Early biomarkers related to secondary primary cancer risk in radiotherapy treated prostate cancer patients: IMRT versus IMAT.

PURPOSE: To investigate whether rotational techniques (Volumetric Modulated Arc Therapy - VMAT) are associated with a higher risk for secondary primary malignancies compared to step-and-shoot Intensity Modulated Radiation Therapy (ss-IMRT). To this end, radiation therapy (RT) induced DNA double-strand-breaks and the resulting chromosomal damage were assessed in peripheral blood T-lymphocytes of prostate cancer (PCa) patients applying γH2AX foci and G0 micronucleus (MN) assays. METHODS AND MATERIALS: The study comprised 33PCa patients. A blood sample was taken before start of therapy and after the 1st and 3rd RT fraction to determine respectively the RT-induced γH2AX foci and MN. The equivalent total body dose (D(ETB)) was calculated based on treatment planning data. RESULTS: A linear dose response was obtained for γH2AX foci yields versus D(ETB) while MN showed a linear-quadratic dose response. Patients treated with large volume (LV) VMAT show a significantly higher level of induced γH2AX foci and MN compared to IMRT and small volume (SV) VMAT (p<0.01). Assuming a linear-quadratic relationship, a satisfactory correlation was found between both endpoints (R(2) 0.86). CONCLUSIONS: Biomarker responses were governed by dose and irradiated volume of normal tissues. No significant differences between IMRT and rotational therapy inherent to the technique itself were observed.

A predictive model for dysphagia following IMRT for head and neck cancer: introduction of the EMLasso technique.

BACKGROUND AND PURPOSE: Design a model for prediction of acute dysphagia following intensity-modulated radiotherapy (IMRT) for head and neck cancer. Illustrate the use of the EMLasso technique for model selection. MATERIAL AND METHODS: Radiation-induced dysphagia was scored using CTCAE v.3.0 in 189 head and neck cancer patients. Clinical data (gender, age, nicotine and alcohol use, diabetes, tumor location), treatment parameters (chemotherapy, surgery involving the primary tumor, lymph node dissection, overall treatment time), dosimetric parameters (doses delivered to pharyngeal constrictor (PC) muscles and esophagus) and 19 genetic polymorphisms were used in model building. The predicting model was achieved by EMLasso, i.e. an EM algorithm to account for missing values, applied to penalized logistic regression, which allows for variable selection by tuning the penalization parameter through crossvalidation on AUC, thus avoiding overfitting. RESULTS: Fifty-three patients (28%) developed acute ≥ grade 3 dysphagia. The final model has an AUC of 0.71 and contains concurrent chemotherapy, D2 to the superior PC and the rs3213245 (XRCC1) polymorphism. The model's false negative rate and false positive rate in the optimal operation point on the ROC curve are 21% and 49%, respectively. CONCLUSIONS: This study demonstrated the utility of the EMLasso technique for model selection in predictive radiogenetics.

Acute radiation-induced nocturia in prostate cancer patients is associated with pretreatment symptoms, radical prostatectomy, and genetic markers in the TGFß1 gene.

PURPOSE: After radiation therapy for prostate cancer, approximately 50% of the patients experience acute genitourinary symptoms, mostly nocturia. This may be highly bothersome with a major impact on the patient's quality of life. In the past, nocturia is seldom reported as a single, physiologically distinct endpoint, and little is known about its etiology. It is assumed that in addition to dose-volume parameters and patient- and therapy-related factors, a genetic component contributes to the development of radiation-induced damage. In this study, we investigated the association among dosimetric, clinical, and TGFß1 polymorphisms and the development of acute radiation-induced nocturia in prostate cancer patients. METHODS AND MATERIALS: Data were available for 322 prostate cancer patients treated with primary or postoperative intensity modulated radiation therapy (IMRT). Five genetic markers in the TGFß1 gene (-800 G>A, -509 C>T, codon 10 T>C, codon 25 G>C, g.10780 T>G), and a high number of clinical and dosimetric parameters were considered. Toxicity was scored using an symptom scale developed in-house. RESULTS: Radical prostatectomy (P<.001) and the presence of pretreatment nocturia (P<.001) are significantly associated with the occurrence of radiation-induced acute toxicity. The -509 CT/TT (P=.010) and codon 10 TC/CC (P=.005) genotypes are significantly associated with an increased risk for radiation-induced acute nocturia. CONCLUSIONS: Radical prostatectomy, the presence of pretreatment nocturia symptoms, and the variant alleles of TGFß1 -509 C>T and codon 10 T>C are identified as factors involved in the development of acute radiation-induced nocturia. These findings may contribute to the research on prediction of late nocturia after IMRT for prostate cancer.

Lack of a correlation between γH2AX foci kinetics in lymphocytes and the severity of acute normal tissue reactions during IMRT treatment for head and neck cancer.

PURPOSE: To investigate the phosphorylated histone H2A isoform X (γH2AX) foci kinetics as an indicator for the development of acute normal tissue complications during Intensity-Modulated Radiotherapy (IMRT) for head and neck cancer (HNC) patients. MATERIALS AND METHODS: Microscopic scoring of the γH2AX foci was used to evaluate the DNA-double-strand-break repair capacity in from Ataxia-Telangiectasia (A-T) patients derived lymphoblastoid cell lines (LCL) and T-lymphocytes isolated from 31 IMRT treated HNC patients. Cells were irradiated in vitro with 0.5 Gy given at high-dose-rate (HDR) and examined at several times up to 24 h after irradiation. The patients were subdivided in three groups showing mild, moderate and severe acute normal tissue complications based on their Common Toxicity Criteria grades for dysphagia, mucositis and dermatitis during the radiotherapy course. RESULTS: For the ATM (Ataxia-Telangiectasia-Mutated) defective LCL, a lower number of radiation-induced foci and a somewhat less efficient repair capacity was observed. No correlation was found between the γH2AX foci kinetics pattern and the risk for acute normal tissue complications among the three patient subgroups. CONCLUSIONS: Scoring of γH2AX foci after in vitro irradiation of isolated T-lymphocytes of HNC patients cannot be applied to predict for the development of acute normal tissue complications.

Genetic variation in three candidate genes and nicotine dependence, withdrawal and smoking cessation in hospitalized patients.

AIMS: This study evaluates the relationship of six polymorphisms found in the CHRNA3, DRD2 and COMT genes with nicotine dependence, the ability to quit smoking and the occurrence of withdrawal symptoms after short-term use of nicotine patch in hospitalized patients. MATERIALS & METHODS: The study included 233 participants from a double-blind, placebo-controlled trial of nicotine patch substitution with a 6-month follow-up period. Nicotine dependence was assessed by the Fagerström Test for Nicotine Dependence (FTND) questionnaire, withdrawal symptoms by the Minnesota Nicotine Withdrawal Scale questionnaire and smoking cessation by self-reported abstinence at 1 week, 1 month and 6 months after treatment. RESULTS: After correcting for multiple testing, three polymorphisms in the DRD2 gene (Taq1A, Taq1B and Pro319Pro) were significantly associated with nicotine dependence (p = 0.018, p = 0.048 and p = 0.006, respectively). Using a cutoff point for the FTND score, the CHRNA3 Tyr215Tyr (rs1051730) polymorphism was also associated with nicotine dependence (p = 0.037 and p = 0.074 after correction for multiple testing). No association of any of the studied polymorphisms was observed with either smoking cessation or the occurrence of withdrawal symptoms. CONCLUSION: This study confirms the reported association of the CHRNA3 locus with nicotine dependence and shows the involvement of two independent DRD2 polymorphisms in nicotine dependence.

Dose response and repair kinetics of gamma-H2AX foci induced by in vitro irradiation of whole blood and T-lymphocytes with X- and gamma-radiation.

PURPOSE: Dose response and repair kinetics of phosphorylated histone H2A isoform X (gamma-H2AX) foci in T-lymphocytes were investigated in the low-dose range after in vitro irradiation of whole blood and T-lymphocytes with 100 kVp X-rays and (60)Co gamma-rays. MATERIALS AND METHODS: Whole blood or isolated T-lymphocytes were irradiated in vitro and gamma-H2AX foci were scored. Dose response was determined in the 0-500 mGy dose range. Foci kinetics were studied at doses of 5 and 200 mGy up to 24 h post-irradiation. RESULTS: After X-irradiation, the dose response for whole blood shows a biphasic behaviour with a low-dose hypersensitivity, which is less pronounced for isolated T-lymphocytes. In contrast, gamma-radiation shows a linear dose response for both irradiation conditions. Concerning repair kinetics, delayed repair was found after X-ray whole blood irradiation (5 and 200 mGy) with 40% of the foci persisting 24 h post-irradiation. This number of foci is reduced to 10% after irradiation of isolated T-lymphocytes with 200 mGy X-rays. On the contrary, gamma-H2AX foci are reduced to background levels 24 h post-irradiation with 200 mGy (60)Co gamma-rays. CONCLUSION: gamma-H2AX foci response and repair kinetics depend on irradiation conditions and radiation quality, possibly linked to Bystander response.

Prediction of late normal tissue complications in RT treated gynaecological cancer patients: potential of the gamma-H2AX foci assay and association with chromosomal radiosensitivity.

In the present study, the gamma-H2AX assay was investigated as a predictive test for the development of late normal tissue complications. Therefore, phosphorylated histone H2AX (gamma-H2AX) foci were scored in peripheral blood T-lymphocytes of gynaecological radiotherapy patients, irradiated in vitro with a high dose rate (HDR) and a low dose rate (LDR) protocol. The G2 chromatid break assay was used to compare chromosomal radiation sensitivity with DNA double-strand-break (DSB) repair capacity. Late normal tissue reactions were scored according to the Common Terminology Criteria for Adverse Events (CTCAE) version 3.0 scale. In our analyses, no differences in foci kinetics were found between the non to mild and moderate to severe patient groups after HDR irradiation. Furthermore, no relation was observed between the level of residual gamma-H2AX foci and CTC score after LDR irradiation. On the contrary, the number of chromatid breaks was associated with late clinical radiation sensitivity. Comparison of G2 chromatid break assay data with the residual number of radiation-induced foci after LDR irradiation and repair times after HDR irradiation showed no relationship between the assays. From this study we can conclude that scoring of gamma-H2AX foci after in vitro irradiation of isolated T-lymphocytes of patients is not predictive for late radiotoxicity. This applies as well to the assessment of the repair kinetics after an HDR dose as to the determination of the number of residual foci after a LDR dose.

gamma-H2AX foci as a biomarker for patient X-ray exposure in pediatric cardiac catheterization: are we underestimating radiation risks?

BACKGROUND: A better knowledge of patient x-ray dose and the associated radiation risk in pediatric interventional cardiology is warranted in view of the extensive use of x-rays and the higher radiosensitivity of children. In the present study, gamma-H2AX foci were used as a biomarker for radiation-induced effects. Patient-specific dose was assessed and radiation risks were estimated according to the linear-no-threshold model, commonly used in radiation protection, and the gamma-H2AX foci data. METHODS AND RESULTS: In 49 pediatric patients (median age, 0.75 years) with congenital heart disease who underwent cardiac catheterization procedures, blood samples were taken before and shortly after the procedure. gamma-H2AX foci were determined in peripheral blood T lymphocytes. In each patient, a net increase in gamma-H2AX foci, representing DNA double-strand breaks induced by interventional x-rays, was observed. In addition, a patient-specific Monte Carlo simulation of the procedure was performed, resulting in individual blood, organ, and tissue doses. Plotting of gamma-H2AX foci versus blood dose indicated a low-dose hypersensitivity. Median effective doses calculated according to the International Commission on Radiological Protection 60 and 103 publications are 5.6 and 6.4 mSv, respectively. The lifetime-attributable risk of cancer mortality was calculated from the linear-no-threshold model and the gamma-H2AX foci data. This resulted in lifetime-attributable risk values of 1% and 4%, respectively, for the patient population under study. CONCLUSIONS: gamma-H2AX foci as a biomarker for DNA damage indicate that radiation risk estimates according to the linear-no-threshold hypothesis are possibly underestimates. Great care should be taken to minimize and optimize patient radiation exposure.

Acute normal tissue reactions in head-and-neck cancer patients treated with IMRT: influence of dose and association with genetic polymorphisms in DNA DSB repair genes.

PURPOSE: To investigate the association between dose-related parameters and polymorphisms in DNA DSB repair genes XRCC3 (c.-1843A>G, c.562-14A>G, c.722C>T), Rad51 (c.-3429G>C, c.-3392G>T), Lig4 (c.26C>T, c.1704T>C), Ku70 (c.-1310C>G), and Ku80 (c.2110-2408G>A) and the occurrence of acute reactions after radiotherapy. MATERIALS AND METHODS: The study population consisted of 88 intensity-modulated radiation therapy (IMRT)-treated head-and-neck cancer patients. Mucositis, dermatitis, and dysphagia were scored using the Common Terminology Criteria (CTC) for Adverse Events v.3.0 scale. The population was divided into a CTC0-2 and CTC3+ group for the analysis of each acute effect. The influence of the dose on critical structures was analyzed using dose-volume histograms. Genotypes were determined by polymerase chain reaction (PCR) combined with restriction fragment length polymorphism or PCR-single base extension assays. RESULTS: The mean dose (D(mean)) to the oral cavity and constrictor pharyngeus (PC) muscles was significantly associated with the development of mucositis and dysphagia, respectively. These parameters were considered confounding factors in the radiogenomics analyses. The XRCC3c.722CT/TT and Ku70c.-1310CG/GG genotypes were significantly associated with the development of severe dysphagia (CTC3+). No association was found between the investigated polymorphisms and the development of mucositis or dermatitis. A risk analysis model for severe dysphagia, which was developed based on the XRCC3c.722CT/TT and Ku70c.-1310CG/GG genotypes and the PC dose, showed a sensitivity of 78.6% and a specificity of 77.6%. CONCLUSIONS: The XRCC3c.722C>T and Ku70c.-1310C>G polymorphisms as well as the D(mean) to the PC muscles were highly associated with the development of severe dysphagia after IMRT. The prediction model developed using these parameters showed a high sensitivity and specificity.

Single-nucleotide polymorphisms in DNA double-strand break repair genes: association with head and neck cancer and interaction with tobacco use and alcohol consumption.

We investigated the effect of different levels of smoking and drinking on the development of squamous cell carcinoma of head and neck (HNSCC) and performed analyses to evaluate possible differences in cancer susceptibility among the anatomical subregions of head and neck. Moreover, we investigated the association between 5 single nucleotide polymorphisms (SNPs) in the homologous recombination DNA repair pathway (XRCC3 c.-1843 A>G, XRCC3 c.562-14 A>G, XRCC3 c.722 C>T, Rad51 c.-3429 G>C, Rad51 c.-3392 G>T) and 4 SNPs in the non- homologous end joining DNA repair pathway (Lig4 c.26 C>T, Lig4 c.1704 T>C, Ku70 c.-1310 C>G and Ku80 c.2110-2408 G>A) on one hand and the risk of the development of HNSCC on the other hand in a case- control setting in a Caucasian population. The study population consisted out of 152 HNSCC patients and 157 healthy controls, matched for age and gender. Polymorphic regions were analysed using the PCR-RFLP and PCR-single base extension assays. Stratification of the populations according to smoking habits and alcohol consumption highlighted the importance of tobacco and alcohol as two risk factors for the development of HNSCC (OR=11.81, p<0.01 and OR=4.66, p<0.01 for high exposure to tobacco and alcohol respectively). A stratification according to the anatomical region of the tumour showed site specific differences in sensitivity to tobacco smoke, with an increase in cancer susceptibility from the oral cavity down to the pharynx and larynx (OR=6.86, p<0.01; OR=9.83, p<0.01 and 36.57, p<0.01 for >25PY). A significant positive association between the XRCC3 c.722 polymorphism and HNSCC was found, with an adjusted odds ratio (OR) of 1.96 (p=0.02). Both the Lig4 c.26 and the Rad51 c.-3429 polymorphisms were associated with a significant reduced risk for HNSCC (OR=0.43, p=0.01; OR=0.43, p=0.05 respectively). Analysis of the gene- smoking interaction revealed no differences in OR for XRCC3 c.722 among the smoking groups. The protective effect seen for the Rad51 c.-3429 and polymorphism was most prominent among the group of heavy smokers (>25 PY). No associations with risk for HNSCC were found for the other SNPs in genes of the DNA DSB repair pathways.

Polymorphisms in nonhomologous end-joining genes associated with breast cancer risk and chromosomal radiosensitivity.

As enhanced chromosomal radiosensitivity (CRS) results from non- or misrepaired double strand breaks (DSBs) and is a hallmark for breast cancer and single nucleotide polymorphisms (SNPs) in DSB repair genes, such as non homologous end-joining (NHEJ) genes, could be involved in CRS and genetic predisposition to breast cancer. In this study, we investigated the association of five SNPs in three different NHEJ genes with breast cancer in a population-based case-control setting. The total patient population composed of a selected group of patients with a family history of the disease and an unselected group, consisting mainly of sporadic cases. SNP analysis showed that the c.2099-2408G>A SNP (XRCC5Ku80) [corrected] has a significant, positive odds ratio (OR) of 2.81 (95% confidence interval (CI): 1.30-6.05) for the heterozygous (He) and homozygous variant (HV) genotypes in the selected patient group. For the c.-1310 C>G SNP (XRCC6Ku70)[corrected] a significant OR of 1.85 (95%CI: 1.01-3.41) was found for the He genotype in the unselected patient group. On the contrary, the HV genotype of c.1781G>T (XRCC6Ku70) [corrected] displays a significant, negative OR of 0.43 (95%CI: 0.18-0.99) in the total patient population. The He+HV genotypes of the c.2099-2408G>A SNP (XRCC5Ku80) [corrected] also showed high and significant ORs in the group of "radiosensitive," familial breast cancer patients. In conclusion, our results provide preliminary evidence that the variant allele of c.-1310C>G (XRCC6Ku70) [corrected]and c.2099-2408G>A (XRCC5Ku80) [corrected] are risk alleles for breast cancer as well as CRS. The HV genotype of c.1781G>T (XRCC6Ku70) [corrected] on the contrary, seems to protect against breast cancer and ionizing radiation induced micronuclei.