Genetic Alterations of TP53 Gene in Brain Astrocytic

Tumours

Methodology 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-f ixed 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 statist ical and genetic methods.

BackgroundSuppressor 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.

astrocytoma- is a brain tumour that arises from star-shaped glial cells called astrocytes. astrocytoma can invade surrounding healthy brain t issue. 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)

ABSTRACT: 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%), fol lowed 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 signif icantly 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 classif ied 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.

HypothesisBrain Astrocytic tumours could be caused by mult iple 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.

Exon 5 Exon 6 Exon 7 Exon 8 Exon 9

ObjectivesTo determine the genetic alterations in TP53 in patients with brain astrocytoma from 2006 to 2008.

ConclusionsΘ TP53 mutations were signif icantly involved in brain astrocytoma.

Θ TP53 mutational patterns varied signif icantly at various disease stages: G II versus G IV (more aggressive).

Θ TP53 mutations, patients age & grade of tumour are possible potent prognostic factors in astrocytoma outcome.

Acknowledgement

Dr. Hassan M. Naif

Molecular Pathology Head , Children’s Hospital @ Westmead Sydney, NSW

Ammira H Al-Shabeeb

University of Sydney

Faculty of Medicine

Children’s Hospital at Westmead

Molecular Pathology Department

Corresponding Author: ammira@centenary.org.au

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).

ResultsA B C

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.

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.

Table 3: Histopathological data of astrocytoma patients. Eighty-three 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.

Results

Variables No Mean ± SE a Median SD b Mini Value Maxi Value 99% CI c Correlation ValueMALE 24Age 52.8 ± 5.01 50 25 6 85 38.2-66.8 -0.012TP53 Mutations 2.08 ± 0.29 2 1.47 0 4 1.25-2.90FEMALE 6Age 32.8 ± 11.6 24 28.4 6 80 0-75.7 ** - 0.88TP53 Mutations 0.66 ± 0.21 1 0.51 0 1 0-1.43

P value p < 0.001 p < 0.001

a= SE of Mean b= SD, c = CI, ** Highly Significant

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.

Astrocytoma's Total Number of Total Number of TP53 Total Number of Histopathology TP53 Mutated Non-Mutated TP53 Exons

Grades Exons Exons O - Value

G I O a 5 0 5 E b 2.8 0G II O 18 52 70 E 20.8 48.2G IV O 33 42 75 E 30.5 43.9

Total Number of 56 94 150 *TP53 Exons X2= 2.257 (P > 0.05) DF = 2( O - Value)

a = Observed Value, b = Expected Value, 150*= 30 patients × 5 TP53 Exons = 150 Exons, DF = Degrees of Freedom

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.

Variables Patients (No= 30)

AgeMean ± SE a 52.3 ± 4.55Median 50.1SD b 24.9Range Jun-85

SexM : F 24 - Six

Recurrent 3Family History 1

a Standard Error b Standard Deviation

P* TP53 Mutated Astrocytoma Astrocytoma TP53 Exon Mutation TP53 Exon Mutation TP53 Exons Mutation TP63 Exons Mutation TP53/Tumor GradesExons Histopathology Location Frequency in 23 Frequency in GIV/15 Frequency in GII/14 Frequency in GI/1 Mutation Frequency

Grades Patients Patients Patients Patients

1 6,8 GIV Supra** Exon 5 Exon 5 Exon 5 Exon 5 Grade I2 5,7 GIV supra 16/23 10/15 5/14 1/1 1/13 5,7,8,9 GII Infra*** 69% 66.70% 35.70%4 WT GII Supra Exon 6 Exon 6 Exon 6 Exon 6 Grade II5 6 GIV Supra 14/23 10/15 3/14 1/1 7/146 6 GII Infra 60.90% 66.70% 21.40% 50%7 6, 8 GIV Supra Exon 7 Exon 7 Exon 7 Exon 7 Grade IV8 5,7 GII Supra 15/23 9/15 5/14 1/1 15/159 5,7 GIV Supra 65.20% 60% 35.70% 100%

10 6 GIV Supra Exon 8 Exon 8 Exon 8 Exon 811 5,6,7,8 GIV® Supra 9/23 4/15 3/14 1/112 5,6,7 GIV® Supra 39.10% 33.30% 21.40%13 WT GII Supra Exon 9 Exon 9 Exon 9 Exon 914 5 GIV Supra 4/23 1/15 2/14 1/115 WT GII Infra 17.40% 6.60% 14.30%16 5,6,7,8,9 GI Supra17 WT GII Supra18 5,6,7,9 GIV Supra19 WT GII Infra * = Patient

20 5,7 GIV Supra ** = Supratentorial

21 7 GII Supra *** = Infratentorial

22 5,9 GII Supra WT = Wild Type

23 7 GIV Supra ® = Recurrent Tumor

24 5,6,7,8 GIV Supra High Mutation Rates

25 5,6,7,8 GII Supra26 WT GII Supra27 WT GII Infra28 5,6,7,8 GII Infra29 5,6,7,8 GIV® Supra30 5,6 GIV Supra

Primers Sequences MW bpE5 Forward 5' ACC CTG GGC AAC CAG CCC TG 3' 6128 152E5 Reverse 5' TTT ATC TGT TCA CTT GTG CCC 3' 6409E6 Forward 5' CTC CCA GAG ACC CCA GTT GC 3' 6103 167E6 Reverse 5' TCA GAT AGC GAT GTG AGC AG 3' 6286E7 Forward 5' CAG TGT GCA GGG TGG CAA GT 3' 6318 132E7 Reverse 5' GCC ACA GGT CTC CCC AAG GC 3' 6128E8 Forward 5' CCA CCG CTT CTT GTC CTG CT 3' 6051 136E8 Reverse 5' GAC CTG ATT TCC TTA CTG CC 3' 6099E9 Forward 5' GGA GAC CAA GGG TGC AGT TAT 3' 6120 149E9 Reverse 5' GTT AGT TAG CTA CAA CCA GGA GCC 3' 6308

Table 1: TP53 Single Strand Conformational Polymorphism SSCP primers sequence , exons 5, 6, 7, 8, and 9 respectively.

Wild Type

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.

Patients Mutations Exon 5 Exon 6 Exon 7 Exon 8 Exon 9WHO GI 1/1 50 - 85 0/1 0 0 0 0 0

TP53 +ve 20 - 50 1/1 1/1 1/1 1/1 1/1 1/11 - 20 0/1 0 0 0 0 0

WHO GII 14 50 - 85 4/7 (57.1%) 2/4 (50%) 2/4 (50%) 3/4 (75%) 1/4 (25%) 0/4TP53 +ve /7 20 - 50 1/7 (14.3%) 1/1 0/1 1/1 1/1 1/1TP53 -ve /7 1 - 20 2/7 (28.5%) 2/2 1/2 1/2 1/2 1/2

WHO GIV 15 50 - 85 8/15 (53.3%) 5/8 (62.5%) 6/8 (75%) 4/8 (50%) 4/8 (50%) 1/8 (12.5%)TP53+ve /15 20 - 50 7/15 (46.7%) 5/7 (46.7%) 4/7 (57%) 5/7 (71.4%) 1/7 (17%) 0/7

1 - 20 0/15 0 0 0 0 0

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.

A

B

C

D

EVariables Patient Number Age / Y

No = 83, N % Tumor Types Mean RangeAstrocytoma 38 (45.8)G I 1 (2.6) / /G II 14 (36.8) 50.9 6 - 8G III 8 (21.1) 53.3 20 - 77G IV 15 (39.1) 52.2 30 - 85Meningioma 16 (19.3)Pituitary Adenoma 5 (6.0)Oligodendroglioma 5 (6.0)Medulloblastoma 5 (6.0)Haemangioblastoma 5 (6.0)Ependymomas 4 (4.8)Schwanoma 3 (3.6)Craniopharyngioma 2 (2.4)

A

B

C

D E

Results

7th Australian Mutation Detection Workshop, 29 July to 1st August 2008,

Broome, Western Australia