Ghazi Alsbeih

Association between Normal Tissue Complications after Radiotherapy and Polymorphic Variations in TGFB1 and XRCC1 Genes

Abstract Genetic predictive biomarkers of radiosensitivity are being sought to individualize radiation treatment of cancer patients. In this pilot case-control study, we tested the association between TGFB1 T869C codon 10 Leu/Pro (rs1982073), XRCC1 G28152A codon 399 Arg/Gln (rs25487), and XRCC3 C18067T codon 241 Thr/Met (rs861539) single-nucleotide polymorphisms (SNPs) and late reaction to radiotherapy in 60 nasopharyngeal cancer patients. Subcutaneous and deep tissue fibrosis was scored using the RTOG/EORTC grading system. Patients with moderate to severe fibrosis (radiosensitive cases, G2–3, n  =  30) were matched and compared to those with little or no reaction (controls, G0–1, n  =  30). The three nonsynonymous SNPs were genotyped by direct DNA sequencing. Significant association was observed for TGFB1 T869C and XRCC1 G28152A genotypes (P ≤ 0.05). Both variant alleles, TGFB1 869C and XRCC1 28152A, were associated with a lower grade of fibrosis (odds ratios were 0.41, 95% CI: 0.20–0.86, P  =  0.02 and 0.30, 95% CI: 0.10–0.89, P  =  0.02, respectively), and therefore the wild-types were the risk alleles. Interestingly, there was a significant difference in the median number of risk alleles between the radiosensitive and the control groups (P  =  0.006). We conclude that radiotherapy complications are associated with genetic variations in our nasopharynx cancer patients. Our findings support the assumption of the combined effects of multiple SNPs. Large-scale studies are required to confirm these findings before polymorphisms can be used as predictive markers to individualize radiation therapy on genetic bases.

Correlation between normal tissue complications and in vitro radiosensitivity of skin fibroblasts derived from radiotherapy patients treated for variety of tumors

PURPOSE To assess the relationship between fibroblast intrinsic radiosensitivity in vitro and late reactions of normal tissues in patients treated by definitive radiotherapy for variety of tumors. PATIENTS AND METHODS Ten patients were selected for this study. They were treated by radical radiotherapy for variety of tumors, including non-Hodgkins lymphoma, prostate, glottic larynx, anal canal, cervix, bladder, thyroid gland, and tonsil pillar. Five patients did not develop any significant late reactions (normally sensitive group, NS). The other five developed late complications in different normal tissues and organs that proved to be fatal in one patient (clinically hyper-sensitive group, HS). Fibroblast cultures were established from punch skin biopsy and radiosensitivity in vitro was measured. The survival fraction at 2 Gy (SF2) was calculated and compared between the two groups. RESULTS SF2 ranged between 0.10 and 0.38 with a mean of 0.24. The mean SF2 for each of the NS and the HS groups were 0.31 and 0.17, respectively. The non-parametric rank test of Mann-Whitney shows that the difference between the two groups is statistically significant (p = 0.01). CONCLUSION This study indicates that the in vitro radiosensitivity of skin fibroblasts is correlated with late complications in different organs and normal tissues following radiotherapy for variety of tumors. It also lends support to the existence of a common genetic component determining the radiosensitivity of cells targeted by the late effects of ionizing radiation.

Enhanced in vitro radiosensitivity of skin fibroblasts in two patients developing brain necrosis following AVM radiosurgery: a new risk factor with potential for a predictive assay

PURPOSE Radiosurgery is an effective treatment for arteriovenous malformations (AVM) with a low risk of developing brain necrosis. Models have been developed to predict the risk of complications. We postulated that genetic differences in radiosensitivity may also be a risk factor. METHODS AND MATERIALS Fibroblast cultures were established from skin biopsies in two AVM patients developing radiation necrosis. The results of clonogenic survival assays were compared to a parallel study with two groups of cancer patients treated with radiation: 1) patients without late side effects; 2) patients experiencing severe late sequelae. RESULTS The survival fraction at 2 Gy (SF2) of the 2 AVM patients was 0.17 (0.14-0.19) and 0.18 (0.14-0.22). The SF2s of the cancer patients ranged between 0.25-0.38 (mean = 0.31) for the control group, and between 0. 10-0.20 (mean = 0.17) for the hypersensitive group. The SF2s of the AVM patients who developed brain necrosis were comparable to that of the hypersensitive group (p = 0.85) but significantly lower than the control group (p = 0.05). CONCLUSION The two patients who developed radiation necrosis demonstrate increased fibroblast radiosensitivity. The SF2 of skin fibroblasts may potentially be used as a predictive assay to detect patients at risk for brain necrosis.

Involvement of Single-Nucleotide Polymorphisms in Predisposition to Head and Neck Cancer in Saudi Arabia

AIM Individuals differ in their inherited tendency to develop cancer. This has been suggested to be due to genetic variations between individuals. Single-nucleotide polymorphisms (SNPs) are the most common form of genetic variations found in the human population. The aim of this study was to investigate the association between 10 SNPs in genes involved in cell cycle control and DNA repair (p21 C31A, p53 G72C, ATM G1853A, XRCC1 G399A, XRCC3 C241T, Ku80 A2790G, DNA Ligase IV C9T, DNA-PKcs A3434G, TGF-beta T10C, MDM2 promoter T309G) and the risk to develop head and neck cancer. MATERIALS AND METHODS A cohort of 407 individuals (156 cancer patients and 251 controls) was included. DNA was extracted from peripheral blood. SNPs were genotyped by direct sequencing. RESULTS Data showed significant allelic associations for p21 C31A (p=0.04; odds ratio [OR]=1.44; confidence interval [CI]: 1.02-2.03), Ku80 A2790G (p=0.04; OR=1.5; CI: 1.01-2.23), and MDM2 T309G (p=0.0003; OR=0.58; CI: 0.43-0.78) and head and neck cancer occurrence. Both cancer cases and controls were in Hardy-Weinberg equilibrium. CONCLUSION SNPs can be associated with head and neck cancer in the Saudi population. The p21 C31A, Ku80 A2790G, and MDM2 T309G SNPs could be used as genetic biomarkers to screen individuals at high cancer risk.

Involvement of mitochondrial DNA sequence variations and respiratory activity in late complications following radiotherapy.

Purpose: Mitochondria and ionizing radiation overlap in a number of features; for instance, both generate harmful reactive oxygen species, and that radiation can induce cell death through the intermediary of mitochondria. Because a number of genetic variations in nuclear genes are frequently associated with response to cancer treatment, the aim of this case-control study was to test the hypothesis that mitochondrial DNA (mtDNA) genetic variations can contribute to patient-to-patient variability in normal tissue response to radiotherapy. Experimental Design: Thirty-two nasopharyngeal carcinomas patients treated with definitive radiotherapy were included. The grade (G) of s.c. and deep tissue fibrosis was scored according to the Radiation Therapy Oncology Group/European Organization for Research and Treatment of Cancer grading system. Coding and RNA mtDNA (between 611 and 15,978 bp) were sequenced, and genetic variations were scored. Mitochondrial respiratory activity was measured by resazurin reduction assay. Results: Data showed a significantly (P = 0.003) higher number of nonsynonymous genetic variations in the radiosensitive (G2-G3; 16 patients) as compared with the control (G0-G1; 16 patients) groups. The nonsynonymous A10398G variation in the ND3 gene was significantly associated with fibrotic reaction (P = 0.01). The radiosensitive patients had a 7-fold (95% confidence interval, 1.16-51.65) higher risk of developing moderate to severe fibrosis (G2-G3) following radiotherapy. This was significantly correlated with lower mitochondrial respiratory activity (P = 0.001). Conclusion: Mitochondria contribute to radiation sensitivity, and genetic variations can be associated with late reactions to radiotherapy. Predictive markers of radiosensitivity should take into account mtDNA genetic variations in addition to variations in nuclear genes. (Clin Cancer Res 2009;15(23):7352–60)

SNPs in genes implicated in radiation response are associated with radiotoxicity and evoke roles as predictive and prognostic biomarkers

BackgroundBiomarkers are needed to individualize cancer radiation treatment. Therefore, we have investigated the association between various risk factors, including single nucleotide polymorphisms (SNPs) in candidate genes and late complications to radiotherapy in our nasopharyngeal cancer patients.MethodsA cohort of 155 patients was included. Normal tissue fibrosis was scored using RTOG/EORTC grading system. A total of 45 SNPs in 11 candidate genes (ATM, XRCC1, XRCC3, XRCC4, XRCC5, PRKDC, LIG4, TP53, HDM2, CDKN1A, TGFB1) were genotyped by direct genomic DNA sequencing. Patients with severe fibrosis (cases, G3-4, n = 48) were compared to controls (G0-2, n = 107).ResultsUnivariate analysis showed significant association (P < 0.05) with radiation complications for 6 SNPs (ATM G/A rs1801516, HDM2 promoter T/G rs2279744 and T/A rs1196333, XRCC1 G/A rs25487, XRCC5 T/C rs1051677 and TGFB1 C/T rs1800469). In addition, Kaplan-Meier analyses have also highlighted significant association between genotypes and length of patients’ follow-up after radiotherapy. Multivariate logistic regression has further sustained these results suggesting predictive and prognostic roles of SNPs.ConclusionsUnivariate and multivariate analysis suggest that radiation toxicity in radiotherapy patients are associated with certain SNPs, in genes including HDM2 promoter studied for the 1st time. These results support the use of SNPs as genetic predictive markers for clinical radiosensitivity and evoke a prognostic role for length of patients’ follow-up after radiotherapy.

Chromosomal fragility syndrome and family history of radiosensitivity as indicators for radiotherapy dose modification

Beside a few known radiosensitive syndromes, a patients reaction to radiotherapy is difficult to predict. In this report we describe the management of a pediatric cancer patient presented with a family history of radiosensitivity and cancer proneness. Laboratory investigations revealed a chromosomal fragility syndrome and an increased cellular radiosensitivity in vitro. AT gene sequencing revealed no mutations. The patient was treated with reduced radiation doses to avoid the presumed increased risks of toxicity to normal tissues. The patient tolerated well the treatment with no significant acute or late radiation sequelae. Five years later, the patient remains both disease and complications free. While an accurate laboratory test for radiosensitivity is still lacking, assessments of chromosomal fragility, cell survival and clinical medicine will continue to be useful for a small number of patients.

Skin fibroblasts in vitro radiosensitivity can predict for late complications following AVM radiosurgery

BACKGROUND AND PURPOSE A small proportion of patients undergoing radiotherapy display heightened normal tissue reactions. We have set out to determine whether this sensitivity is genetic in nature and can be assessed using an in vitro skin fibroblast assay in order to predict and avoid excessive normal tissue complications. PATIENTS AND METHODS In this study we compared five arteriovenous malformation (AVM) patients who were treated with radiotherapy and showed severe normal tissue reactions (necrosis) to two AVM patients who showed normal reactions. Fibroblasts taken from patients were cultured in vitro and irradiated. RESULTS The results showed that the fibroblasts from the sensitive patients were also more radiosensitive in vitro than the cells from the normally responding patients. CONCLUSIONS The results suggest underlying genetic radiosensitivity and that such an assay may be used for prediction of severe radiosensitivity in AVM patients.

Association between TP53 codon 72 single-nucleotide polymorphism and radiation sensitivity of human fibroblasts.

Abstract Alsbeih, G., Al-Harbi, N., Al-Buhairi, M., Al-Hadyan, K. and Al-Hamed, M. Association between TP53 Codon 72 Single-Nucleotide Polymorphism and Radiation Sensitivity of Human Fibroblasts. Radiat. Res. 167, 535–540 (2007). Inherent radiosensitivity varies widely between individuals. We hypothesized that amino acid substitution variants in two highly radiation-responsive proteins, TP53 (p53) and CDKN1A (p21, Waf1, Cip1), are associated with and could explain individual variations in radiosensitivity. The two non-synonymous single-nucleotide polymorphisms (SNPs) TP53 codon 72 Arg/Pro G>C and CDKN1A codon 31 Ser/Arg C>A were genotyped in 92 normal fibroblast cell strains of different radiosensitivity. The clonogenic surviving fraction at 2 Gy (SF2) ranged between 0.15 and 0.50 (mean = 0.34, SD = 0.08). The mean SF2 was used to divide the cell strains into radiosensitive (45) and normal groups (47). A significant association was observed between SF2 and the TP53 codon 72 haplotype (C compared to G, P = 0.01). No association was observed between CDKN1A codon 31 haplotype and radiosensitivity (P = 0.86). The variant TP53 Arg72 allele was associated with a decrease in radiosensitivity, presumably due to suboptimal function leading to less stringent control of cell division. We conclude that certain SNPs in susceptible genes can influence cellular radiation response. Such risk alleles could ultimately be used as predictive markers for radiosensitivity to help stratifying individuals during assessment of risk of radiation exposure.

Loss of Wild-Type Trp53 Protein in Mouse Fibroblasts Leads to Increased Radioresistance with Consequent Decrease in Repair of Potentially Lethal Damage

Abstract Alsbeih, G., Torres, M., Al-Harbi, N. and Alsubael, M. Loss of Wild-Type Trp53 Protein in Mouse Fibroblasts Leads to Increased Radioresistance with Consequent Decrease in Repair of Potentially Lethal Damage. Radiat. Res. 161, 185–192 (2004). It has been reported that the loss of function of Trp53 protein is associated with a reduction in the expression of radiation-induced potentially lethal damage (PLD). These studies, however, were carried out using either transformed or transfected cell lines, and other factors may have existed that could interfere with PLD repair. In this study, we used isogenic fibroblasts derived from Trp53 knockout mice to study radiation sensitivity, PLD repair, and repair of DNA double-strand breaks (DSBs). Experiments were carried out using wild-type (Trp53+/+), heterozygous (Trp53+/−) and homozygous mutant (Trp53−/−) cells. This is an ideal system because the only difference in the three cell strains is the status of the Trp53 protein. DSB repair was measured by pulsed-field-gel electrophoresis (PFGE), while radiosensitivity and PLD repair were studied using the clonogenic survival assay. Cells were irradiated in plateau phase and then trypsinized and plated either immediately or 24 h later to allow for PLD repair. The results of Western blot analyses showed that Trp53−/− cells expressed a putative mutant form of Trp53 that was unable to transcriptionally activate Cdkn1a (p21) protein in response to irradiation. The Trp53−/− cells were significantly more radioresistant than the Trp53+/+ cells, and this was associated with a moderate reduction in PLD repair. DNA repair experiments showed no difference in DSB rejoining capability between the two cell lines. In conclusion, our results show that loss of wild-type Trp53 leads to increased radioresistance with consequent reduction in PLD repair but with no effect on DNA DSB repair.