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Combined low initial DNA damage and high radiation-induced apoptosis confers clinical resistance to long-term toxicity in breast cancer patients treated with high-dose radiotherapy

Combined low initial DNA damage and high radiation-induced apoptosis confers clinical resistance... Background: Either higher levels of initial DNA damage or lower levels of radiation-induced apoptosis in peripheral blood lymphocytes have been associated to increased risk for develop late radiation-induced toxicity. It has been recently published that these two predictive tests are inversely related. The aim of the present study was to investigate the combined role of both tests in relation to clinical radiation-induced toxicity in a set of breast cancer patients treated with high dose hyperfractionated radical radiotherapy. Methods: Peripheral blood lymphocytes were taken from 26 consecutive patients with locally advanced breast carcinoma treated with high-dose hyperfractioned radical radiotherapy. Acute and late cutaneous and subcutaneous toxicity was evaluated using the Radiation Therapy Oncology Group morbidity scoring schema. The mean follow-up of survivors (n = 13) was 197.23 months. Radiosensitivity of lymphocytes was quantified as the initial number of DNA double-strand breaks induced per Gy and per DNA unit (200 Mbp). Radiation-induced apoptosis (RIA) at 1, 2 and 8 Gy was measured by flow cytometry using annexin V/propidium iodide. Results: Mean DSB/Gy/DNA unit obtained was 1.70 ± 0.83 (range 0.63-4.08; median, 1.46). Radiation-induced apoptosis increased with radiation dose (median 12.36, 17.79 and 24.83 for 1, 2, and 8 Gy respectively). We observed that those “expected resistant patients” (DSB values lower than 1.78 DSB/Gy per 200 Mbp and RIA values over 9.58, 14.40 or 24.83 for 1, 2 and 8 Gy respectively) were at low risk of suffer severe subcutaneous late toxicity (HR 0.223, 95%CI 0.073-0.678, P = 0.008; HR 0.206, 95%CI 0.063-0.677, P = 0.009; HR 0.239, 95%CI 0.062-0.929, P = 0.039, for RIA at 1, 2 and 8 Gy respectively) in multivariate analysis. Conclusions: A radiation-resistant profile is proposed, where those patients who presented lower levels of initial DNA damage and higher levels of radiation induced apoptosis were at low risk of suffer severe subcutaneous late toxicity after clinical treatment at high radiation doses in our series. However, due to the small sample size, other prospective studies with higher number of patients are needed to validate these results. * Correspondence: lhenriquez@dcc.ulpgc.es Radiation Oncology Department, Hospital Universitario de Gran Canaria Dr. Negrín, Spain Full list of author information is available at the end of the article © 2011 Henríquez-Hernández et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Henríquez-Hernández et al. Radiation Oncology 2011, 6:60 Page 2 of 8 http://www.ro-journal.com/content/6/1/60 Table 1 Characteristics of patients studied Background Locally advanced breast cancer (LABC) is a relatively N (%) Mean ± SD Median (Range) infrequently tumour which poses a significant clinical Age 57.62 ± 12.9 60 (30-83) challenge. The management of LABC has evolved <60 years 12 (46.2) considerably. Initially, patients with LABC were treated ≥60 years 14 (53.8) with radical mastectomy [1,2]; thereafter, systemic ther- Menopause apy was subsequently incorporated along with surgery Premenopausal 8 (30.8) and radiotherapy (RT) [3]. However, even with such Postmenopausal 18 (69.2) combined modality therapy, the long-term survival rate Tumor type is approximately 50% among patients with LABC [4]. In Inflammatory 7 (26.9) cases with inadequate response to neoadjuvant systemic Non-inflammatory 19 (73.1) therapies and inability to perform surgery, RT is the Tumor size only possible treatment [5]. T3 1 (3.8) Better local control outcomes, with acceptable toxicity, T4a-T4b 18 (69.2) have been obtained by using high total doses of radia- T4c-T4d 7 (27.0) tion administered in two small fractions per day (hyper- Nodes fractionation, HF) [6]. HF allows escalation of the N0 18 (69.2) biologically effective dose to the tumour without a sig- N1-N2 8 (30.8) nificant increase in late complications [7]. The radio Metastasis therapeutic doses received by the patient are limited by M0 24 (92.3) the tolerance of the normal tissues. Different patients M1 2 (7.7) given a standardized treatment can exhibit a range of Bra size 100 ± 10.6 100 (80-120) normalacuteand/orlatetissuereactions[8,9].Thus, <100 9 (34.6) there is both a dose dependence and a variability in ≥100 17 (65.4) individual radiosensitivity, where genetic [10,11] and Systemic treatment constitutional factors [9,12] inherit to each patient could Chemotherapy 4 (15.4) exert an influence. Hormonal therapy 5 (19.2) The prediction of radiation-induced toxicity could help Chemotherapy-hormonal 17 (65.4) to select the most appropriate treatment for each patient. therapy Many predictive factors have been described, including Received dose (Gy) 78.48 ± 5.7 81.60 initial DNA damage [13], cell apoptosis [14], or gene (64.8-81.6) expression patterns [15,16]. In previous studies, we have <81.6 7 (26.9) reported an association between the initial number of ≥81.6 19 (73.1) DNA double-strand breaks (DSB) induced by x-rays in Maximum dose (Gy) 87.36 ± 8.8 89.76 peripheral blood lymphocytes (PBL) and radiation-toxicity (62.8-101.7) [17,18]. Thus, increasing numbers of radiation induced <89.8 15 (57.7) DSB were related to severe late subcutaneous toxicity in ≥89.8 11 (42.3) LABC patients treated with HF [18]. In the other hand, determination of radiation-induced apoptosis (RIA) in Methods PBL by flow cytometry analysis has also been proposed as Characteristics of Patients an approach for predicting normal tissue responses follow- Twenty-six consecutive patients diagnosed in our institu- ing radiotherapy [19,20]. Patients suffering of late toxicity tion with locally advanced/inflammatory breast cancer after RT showed reduced rates of RIA in several tumour were recruited prospectively for the study after they locations [20-22]. Moreover, we have recently reported an signed informed consent to their participation. The study inverse association between the initial DNA damage and was approved by the Research and Ethics Committee of RIA in LABC patients [23]. our Institution. All patients were treated between 1992 Taking into account the above background and our and 1997; blood samples for radiosensitivity testing were previously observations, we explored the clinical associa- extracted between February and December 1998. All the tion between initial DNA damage and RIA in relation to analyses were double-blinded to ensure their reliability. radiation-induced toxicity in the set of LABC patients Mean age of patients was 57.62 ± 12.9 years (range treated with high dose HF radical RT with long-term 30-83). The majority of patients were postmenopausal follow-up where this association have been previously (69.2%), presented bra size over 100 (65.4%), and observed [23]. Henríquez-Hernández et al. Radiation Oncology 2011, 6:60 Page 3 of 8 http://www.ro-journal.com/content/6/1/60 non-inflammatory LABC (73.1%). Characteristics of Table 3 Apoptosis data obtained after the irradiation of PBL at 1, 2 and 8 Gy patients are detailed in Table 1. Evaluation of clinical toxi- city was made in each visit. The Radiotherapy Oncology Mean ± SD Median (range) Tertiles P Group (RTOG) morbidity score system was used to clas- DSB/Gy/DNA unit 1.70 ± 0.83 1.46 (0.63-4.08) 1.28-1.78 0.290 sify the toxicity of patients. Acute toxicity was evaluated RIA 1Gy 13.33 ± 7.26 12.36 (2.51-29.00) 9.58-15.52 0.971 during and at the end of RT. Late cutaneous and subcuta- RIA 2Gy 18.20 ± 7.82 17.79 (4.17-32.08) 14.40-22.43 0.996 neous toxicity was evaluated every three months during RIA 8Gy 29.70 ± 10.05 30.44 (9.02-44.10) 24.83-34.40 0.977 the first two years, every six months to five years, and a 13.08 ± 7.21 12.64 (1.64-26.63) 9.91-15.63 0.994 thereafter annually. At the end of the analysis (January b 7.93 ± 2.68 7.85 (3.18-12.57) 7.14-9.29 0.943 2011), the mean clinical follow-up of survivors (n = 13) Abbreviations: DSB/Gy/DNA unit = double-strand breaks induced per Gy and was 197.23 months (range 155-228). The time point finally per 200 Mbp; RIA = radiation-induced apoptosis at 1, 2 and 8 Gy after 24 hours. a and b are the constants that define the model. P values were used for analysis corresponds to the last evaluation. obtained after a Kolmogorov-Smirnov test. Clinical toxicities of patients are detailed in Table 2. Radiation Treatment by pulsed-field gel electrophoresis (PFGE) as previously Patients were treated with a dose-escalation radiation ther- described [24], and data are summarized in Table 3. apy schedule using hyperfractionation. All patients received 60 Gy to the whole breast over a period of 5 Apoptosis assay and flow cytometry weeks in two daily fractions of 1.2 Gy, separated by at least RIA analyses were performed as previously reported 6 h on 5 days each week. A boost covering the tumour [21,22]. PBL were irradiated with 0, 1, 2 and 8 Gy. After plus margins was prescribed at a dose of 9.4-21.6 Gy [17]. irradiation, samples were incubated for 24 hours at 37°C Peripheral nodes were treated by conventional fractiona- and 5% CO . After extraction of cellular pellet, it was tion (1.8/2Gy/day) at doses of 50-70 Gy. Supraclavicular resuspended in 100 μl Annexin V buffer Kit (Pharmin- and axillary lymph node areas were treated with an ante- gen, Becton Dickinson). After the addition of 4 μlof rior field and a posterior axillary compensating field. Annexin-V-FITC and 10 μl of propidium iodure (PI), Doses were prescribed to the mid-plane of the axilla and cells were incubated during 15 minutes at room tem- at a depth of 3 cm in the supraclavicular area. The internal perature in the dark. Finally, 400 μl of Annexin V buffer mammary chain was treated by a direct anterior field with Kit were added. Every assay was made in triplicate. the dose prescribed at depth of 3 cm. Doses to the breast The flow cytometry analysis was performed in a ranged from 64.8 Gy to 81.6 Gy (mean 77.5 ± 5.7 Gy; FACScalibur (Becton Dickinson,San José,CA)usinga median 81.6 Gy). Maximum point doses ranged from 62.8 488 nm argon laser, and each sample was analyzed in a to 101.7 Gy (mean 87.4 ± 8.8; median 89.7 Gy). Macintosh Quadra 650 minicomputer (Apple computer Inc., Cupertino, CA) as previously reported [25]. Data Analysis of Initial DNA Damage were analyzed using the CellQuest program (Becton Data related to initial DNA damage were obtained from Dickinson, San José, CA) calculating early and late our files [17]. Shortly, mononuclear cells were isolated apoptosis levels. RIA is defined as the percentage of from blood of patients, resuspended in cold DMEM, total PBL death induced by the radiation dose minus and mixed with 1% ultra-low-melting-point agarose to the spontaneous cell death (control, 0 Gy). obtain 250 μl plugs. Irradiation on ice was performed using a Co source (rate dose 1.5 Gy/min, approxi- Statistical analyses mately) as previously reported [17]. Plugs were held 1 Statistical analyses were performed using the SPSS Statis- hour at 4°C and incubated at 37°C for 24 hours. Initial tical Package (version 15.0 for Windows). The cut-off radiation-induced DNA damage in PBL was measured values for continuous variables were the median and the tertiles of the distribution, as previously reported [17,23]. Univariate and multivariate analyses were performed Table 2 Number of patients who developed acute/late using Cox regression. All tests were two sided and statis- toxicity due to radiotherapy tical significance level was established for a P value less Acute Toxicity Late Toxicity than 0.05. All samples were processed anonymously. Grade Cutaneous Cutaneous Subcutaneous 1 6 (23.1) 0 (0.0) 1 (3.8) Results and Discussion 2 12 (46.2) 16 (61.5) 5 (19.3) Radiation-induced toxicity in breast cancer patients 3 8 (30.8) 10 (38.5) 19 (73.1) The actuarial probability of being free of severe late 4 0 (0.0) 0 (0.0) 1 (3.8) cutaneous toxicity, nine-teen years after radiation ther- Numbers in brackets represent the percentage. apy, was 61.5%, while only 19.2% were free of severe late Henríquez-Hernández et al. Radiation Oncology 2011, 6:60 Page 4 of 8 http://www.ro-journal.com/content/6/1/60 subcutaneous toxicity. In a previous observation, patients [17,18]. However, other molecular events such 10 years after RT [17], 65% of patients were free of as DNA repair foci or DNA-loops should be taken into severe late cutaneous toxicity (c test, P =0.463); while account for the correct interpretation of data. It has 29% were free of severe late subcutaneous toxicity (c been observed that DNA DSB in residual foci and test, P = 0.031). Severe subcutaneous toxicity is related relaxation of DNA-loops may be linked to induction of to breast shrinkage, fibrosis and sometimes pain. Late radiation-induced apoptosis in lymphocytes [33-35]. radiation-induced reaction occurs after a latency period We have previously demonstrated a relation between of >90 days (typical range 0.5-5 years). The latency per- the sensitivity of in vitro-irradiated peripheral blood lymphocytes and the risk of developing late toxic effects iod in animals is known to be shorter after higher doses, and in humans, it is even >5 years for moderate doses after RT in the present set of patients [17]. However, or for very late reacting tissues. Late damage progresses the predictive value of initial DNA damage is controver- over time, and it is important to highlight that doses sial and different findings have been reported on this believed safe at 5 years may result in serious late side regard. Thus, we agree with some authors [28,30,36] effects beyond the 5-year period with any treatment pro- and we disagree with some others [37]. Moreover, more tocol [26]. For this, the ability to predict late effects in initial DSB have been detected in lymphocytes from the treated breast is of great importance, especially normal patients as compared to radiosensitive [38]. In when an unconventional treatment schedule is pre- our opinion, it is important to highlight that the predic- scribed. In univariate analysis (simple Cox regression), tive role of initial DNA damage was observed in patients severe subcutaneous late toxicity (grades 3-4) was treated with high-dose of radiation, where the toxicity related to bra size-estimated breast volume (P = 0.037) reactions are more evident. Differences in the protocol (Table 4). Breast size is strongly related to late changes treatment (RT schedule: dose and type of fractionation) in breast appearance possible because greater radiation and in the methodology used (PFGE, comet assay, changes are related to greater dose inhomogeneity in gamma-H2AX induction) could help to explain the women with large breasts [12,17,27]. discrepancies observed. Initial DNA damage levels in breast cancer patients Radiation-induced apoptosis in breast cancer patients Initial DNA damage was determined as radiation- Data of RIA were available in all 26 breast cancer induced double-strand breaks (DSB) in irradiated lym- patients as shown in Table 3. RIA increased with radia- phocyte from all 26 LABC patients. There was a wide tion dose and data fitted to a semi logarithmic model as variation in DSB among patients (Table 3) with a mean follows: RIA = b ln(Gy) + a. This mathematical model value of 1.70 ± 0.83 DSB/Gy per 200 Mbp (median, was defined by two constants: the coefficient in origin a 1.46; range, 0.63-4.08). These results support the sugges- (determining the spontaneous apoptosis) and the coeffi- tion that variation in cell radiosensitivity can be detected cient b (defining the slope of the curve) [21,22,25,39]. in vitro using radiosensitivity assays on lymphocytes As expected, RIA at 1, 2 and 8 Gy, as well as a and derived from normal tissues of cancer patients prior to b constants followed a normal distribution (Kolmo- radiotherapy [18,28-30]. This wide variation in DNA gorov-Smirnov test, P > 0.05). There is an important DSB can be attributed to variation between individuals variationinthe ex vivo susceptibility of normal cells more than to variation due to technical or sampling against ionizing radiation. It has been suggested that the errors [18,31,32]. Initial DNA damage followed a normal radiation-induced damage measured on lymphocytes distribution (Kolmogorov-Smirnov test, P >0.05),and could be proportional to the acute damage evaluated on data obtained from the present group of patients the skin of treated patients [40]. Anyhow, it is possible matched previously published results for breast cancer to estimate the cellular radiosensitivity of PBL of patients analyzing the RIA rate by annexin V/PI staining flow cytometric analysis, defining an intrinsic individual Table 4 Distribution of patients according to expected value of radiosensitivity inherit to each patient. radiation sensitivity after the irradiation of peripheral Radiation-induced apoptosis has been proposed as a blood lymphocytes at 1, 2 and 8 Gy reliable method for prediction of normal tissue toxicity Expected radiation sensitivity RIA 1 Gy RIA 2 Gy RIA 8 Gy after radiotherapy by us [21,22] and other authors High (↑ DSB, ↓ RIA) 2 1 3 [14,19,20]. However, some other studies reported no cor- Intermediate* 13 15 10 relations between individual radiosensitivity of cancer Low (↓ DSB, ↑ RIA) 11 10 13 patients and radiation-induced apoptosis in PBLs [41,42]. 26 26 26 The lack of uniformity in experimental design helps to Abbreviations: DSB = DNA double-strand breaks; RIA = radiation-induced understand these differences. Thus, the cells used in the apoptosis. assay (total PBL, Epstein-Barr virus-transformed *Intermediate: patients showing ↑ DSB, ↑ RIA; or ↓ DSB, ↓ RIA. Henríquez-Hernández et al. Radiation Oncology 2011, 6:60 Page 5 of 8 http://www.ro-journal.com/content/6/1/60 lymphoblastoid cell lines, CD(3+) lymphocytes), the distribution) and RIA values over 9.58, 14.40 or 24.83 radiation protocol, or the analysis strategy are critical to for 1, 2 and 8 Gy respectively (two upper thirds of the make possible the comparison among studies. distribution) (Table 3). We did not observe any associa- tion with late toxicity in the whole series, in univariate Association of initial DNA damage and radiation-induced analysis. However, order to the higher received dose apoptosis with normal tissue toxicity (≥81.6 Gy), we observed that severe subcutaneous late toxicity (grades 3-4) was related to this radiation- As previously published, increasing numbers of radiation resistance profile in patients treated with higher dose of induced DSB were related to severe late toxicity in radiation (simple Cox regression, Table 5). Those breast cancer patients [17]. Thus, among patients receiv- ing the highest radiation doses (81.6 Gy), those who patients treated at very high doses (≥81.6 Gy) and who showed higher levels of initial DNA damage had a presented this radiation-resistance pattern were at low greater risk of severe subcutaneous toxicity. In the pre- risk of suffer severe subcutaneous late toxicity (Table 5). sent set of patients, no association was observed Furthermore, in multivariate analysis in the whole series, between DNA DSB or RIA (at any radiation dose), a or severe subcutaneous late toxicity was related to the b constants and normal tissue toxicity, possibly due to received dose (HR 1.138, 95%CI 1.003-1.291, P = 0.045), the small sample size (data not shown). An association the bra size-estimated volume (HR 1.073, 95%CI between the initial DNA damage and the radiation- 1.004-1.147, P = 0.038), and with this radiation-resistant induced apoptosis, as a consequence of x-ray, may exist profile (HR 0.223, 95%CI 0.073-0.678, P = 0.008; HR [43,44]. DNA DSB are assumed to be the most impor- 0.206, 95%CI 0.063-0.677, P = 0.009; HR 0.239, 95%CI tant lesion to induce apoptosis [45]. Depending on the 0.062-0.929, P = 0.039, for RIA at 1, 2 and 8 Gy, respec- severity of the DNA damage and the cell type involved, tively) (Table 6). Thus, those patients who presented cells may undergo apoptosis instead of attempting to lower levels of initial DNA damage and higher levels of repair the damage [46]. Lymphocytes are particularly radiation induced apoptosis were at low risk of suffer sensitive to apoptosis, partly because they induce Bax severe subcutaneous late toxicity. No relation was found expression in response to ionizing radiation exposure with acute or late cutaneous toxicity. The close relation [46]. Lymphocytes from patients who suffered Ataxia- between chromosome fragment production and killing telangiectasia, Bloom syndrome, or Fanconi anaemia in many cell systems has been important in linking showed absence of induction of p53 and lower levels of DNA DSB to death, because it is a natural step to relate DNA strand breakage to chromosome breakage. Bax [47-49]. Apoptosis is initiated following DSB through However, the recognition that apoptosis may be an an ATM-directed pathway [50]. This could explain the fact that patients affected by the Ataxia-Telangiectasia important mode of radiation-induced death in some cell syndrome show the lowest rates of RIA. In that sense, we types raise the possibility that other types of damage have recently reported an inverse association between the may induce apoptosis [13]. A significant association was initial DNA damage and RIA in LABC patients [23]. Defective apoptotic response to radiation in PBLs could help to explain this inverse relation [14]. Table 5 Univariate analysis for grades 3-4 late subcutaneous toxicity in the whole series of patients According to the above observations, high initial DNA (n = 26) and in patients who received higher doses damage [17] or low radiation-induced apoptosis of RT (n = 19) [14,20-22,25,51] would confer sensitivity to long-term HR (95% CI) P toxicity, separately. In the present study, we tried to dis- Whole series close the predictive value of both parameters in a com- Age 1.012 (0.975-1.012) 0.535 bined form. The percentage of patients developing Received dose 1.079 (0.980-1.189) 0.123 severe late toxicity determines the maximum acceptable Maximum dose 1.054 (0.991-1.121) 0.096 radiation dose. Generally, an adverse effect frequency of Bra size 1.056 (1.003-1.111) 0.037 5%-10% is considered acceptable [52]. We observed that Systemic treatment 1.084 (0.351-3.347) 0.888 7.6% (range 3.8-11.5%) of our patients suffered from Low DSB-High RIA 1Gy 0.564 (0.233-1.370) 0.206 severe complications (2, 1, and 3 out of 26 patients ana- Low DSB-High RIA 2Gy 0.510 (0.204-1.277) 0.150 lyzed at 1, 2 and 8 Gy respectively) (Table 4). Because Low DSB-High RIA 8Gy 0.642 (0.270-1.523) 0.314 this subset of patients is too small, we focused on the Higher dose (≥81.6Gy) expected most resistant patients to RT: those who Low DSB-High RIA 1Gy 0.252 (0.077-0.826) 0.023 presented low initial DNA damage and high radiation- Low DSB-High RIA 2Gy 0.197 (0.053-0.735) 0.016 induced apoptosis (Table 4). Thus, we considered “resis- Low DSB-High RIA 8Gy 0.240 (0.074-0.778) 0.017 tant patients” those who presented DSB values lower than 1.78 DSB/Gy per 200 Mbp (two lower thirds of the Abbreviations: HR = hazard ratio; CI = confidence interval. Henríquez-Hernández et al. Radiation Oncology 2011, 6:60 Page 6 of 8 http://www.ro-journal.com/content/6/1/60 Authors’ contributions Table 6 Multivariate analysis for grades 3-4 late LAHH has written the manuscript, has participated in the statistical analysis, subcutaneous toxicity in the whole series of has made tables and has been involved in type of packaging likewise in the patients (n = 26) submission process. RCV has made the last revision of patients as well as the HR (95% CI) P update of the medical records. BP and ML have made the selection of patients, the evaluation of clinical variables and grade of toxicity as well as Whole series all the aspects related with the patients selected, including the treatment. EB Age 1.044 (0.986-1.106) 0.139 and CRG have made the cell experiments with lymphocytes, irradiation of cells, flow cytometry experiments and data acquisition. MIN has been Received dose 1.138 (1.003-1.291) 0.045 involved in conception and design of the study and has made the DNA-DSB Bra size 1.073 (1.004-1.147) 0.038 experiments and analyses. PCL has been involved in conception and design Systemic treatment 1.155 (0.199-6.697) 0.873 of the study and in drafting the manuscript and has given final approval of the version to be published. All authors read and approved the final Low DSB-High RIA 1Gy 0.223 (0.073-0.678) 0.008 manuscript. Low DSB-High RIA 2Gy 0.206 (0.063-0.677) 0.009 Low DSB-High RIA 8Gy 0.239 (0.062-0.929) 0.039 Competing interests The authors report no conflicts of interest. The authors alone are responsible Abbreviations: HR = hazard ratio; CI = confidence interval. for the content and writing of the paper. Received: 26 January 2011 Accepted: 6 June 2011 observed for the first time between these variables, both Published: 6 June 2011 considered as predictive factors for radiation toxicity, and normal tissue damage. 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Dikomey E, Brammer I, Johansen J, Bentzen SM, Overgaard J: Relationship Gallego C, Lloret M: Prediction of clinical toxicity in locally advanced between DNA double-strand breaks, cell killing, and fibrosis studied in head and neck cancer patients by radio-induced apoptosis in peripheral confluent skin fibroblasts derived from breast cancer patients. Int J blood lymphocytes (PBLs). Radiat Oncol 2010, 5:4. Radiat Oncol Biol Phys 2000, 46:481-490. 23. Pinar B, Henriquez-Hernandez LA, Lara PC, Bordon E, Rodriguez-Gallego C, 41. Greve B, Dreffke K, Rickinger A, Konemann S, Fritz E, Eckardt- Lloret M, Nunez MI, De Almodovar MR: Radiation induced apoptosis and Schupp F, Amler S, Sauerland C, Braselmann H, Sauter W, et al: initial DNA damage are inversely related in locally advanced breast Multicentric investigation of ionising radiation-induced cell death cancer patients. Radiat Oncol 2010, 5:85. as a predictive parameter of individual radiosensitivity. Apoptosis 24. 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Crompton NE, Shi YQ, Emery GC, Wisser L, Blattmann H, Maier A, Li L, Schindler D, Ozsahin H, Ozsahin M: Sources of variation in patient response to radiation treatment. Int J Radiat Oncol Biol Phys 2001, 49:547-554. 52. Svensson JP, Stalpers LJ, Esveldt-van Lange RE, Franken NA, Haveman J, Klein B, Turesson I, Vrieling H, Giphart-Gassler M: Analysis of gene expression using gene sets discriminates cancer patients with and without late radiation toxicity. PLoS Med 2006, 3:e422. doi:10.1186/1748-717X-6-60 Cite this article as: Henríquez-Hernández et al.: Combined low initial DNA damage and high radiation-induced apoptosis confers clinical resistance to long-term toxicity in breast cancer patients treated with high-dose radiotherapy. Radiation Oncology 2011 6:60. 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Combined low initial DNA damage and high radiation-induced apoptosis confers clinical resistance to long-term toxicity in breast cancer patients treated with high-dose radiotherapy

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Springer Journals
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Copyright © 2011 by Henríquez-Hernández et al; licensee BioMed Central Ltd.
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Medicine & Public Health; Oncology; Radiotherapy
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1748-717X
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10.1186/1748-717X-6-60
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21645372
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Abstract

Background: Either higher levels of initial DNA damage or lower levels of radiation-induced apoptosis in peripheral blood lymphocytes have been associated to increased risk for develop late radiation-induced toxicity. It has been recently published that these two predictive tests are inversely related. The aim of the present study was to investigate the combined role of both tests in relation to clinical radiation-induced toxicity in a set of breast cancer patients treated with high dose hyperfractionated radical radiotherapy. Methods: Peripheral blood lymphocytes were taken from 26 consecutive patients with locally advanced breast carcinoma treated with high-dose hyperfractioned radical radiotherapy. Acute and late cutaneous and subcutaneous toxicity was evaluated using the Radiation Therapy Oncology Group morbidity scoring schema. The mean follow-up of survivors (n = 13) was 197.23 months. Radiosensitivity of lymphocytes was quantified as the initial number of DNA double-strand breaks induced per Gy and per DNA unit (200 Mbp). Radiation-induced apoptosis (RIA) at 1, 2 and 8 Gy was measured by flow cytometry using annexin V/propidium iodide. Results: Mean DSB/Gy/DNA unit obtained was 1.70 ± 0.83 (range 0.63-4.08; median, 1.46). Radiation-induced apoptosis increased with radiation dose (median 12.36, 17.79 and 24.83 for 1, 2, and 8 Gy respectively). We observed that those “expected resistant patients” (DSB values lower than 1.78 DSB/Gy per 200 Mbp and RIA values over 9.58, 14.40 or 24.83 for 1, 2 and 8 Gy respectively) were at low risk of suffer severe subcutaneous late toxicity (HR 0.223, 95%CI 0.073-0.678, P = 0.008; HR 0.206, 95%CI 0.063-0.677, P = 0.009; HR 0.239, 95%CI 0.062-0.929, P = 0.039, for RIA at 1, 2 and 8 Gy respectively) in multivariate analysis. Conclusions: A radiation-resistant profile is proposed, where those patients who presented lower levels of initial DNA damage and higher levels of radiation induced apoptosis were at low risk of suffer severe subcutaneous late toxicity after clinical treatment at high radiation doses in our series. However, due to the small sample size, other prospective studies with higher number of patients are needed to validate these results. * Correspondence: lhenriquez@dcc.ulpgc.es Radiation Oncology Department, Hospital Universitario de Gran Canaria Dr. Negrín, Spain Full list of author information is available at the end of the article © 2011 Henríquez-Hernández et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Henríquez-Hernández et al. Radiation Oncology 2011, 6:60 Page 2 of 8 http://www.ro-journal.com/content/6/1/60 Table 1 Characteristics of patients studied Background Locally advanced breast cancer (LABC) is a relatively N (%) Mean ± SD Median (Range) infrequently tumour which poses a significant clinical Age 57.62 ± 12.9 60 (30-83) challenge. The management of LABC has evolved <60 years 12 (46.2) considerably. Initially, patients with LABC were treated ≥60 years 14 (53.8) with radical mastectomy [1,2]; thereafter, systemic ther- Menopause apy was subsequently incorporated along with surgery Premenopausal 8 (30.8) and radiotherapy (RT) [3]. However, even with such Postmenopausal 18 (69.2) combined modality therapy, the long-term survival rate Tumor type is approximately 50% among patients with LABC [4]. In Inflammatory 7 (26.9) cases with inadequate response to neoadjuvant systemic Non-inflammatory 19 (73.1) therapies and inability to perform surgery, RT is the Tumor size only possible treatment [5]. T3 1 (3.8) Better local control outcomes, with acceptable toxicity, T4a-T4b 18 (69.2) have been obtained by using high total doses of radia- T4c-T4d 7 (27.0) tion administered in two small fractions per day (hyper- Nodes fractionation, HF) [6]. HF allows escalation of the N0 18 (69.2) biologically effective dose to the tumour without a sig- N1-N2 8 (30.8) nificant increase in late complications [7]. The radio Metastasis therapeutic doses received by the patient are limited by M0 24 (92.3) the tolerance of the normal tissues. Different patients M1 2 (7.7) given a standardized treatment can exhibit a range of Bra size 100 ± 10.6 100 (80-120) normalacuteand/orlatetissuereactions[8,9].Thus, <100 9 (34.6) there is both a dose dependence and a variability in ≥100 17 (65.4) individual radiosensitivity, where genetic [10,11] and Systemic treatment constitutional factors [9,12] inherit to each patient could Chemotherapy 4 (15.4) exert an influence. Hormonal therapy 5 (19.2) The prediction of radiation-induced toxicity could help Chemotherapy-hormonal 17 (65.4) to select the most appropriate treatment for each patient. therapy Many predictive factors have been described, including Received dose (Gy) 78.48 ± 5.7 81.60 initial DNA damage [13], cell apoptosis [14], or gene (64.8-81.6) expression patterns [15,16]. In previous studies, we have <81.6 7 (26.9) reported an association between the initial number of ≥81.6 19 (73.1) DNA double-strand breaks (DSB) induced by x-rays in Maximum dose (Gy) 87.36 ± 8.8 89.76 peripheral blood lymphocytes (PBL) and radiation-toxicity (62.8-101.7) [17,18]. Thus, increasing numbers of radiation induced <89.8 15 (57.7) DSB were related to severe late subcutaneous toxicity in ≥89.8 11 (42.3) LABC patients treated with HF [18]. In the other hand, determination of radiation-induced apoptosis (RIA) in Methods PBL by flow cytometry analysis has also been proposed as Characteristics of Patients an approach for predicting normal tissue responses follow- Twenty-six consecutive patients diagnosed in our institu- ing radiotherapy [19,20]. Patients suffering of late toxicity tion with locally advanced/inflammatory breast cancer after RT showed reduced rates of RIA in several tumour were recruited prospectively for the study after they locations [20-22]. Moreover, we have recently reported an signed informed consent to their participation. The study inverse association between the initial DNA damage and was approved by the Research and Ethics Committee of RIA in LABC patients [23]. our Institution. All patients were treated between 1992 Taking into account the above background and our and 1997; blood samples for radiosensitivity testing were previously observations, we explored the clinical associa- extracted between February and December 1998. All the tion between initial DNA damage and RIA in relation to analyses were double-blinded to ensure their reliability. radiation-induced toxicity in the set of LABC patients Mean age of patients was 57.62 ± 12.9 years (range treated with high dose HF radical RT with long-term 30-83). The majority of patients were postmenopausal follow-up where this association have been previously (69.2%), presented bra size over 100 (65.4%), and observed [23]. Henríquez-Hernández et al. Radiation Oncology 2011, 6:60 Page 3 of 8 http://www.ro-journal.com/content/6/1/60 non-inflammatory LABC (73.1%). Characteristics of Table 3 Apoptosis data obtained after the irradiation of PBL at 1, 2 and 8 Gy patients are detailed in Table 1. Evaluation of clinical toxi- city was made in each visit. The Radiotherapy Oncology Mean ± SD Median (range) Tertiles P Group (RTOG) morbidity score system was used to clas- DSB/Gy/DNA unit 1.70 ± 0.83 1.46 (0.63-4.08) 1.28-1.78 0.290 sify the toxicity of patients. Acute toxicity was evaluated RIA 1Gy 13.33 ± 7.26 12.36 (2.51-29.00) 9.58-15.52 0.971 during and at the end of RT. Late cutaneous and subcuta- RIA 2Gy 18.20 ± 7.82 17.79 (4.17-32.08) 14.40-22.43 0.996 neous toxicity was evaluated every three months during RIA 8Gy 29.70 ± 10.05 30.44 (9.02-44.10) 24.83-34.40 0.977 the first two years, every six months to five years, and a 13.08 ± 7.21 12.64 (1.64-26.63) 9.91-15.63 0.994 thereafter annually. At the end of the analysis (January b 7.93 ± 2.68 7.85 (3.18-12.57) 7.14-9.29 0.943 2011), the mean clinical follow-up of survivors (n = 13) Abbreviations: DSB/Gy/DNA unit = double-strand breaks induced per Gy and was 197.23 months (range 155-228). The time point finally per 200 Mbp; RIA = radiation-induced apoptosis at 1, 2 and 8 Gy after 24 hours. a and b are the constants that define the model. P values were used for analysis corresponds to the last evaluation. obtained after a Kolmogorov-Smirnov test. Clinical toxicities of patients are detailed in Table 2. Radiation Treatment by pulsed-field gel electrophoresis (PFGE) as previously Patients were treated with a dose-escalation radiation ther- described [24], and data are summarized in Table 3. apy schedule using hyperfractionation. All patients received 60 Gy to the whole breast over a period of 5 Apoptosis assay and flow cytometry weeks in two daily fractions of 1.2 Gy, separated by at least RIA analyses were performed as previously reported 6 h on 5 days each week. A boost covering the tumour [21,22]. PBL were irradiated with 0, 1, 2 and 8 Gy. After plus margins was prescribed at a dose of 9.4-21.6 Gy [17]. irradiation, samples were incubated for 24 hours at 37°C Peripheral nodes were treated by conventional fractiona- and 5% CO . After extraction of cellular pellet, it was tion (1.8/2Gy/day) at doses of 50-70 Gy. Supraclavicular resuspended in 100 μl Annexin V buffer Kit (Pharmin- and axillary lymph node areas were treated with an ante- gen, Becton Dickinson). After the addition of 4 μlof rior field and a posterior axillary compensating field. Annexin-V-FITC and 10 μl of propidium iodure (PI), Doses were prescribed to the mid-plane of the axilla and cells were incubated during 15 minutes at room tem- at a depth of 3 cm in the supraclavicular area. The internal perature in the dark. Finally, 400 μl of Annexin V buffer mammary chain was treated by a direct anterior field with Kit were added. Every assay was made in triplicate. the dose prescribed at depth of 3 cm. Doses to the breast The flow cytometry analysis was performed in a ranged from 64.8 Gy to 81.6 Gy (mean 77.5 ± 5.7 Gy; FACScalibur (Becton Dickinson,San José,CA)usinga median 81.6 Gy). Maximum point doses ranged from 62.8 488 nm argon laser, and each sample was analyzed in a to 101.7 Gy (mean 87.4 ± 8.8; median 89.7 Gy). Macintosh Quadra 650 minicomputer (Apple computer Inc., Cupertino, CA) as previously reported [25]. Data Analysis of Initial DNA Damage were analyzed using the CellQuest program (Becton Data related to initial DNA damage were obtained from Dickinson, San José, CA) calculating early and late our files [17]. Shortly, mononuclear cells were isolated apoptosis levels. RIA is defined as the percentage of from blood of patients, resuspended in cold DMEM, total PBL death induced by the radiation dose minus and mixed with 1% ultra-low-melting-point agarose to the spontaneous cell death (control, 0 Gy). obtain 250 μl plugs. Irradiation on ice was performed using a Co source (rate dose 1.5 Gy/min, approxi- Statistical analyses mately) as previously reported [17]. Plugs were held 1 Statistical analyses were performed using the SPSS Statis- hour at 4°C and incubated at 37°C for 24 hours. Initial tical Package (version 15.0 for Windows). The cut-off radiation-induced DNA damage in PBL was measured values for continuous variables were the median and the tertiles of the distribution, as previously reported [17,23]. Univariate and multivariate analyses were performed Table 2 Number of patients who developed acute/late using Cox regression. All tests were two sided and statis- toxicity due to radiotherapy tical significance level was established for a P value less Acute Toxicity Late Toxicity than 0.05. All samples were processed anonymously. Grade Cutaneous Cutaneous Subcutaneous 1 6 (23.1) 0 (0.0) 1 (3.8) Results and Discussion 2 12 (46.2) 16 (61.5) 5 (19.3) Radiation-induced toxicity in breast cancer patients 3 8 (30.8) 10 (38.5) 19 (73.1) The actuarial probability of being free of severe late 4 0 (0.0) 0 (0.0) 1 (3.8) cutaneous toxicity, nine-teen years after radiation ther- Numbers in brackets represent the percentage. apy, was 61.5%, while only 19.2% were free of severe late Henríquez-Hernández et al. Radiation Oncology 2011, 6:60 Page 4 of 8 http://www.ro-journal.com/content/6/1/60 subcutaneous toxicity. In a previous observation, patients [17,18]. However, other molecular events such 10 years after RT [17], 65% of patients were free of as DNA repair foci or DNA-loops should be taken into severe late cutaneous toxicity (c test, P =0.463); while account for the correct interpretation of data. It has 29% were free of severe late subcutaneous toxicity (c been observed that DNA DSB in residual foci and test, P = 0.031). Severe subcutaneous toxicity is related relaxation of DNA-loops may be linked to induction of to breast shrinkage, fibrosis and sometimes pain. Late radiation-induced apoptosis in lymphocytes [33-35]. radiation-induced reaction occurs after a latency period We have previously demonstrated a relation between of >90 days (typical range 0.5-5 years). The latency per- the sensitivity of in vitro-irradiated peripheral blood lymphocytes and the risk of developing late toxic effects iod in animals is known to be shorter after higher doses, and in humans, it is even >5 years for moderate doses after RT in the present set of patients [17]. However, or for very late reacting tissues. Late damage progresses the predictive value of initial DNA damage is controver- over time, and it is important to highlight that doses sial and different findings have been reported on this believed safe at 5 years may result in serious late side regard. Thus, we agree with some authors [28,30,36] effects beyond the 5-year period with any treatment pro- and we disagree with some others [37]. Moreover, more tocol [26]. For this, the ability to predict late effects in initial DSB have been detected in lymphocytes from the treated breast is of great importance, especially normal patients as compared to radiosensitive [38]. In when an unconventional treatment schedule is pre- our opinion, it is important to highlight that the predic- scribed. In univariate analysis (simple Cox regression), tive role of initial DNA damage was observed in patients severe subcutaneous late toxicity (grades 3-4) was treated with high-dose of radiation, where the toxicity related to bra size-estimated breast volume (P = 0.037) reactions are more evident. Differences in the protocol (Table 4). Breast size is strongly related to late changes treatment (RT schedule: dose and type of fractionation) in breast appearance possible because greater radiation and in the methodology used (PFGE, comet assay, changes are related to greater dose inhomogeneity in gamma-H2AX induction) could help to explain the women with large breasts [12,17,27]. discrepancies observed. Initial DNA damage levels in breast cancer patients Radiation-induced apoptosis in breast cancer patients Initial DNA damage was determined as radiation- Data of RIA were available in all 26 breast cancer induced double-strand breaks (DSB) in irradiated lym- patients as shown in Table 3. RIA increased with radia- phocyte from all 26 LABC patients. There was a wide tion dose and data fitted to a semi logarithmic model as variation in DSB among patients (Table 3) with a mean follows: RIA = b ln(Gy) + a. This mathematical model value of 1.70 ± 0.83 DSB/Gy per 200 Mbp (median, was defined by two constants: the coefficient in origin a 1.46; range, 0.63-4.08). These results support the sugges- (determining the spontaneous apoptosis) and the coeffi- tion that variation in cell radiosensitivity can be detected cient b (defining the slope of the curve) [21,22,25,39]. in vitro using radiosensitivity assays on lymphocytes As expected, RIA at 1, 2 and 8 Gy, as well as a and derived from normal tissues of cancer patients prior to b constants followed a normal distribution (Kolmo- radiotherapy [18,28-30]. This wide variation in DNA gorov-Smirnov test, P > 0.05). There is an important DSB can be attributed to variation between individuals variationinthe ex vivo susceptibility of normal cells more than to variation due to technical or sampling against ionizing radiation. It has been suggested that the errors [18,31,32]. Initial DNA damage followed a normal radiation-induced damage measured on lymphocytes distribution (Kolmogorov-Smirnov test, P >0.05),and could be proportional to the acute damage evaluated on data obtained from the present group of patients the skin of treated patients [40]. Anyhow, it is possible matched previously published results for breast cancer to estimate the cellular radiosensitivity of PBL of patients analyzing the RIA rate by annexin V/PI staining flow cytometric analysis, defining an intrinsic individual Table 4 Distribution of patients according to expected value of radiosensitivity inherit to each patient. radiation sensitivity after the irradiation of peripheral Radiation-induced apoptosis has been proposed as a blood lymphocytes at 1, 2 and 8 Gy reliable method for prediction of normal tissue toxicity Expected radiation sensitivity RIA 1 Gy RIA 2 Gy RIA 8 Gy after radiotherapy by us [21,22] and other authors High (↑ DSB, ↓ RIA) 2 1 3 [14,19,20]. However, some other studies reported no cor- Intermediate* 13 15 10 relations between individual radiosensitivity of cancer Low (↓ DSB, ↑ RIA) 11 10 13 patients and radiation-induced apoptosis in PBLs [41,42]. 26 26 26 The lack of uniformity in experimental design helps to Abbreviations: DSB = DNA double-strand breaks; RIA = radiation-induced understand these differences. Thus, the cells used in the apoptosis. assay (total PBL, Epstein-Barr virus-transformed *Intermediate: patients showing ↑ DSB, ↑ RIA; or ↓ DSB, ↓ RIA. Henríquez-Hernández et al. Radiation Oncology 2011, 6:60 Page 5 of 8 http://www.ro-journal.com/content/6/1/60 lymphoblastoid cell lines, CD(3+) lymphocytes), the distribution) and RIA values over 9.58, 14.40 or 24.83 radiation protocol, or the analysis strategy are critical to for 1, 2 and 8 Gy respectively (two upper thirds of the make possible the comparison among studies. distribution) (Table 3). We did not observe any associa- tion with late toxicity in the whole series, in univariate Association of initial DNA damage and radiation-induced analysis. However, order to the higher received dose apoptosis with normal tissue toxicity (≥81.6 Gy), we observed that severe subcutaneous late toxicity (grades 3-4) was related to this radiation- As previously published, increasing numbers of radiation resistance profile in patients treated with higher dose of induced DSB were related to severe late toxicity in radiation (simple Cox regression, Table 5). Those breast cancer patients [17]. Thus, among patients receiv- ing the highest radiation doses (81.6 Gy), those who patients treated at very high doses (≥81.6 Gy) and who showed higher levels of initial DNA damage had a presented this radiation-resistance pattern were at low greater risk of severe subcutaneous toxicity. In the pre- risk of suffer severe subcutaneous late toxicity (Table 5). sent set of patients, no association was observed Furthermore, in multivariate analysis in the whole series, between DNA DSB or RIA (at any radiation dose), a or severe subcutaneous late toxicity was related to the b constants and normal tissue toxicity, possibly due to received dose (HR 1.138, 95%CI 1.003-1.291, P = 0.045), the small sample size (data not shown). An association the bra size-estimated volume (HR 1.073, 95%CI between the initial DNA damage and the radiation- 1.004-1.147, P = 0.038), and with this radiation-resistant induced apoptosis, as a consequence of x-ray, may exist profile (HR 0.223, 95%CI 0.073-0.678, P = 0.008; HR [43,44]. DNA DSB are assumed to be the most impor- 0.206, 95%CI 0.063-0.677, P = 0.009; HR 0.239, 95%CI tant lesion to induce apoptosis [45]. Depending on the 0.062-0.929, P = 0.039, for RIA at 1, 2 and 8 Gy, respec- severity of the DNA damage and the cell type involved, tively) (Table 6). Thus, those patients who presented cells may undergo apoptosis instead of attempting to lower levels of initial DNA damage and higher levels of repair the damage [46]. Lymphocytes are particularly radiation induced apoptosis were at low risk of suffer sensitive to apoptosis, partly because they induce Bax severe subcutaneous late toxicity. No relation was found expression in response to ionizing radiation exposure with acute or late cutaneous toxicity. The close relation [46]. Lymphocytes from patients who suffered Ataxia- between chromosome fragment production and killing telangiectasia, Bloom syndrome, or Fanconi anaemia in many cell systems has been important in linking showed absence of induction of p53 and lower levels of DNA DSB to death, because it is a natural step to relate DNA strand breakage to chromosome breakage. Bax [47-49]. Apoptosis is initiated following DSB through However, the recognition that apoptosis may be an an ATM-directed pathway [50]. This could explain the fact that patients affected by the Ataxia-Telangiectasia important mode of radiation-induced death in some cell syndrome show the lowest rates of RIA. In that sense, we types raise the possibility that other types of damage have recently reported an inverse association between the may induce apoptosis [13]. A significant association was initial DNA damage and RIA in LABC patients [23]. Defective apoptotic response to radiation in PBLs could help to explain this inverse relation [14]. Table 5 Univariate analysis for grades 3-4 late subcutaneous toxicity in the whole series of patients According to the above observations, high initial DNA (n = 26) and in patients who received higher doses damage [17] or low radiation-induced apoptosis of RT (n = 19) [14,20-22,25,51] would confer sensitivity to long-term HR (95% CI) P toxicity, separately. In the present study, we tried to dis- Whole series close the predictive value of both parameters in a com- Age 1.012 (0.975-1.012) 0.535 bined form. The percentage of patients developing Received dose 1.079 (0.980-1.189) 0.123 severe late toxicity determines the maximum acceptable Maximum dose 1.054 (0.991-1.121) 0.096 radiation dose. Generally, an adverse effect frequency of Bra size 1.056 (1.003-1.111) 0.037 5%-10% is considered acceptable [52]. We observed that Systemic treatment 1.084 (0.351-3.347) 0.888 7.6% (range 3.8-11.5%) of our patients suffered from Low DSB-High RIA 1Gy 0.564 (0.233-1.370) 0.206 severe complications (2, 1, and 3 out of 26 patients ana- Low DSB-High RIA 2Gy 0.510 (0.204-1.277) 0.150 lyzed at 1, 2 and 8 Gy respectively) (Table 4). Because Low DSB-High RIA 8Gy 0.642 (0.270-1.523) 0.314 this subset of patients is too small, we focused on the Higher dose (≥81.6Gy) expected most resistant patients to RT: those who Low DSB-High RIA 1Gy 0.252 (0.077-0.826) 0.023 presented low initial DNA damage and high radiation- Low DSB-High RIA 2Gy 0.197 (0.053-0.735) 0.016 induced apoptosis (Table 4). Thus, we considered “resis- Low DSB-High RIA 8Gy 0.240 (0.074-0.778) 0.017 tant patients” those who presented DSB values lower than 1.78 DSB/Gy per 200 Mbp (two lower thirds of the Abbreviations: HR = hazard ratio; CI = confidence interval. Henríquez-Hernández et al. Radiation Oncology 2011, 6:60 Page 6 of 8 http://www.ro-journal.com/content/6/1/60 Authors’ contributions Table 6 Multivariate analysis for grades 3-4 late LAHH has written the manuscript, has participated in the statistical analysis, subcutaneous toxicity in the whole series of has made tables and has been involved in type of packaging likewise in the patients (n = 26) submission process. RCV has made the last revision of patients as well as the HR (95% CI) P update of the medical records. BP and ML have made the selection of patients, the evaluation of clinical variables and grade of toxicity as well as Whole series all the aspects related with the patients selected, including the treatment. EB Age 1.044 (0.986-1.106) 0.139 and CRG have made the cell experiments with lymphocytes, irradiation of cells, flow cytometry experiments and data acquisition. MIN has been Received dose 1.138 (1.003-1.291) 0.045 involved in conception and design of the study and has made the DNA-DSB Bra size 1.073 (1.004-1.147) 0.038 experiments and analyses. PCL has been involved in conception and design Systemic treatment 1.155 (0.199-6.697) 0.873 of the study and in drafting the manuscript and has given final approval of the version to be published. All authors read and approved the final Low DSB-High RIA 1Gy 0.223 (0.073-0.678) 0.008 manuscript. Low DSB-High RIA 2Gy 0.206 (0.063-0.677) 0.009 Low DSB-High RIA 8Gy 0.239 (0.062-0.929) 0.039 Competing interests The authors report no conflicts of interest. The authors alone are responsible Abbreviations: HR = hazard ratio; CI = confidence interval. for the content and writing of the paper. Received: 26 January 2011 Accepted: 6 June 2011 observed for the first time between these variables, both Published: 6 June 2011 considered as predictive factors for radiation toxicity, and normal tissue damage. 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Radiation OncologySpringer Journals

Published: Jun 6, 2011

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