43.9
5
56
94
O a
E b
O
E
O
E
Total Number of
TP53 Exons
( O - Value)
A
C
70
75
150 *
X2= 2.257 (P > 0.05) DF = 2
Exon 6
Table 5: Relationship between astrocytoma grades and total
number of TP53 mutated and non-mutated exons. There is no
association between TP53 mutations and tumor grade (p>0.05).
There is close similarity between observed and expected numbers
of mutations, this reflect that the exons number and how many
mutation times they are presented play a role in tumors
progression.
Exon 7
Exon 8
Exon 9
Wild Type
A
a
Standard Error
52.3 ± 4.55
50.1
24.9
Jun-85
b
C
24 - Six
3
1
Standard Deviation
Results
Patients
1/1 50 - 85
WHO GI
TP53 +ve 20 - 50
1 - 20
WHO GII
14 50 - 85
TP53 +ve /7 20 - 50
TP53 -ve /7 1 - 20
WHO GIV
15 50 - 85
TP53+ve /15 20 - 50
1 - 20
Mutations
0/1
1/1
0/1
4/7 (57.1%)
1/7 (14.3%)
2/7 (28.5%)
8/15 (53.3%)
7/15 (46.7%)
0/15
Exon 5
0
1/1
0
2/4 (50%)
1/1
2/2
5/8 (62.5%)
5/7 (46.7%)
0
Exon 6
0
1/1
0
2/4 (50%)
0/1
1/2
6/8 (75%)
4/7 (57%)
0
Exon 7
0
1/1
0
3/4 (75%)
1/1
1/2
4/8 (50%)
5/7 (71.4%)
0
Exon 8
0
1/1
0
1/4 (25%)
1/1
1/2
4/8 (50%)
1/7 (17%)
0
Exon 9
0
1/1
0
0/4
1/1
1/2
1/8 (12.5%)
0/7
0
Conclusions
D
Θ TP53 mutations were significantly
involved in brain astrocytoma.
E
Tumor Types
Astrocytoma
GI
G II
G III
G IV
Meningioma
Pituitary Adenoma
Oligodendroglioma
Medulloblastoma
Haemangioblastoma
Ependymomas
Schwanoma
Craniopharyngioma
E
Table 6: Correlation between TP53 Mutations, tumor grade and
patients age. There was strong correlation between TP53
mutated exons and certain age group within astrocytoma grade.
Mutations in exon 7 were detected in 75% of old age group
within GII, but with G IV showed 53.3% within same age group.
B
Table 2: Demographics of astrocytoma patients. Thirty patients, 24
male , 6 females, mean age 52.3± 4.55 years, 3 patients with
recurrent astrocytoma and one patient with family history of brain
astrocytoma WHO grade IV.
Variables
D
B
Patients (No= 30)
Age
Mean ± SE a
Median
SD b
Range
Sex
M : F
Recurrent
Family History
astrocytoma- is a brain tumour that
arises from star-shaped glial cells called
astrocytes. astrocytoma can invade
surrounding healthy brain tissue. The
grade of tumour refers to the stage of the
disease and can be distinguished
morphologically under a microscope.
Cells from high –grade tumours look
more abnormal and grow faster (Figure
1A- E)
a = Observed Value, b = Expected Value, 150*= 30 patients × 5 TP53 Exons = 150 Exons, DF = Degrees of Freedom
Table 4: Association between mutated TP53 and patient age and
sex. The mutation rate in males was 3.2 folds. The 99%
confidence interval for the effect of female age and number of
TP53 mutations was expanded from 0-75.7 for age and 0-1.43
for the number of mutations. There was a highly significant
differences in the number of mutations in males (2.08) than in
females (0.66), p<0.001.
Figure 2: Hematoxylineeosin (H&E) stain of Astrocytic brain
tumors. A: WHO Figure 2: Grade I low grade astrocytoma, B: WHO
Grade II low grade astrocytoma, C: WHO Grade IV astrocytoma,
glioblastoma multiforms.
Variables
Suppressor genes such as TP53 are
essential for cell functions. Mutations in
the TP53 gene can result in the onset of
certain cancers. The TP53 gene has
been mapped to chromosome 17.
Genetic alteration (mutations) in this
genes can either be inherited or
accumulated due to environmental
factors.
Results
P value
B
Background
ABSTRACT:
149
Results
A
To determine the genetic alterations in
TP53 in patients with brain astrocytoma
from 2006 to 2008.
Corresponding Author: [email protected]
Methodology
Primers
E5 Forward
E5 Reverse
E6 Forward
E6 Reverse
E7 Forward
E7 Reverse
E8 Forward
E8 Reverse
E9 Forward
E9 Reverse
Objectives
Patient Number
No = 83, N %
Age / Y
Mean
38 (45.8)
1 (2.6)
14 (36.8)
8 (21.1)
15 (39.1)
16 (19.3)
5 (6.0)
5 (6.0)
5 (6.0)
5 (6.0)
4 (4.8)
3 (3.6)
2 (2.4)
/
50.9
53.3
52.2
Range
/
6-8
20 - 77
30 - 85
Figure 3 : PCR-SSCP analysis of TP53 in Astrocytoma. Exons 5, 6, 7, 8 and 9 of TP53 gene were amplified using genomic DNA
extracted from tumor biopsy as a template. Electrophoresis was carried out in a 2.5% agarose gel at 9.5 V/cm for 45 minutes and
stained with ethidium bromide. Normal bands (non-mutated exon) distinguished from abnormal (shifted mutated exon) bands. (A)
PCR-SSCP analysis results of patients 17, 27, 26, 13, and, 22 for exons 5, 6, 7, 8, and, 9 respectively. (B) PCR-SSCP analysis
results of patients 10, 11, 12, 14, and, 30 for exons 5, 6, 7, 8, and, 9 respectively. (C) PCR-SSCP analysis results of patients 24, 25,
28, 29, and 16 for exons 5, 6, 7, 8 and, 9, respectively. (D) PCR-SSCP analysis results of patients 18, 19, 16, 21 and, 15 for exons 5,
6, 7, 8, and 9, respectively. (E) PCR-SSCP analysis results of patients 1, 5, 6, 4, and, 7 for exons 5, 6, 7, 8, and, 9, respectively, wild
type TP53 exons 5, 6, 7, 8, and 9( right side).
Table 3: Histopathological data of astrocytoma patients. Eightythree brain tumor biopsies, 45.8% astrocytomas, 19.3%
meningioma, 34.9% all other brain tumor types . The mean age of
patients was 50.9, 53.3, and 52.2 years for WHO grade II, III, and
IV, respectively.
Acknowledgement
Dr. Hassan M. Naif
Molecular Pathology Head , Children’s Hospital @ Westmead
Sydney, NSW
P*
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
TP53 Mutated
Exons
6,8
5,7
5,7,8,9
WT
6
6
6, 8
5,7
5,7
6
5,6,7,8
5,6,7
WT
5
WT
5,6,7,8,9
WT
5,6,7,9
WT
5,7
7
5,9
7
5,6,7,8
5,6,7,8
WT
WT
5,6,7,8
5,6,7,8
5,6
Astrocytoma
Histopathology
Grades
GIV
GIV
GII
GII
GIV
GII
GIV
GII
GIV
GIV
GIV®
GIV®
GII
GIV
GII
GI
GII
GIV
GII
GIV
GII
GII
GIV
GIV
GII
GII
GII
GII
GIV®
GIV
Astrocytoma
Location
Supra**
supra
Infra***
Supra
Supra
Infra
Supra
Supra
Supra
Supra
Supra
Supra
Supra
Supra
Infra
Supra
Supra
Supra
Infra
Supra
Supra
Supra
Supra
Supra
Supra
Supra
Infra
Infra
Supra
Supra
TP53 Exon Mutation
Frequency in 23
Patients
Exon 5
16/23
TP53 Exon Mutation
Frequency in GIV/15
Patients
Exon 5
10/15
69%
Exon 6
14/23
Exon 5
5/14
66.70%
Exon 6
10/15
60.90%
Exon 7
15/23
66.70%
65.20%
60%
39.10%
Exon 9
4/23
33.30%
Exon 9
1/15
17.40%
Grade I
1/1
Exon 6
1/1
Grade II
7/14
Exon 7
1/1
Grade IV
15/15
50%
100%
Exon 8
1/1
21.40%
Exon 9
2/14
6.60%
Exon 5
1/1
35.70%
Exon 8
3/14
Exon 9
1/1
14.30%
Θ TP53 mutations, patients age & grade
of tumour are possible potent prognostic
factors in astrocytoma outcome.
TP53/Tumor Grades
Mutation Frequency
21.40%
Exon 7
5/14
Exon 8
4/15
TP63 Exons Mutation
Frequency in GI/1
Patients
35.70%
Exon 6
3/14
Exon 7
9/15
Exon 8
9/23
TP53 Exons Mutation
Frequency in GII/14
Patients
Θ TP53 mutational patterns varied
significantly at various disease stages: G II
versus G IV (more aggressive).
Table 7: Incidence of TP53 mutations within astrocytoma grades. The
study group consists of 30 patients, 7 of them with wild type TP53 and 23
with mutated one. The most frequently mutated exon was exon 5 then
7,6,8, and 9.
Exon 5 was more mutated in GIV (66.7%), in G II, exons 5 & 7 were more
frequent (35.7%), followed by exons 6 & 8 (21.4%), and exon 9 (14.3%).
G II had a mutation rate of 50% while G IV showed higher rate (100%)
among other grades.
* = Patient
** = Supratentorial
*** = Infratentorial
WT = Wild Type
® = Recurrent Tumor
High Mutation Rates
7th Australian Mutation Detection
Workshop, 29 July to 1st August 2008,
Broome, Western Australia