1. 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-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. Background 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. 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) 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%), 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. Hypothesis 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. Exon 5 Exon 6 Exon 7 Exon 8 Exon 9 Objectives To determine the genetic alterations in TP53 in patients with brain astrocytoma from 2006 to 2008. Conclusions Θ TP53 mutations were significantly involved in brain astrocytoma. Θ TP53 mutational patterns varied significantly 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). Results ABC 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 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. 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. 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. 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 E A B C DE Results 7 th Australian Mutation Detection Workshop, 29 July to 1 st August 2008, Broome, Western Australia