Anna Gasinska

Influence of overall treatment time and radiobiological parameters on biologically effective doses in cervical cancer patients treated with radiation therapy alone.

The aim of the study was to examine the influence of overall treatment time (OTT) on the value of calculated biological effective doses (BEDs) for different biological variables. These variables were: tumour proliferation rate, different cell radiosensitivity (α=0.2, 0.3, and 0.4 /Gy), and different start time for repopulation (Tk=21, 28, and 35 days). Also the influence of age (≤50 years >), Hb level (≤116 g/l>), tumor proliferation rate (bromodeoxyuridine labelling index; BrdUrdLI), and DNA ploidy on survival after shorter (≤60 days) or longer (>60 days) OTT was investigated. The study included 229 patients with cervix carcinoma treated entirely by standard radiotherapy (RT) (external beam RT plus low-medium dose-rate (LDR/MDR) brachytherapy (BT) at the Center of Oncology in Krakow. The linear quadratic equation was used to calculate BED, which is proportional to log cell kill. BEDs 10 (for tumours) were calculated with consideration of OTT for each patient and tumour proliferation rate (standardized potential doubling time; standardized Tpot) based on BrdUrdLI assessed on biopsy material before RT. Median OTT was 90 days (range 30–210). The mean calculated total BED for point A for tumour and ‘early reactions’ was equal to 103.0 Gy10. The longest median survival time—52 months—was seen for patients treated with OTT ≤60 days. If OTT exceeded 90 days to more than 120 days, loss in BED10 for relatively radiosensitive tumours (α=0.3–0.4/Gy and Tk=28 days) was equal to 0.37–0.26 Gy/day. However, for radioresistant tumours (α=0.2/Gy) it was 0.6 Gy/day. For fast proliferating tumours (BrdUrdLI >8.8%) BED loss was 1.4 Gy/day and for slowly proliferating tumours (BrdUrdLI ≤8.8%) it was 0.2 Gy/day. Assuming shorter (21 days) or longer (35 days) periods for Tk and relatively radiosensitive tumours similar BED loss of 0.38 Gy/day was observed. Kaplan–Meier analysis revealed that OTT ≤60 days was a significant prognostic factor for overall survival (OS) (p=0.019), disease-free survival (DFS) (p=0.0173), and local control (LC) (p=0.011). BED10 had significant influence on survival (p=0.047). Cox multivariate analysis revealed that for OTT shorter than 60 days the only favourable significant parameters were: age >50 years (p=0.003) and high Hb level (>116 g/l) (p=0.041). For longer treatments (OTT >60 days) the unfavourable parameters were: age ≤50 years (p=0.037), BrdUrdLI ≤8.8% (p=0.003), tumour aneuploidy (p=0.043), and BED10 ≤103 Gy (p=0.017). The examined tumour biological parameters should be taken into account for RT and provide a basis for adjuvant RT.

Comparison of the radiosensitivity of normal-tissue cells with normal-tissue reactions after radiotherapy

Purpose : To investigate whether the in vitro radiosensitivity of normal lymphocytes and fibroblasts evaluated by the micronucleus (MN) assay predicts acute and late reactions after radiotherapy in cancer patients. Materials and methods : Studies were performed on blood samples from 31 cervical and head and neck cancer patients and on skin fibroblasts from eight of the cancer patients. The radiosensitivity of lymphocytes and of fibroblasts was also assessed in 24 and five healthy donors, respectively. Radiosensitivity was measured after in vitro irradiation with doses ranging from 2 to 5 Gy using micronucleus frequency (the number of micronuclei per single binucleated (BN) cell) and the percentage of BN cells with micronuclei. The in vitro results were compared with the maximum grade of acute and late reactions. Results : There was no significant difference in cellular radiosensitivity between cancer patients and healthy donors. Although cancer patients differed considerably in normal-cell radiosensitivity, no correlation was found between radiosensitivity, either of lymphocytes or fibroblasts, and acute and late clinically observed side effects. In addition, no relationship was observed between the radiosensitivity of lymphocytes and fibroblasts derived from the same donors. Conclusion : The data do not support the usefulness of the MN assay in predicting normal-tissue response to radiotherapy in cancer patients.PURPOSE To investigate whether the in vitro radiosensitivity of normal lymphocytes and fibroblasts evaluated by the micronucleus (MN) assay predicts acute and late reactions after radio-therapy in cancer patients. MATERIALS AND METHODS Studies were performed on blood samples from 31 cervical and head and neck cancer patients and on skin fibroblasts from eight of the cancer patients. The radiosensitivity of lymphocytes and of fibroblasts was also assessed in 24 and five healthy donors, respectively. Radiosensitivity was measured after in vitro irradiation with doses ranging from 2 to 5 Gy using micronucleus frequency (the number of micronuclei per single binucleated (BN) cell) and the percentage of BN cells with micronuclei. The in vitro results were compared with the maximum grade of acute and late reactions. RESULTS There was no significant difference in cellular radiosensitivity between cancer patients and healthy donors. Although cancer patients differed considerably in normal-cell radiosensitivity, no correlation was found between radiosensitivity, either of lymphocytes or fibroblasts, and acute and late clinically observed side effects. In addition, no relationship was observed between the radiosensitivity of lymphocytes and fibroblasts derived from the same donors. CONCLUSION The data do not support the usefulness of the MN assay in predicting normal-tissue response to radiotherapy in cancer patients.

Tumour cell kinetics as a prognostic factor in squamous cell carcinoma of the cervix treated with radiotherapy.

PURPOSE Proliferative rate and DNA ploidy status were evaluated by flow cytometry in cervical cancer patients, prior to radiotherapy, to assess their importance as prognostic factors to predict survival rates. MATERIAL AND METHODS Between 1987 and 1995, a total of 260 patients with squamous cell carcinoma (SCC) of the cervix, FIGO stages IB-IIIB were analysed. Tumour samples were incubated with bromodeoxyuridine (BrdUrd) in vitro to measure their total labelling index (totLI) and LI (totLI for diploid and anLI for aneuploid tumours). Proliferation was also assessed by S-phase fraction (SPF) analysis of the DNA profile. Patients had intracavitary therapy (three applications, each of 16 Gy to point A) and XRT of 40-50 Gy given over 4-5 weeks. RESULTS The cervical tumours were characterized by a high proliferation rate which varied within each clinical stage of disease. The totLI ranged from 1.1 to 33.1% with median value of 9.6% whilst the LI ranged from 1.1 to 37.1% with a median value of 10.9%. Univariate analysis identified patients age (cut-offpoint < or = 50&greater; years) and to a lesser extent proliferation (cut-off point, median totLI=9.6%) as significant prognostic factors for 5-year survival. The median survival time for younger patients ( < or = 50 years) with tumours of low proliferation (totLI < or = 9.6%) tumours was 17.5 months compared with 56 months in the faster proliferating tumours (P=0.0354). In the older patient sub-group, proliferation rate had no influence on survival. The median LI value was not a useful parameter in survival. Cox multivariate analysis showed that patient age ( < or = 50 years) and low proliferation of the tumour cells (totLI < or = 9.6) were unfavourable prognostic factors for cervical cancers treated with radiotherapy. DNA ploidy was not significant in this series. CONCLUSIONS These data suggest that further improvements in therapy might be gained by selection of alternative treatments strategies such as neoadjuvant chemotherapy or radiation sensitizers in younger patients with more slowly proliferating tumours.

Comparison of eight commercially available kits for DNA extraction from formalin-fixed paraffin-embedded tissues

A proper extraction method from formalin-fixed paraffin-embedded (FFPE) blocks is essential to obtain DNA of satisfactory quality/quantity. We compared the effectiveness of eight commercially available kits for DNA extraction based on 10 FFPE tissues. Kits differed significantly in terms of DNA yield, purity, and quality. Using the QIAamp DNA FFPE Tissue Kit (Qiagen) and the ReliaPrep FFPE gDNA Miniprep System (Promega), we obtained DNA of the highest quality and acceptable quantity. We also demonstrated that overnight digestion of samples usually improved DNA yield and/or purity. For precious or limited material, double elution is recommended for obtaining up to 42% higher amount of DNA.

Flow cytometric evaluation of the effects of 2.3 MeV neutrons and 240 kV X rays on mouse spermatogenic S-phase cells using bromodeoxyuridine incorporation

A sensitive flow cytometric assay has been developed to assess the effect of X rays or neutrons on the DNA-synthesizing ability of mouse testis cells. The dose to reduce the labelling index (LI) to 50% (ED50) was 25 cGy for X rays and 5 cGy for neutrons, giving a relative biological effectiveness (RBE) of 5.0. A significant reduction of the LI was seen at X-ray doses as low as 10 cGy. Neutron treatment resulted in an exponential decrease in functional S-phase cells while the X-ray response was biphasic. The sensitivity and rapidity of the assay would be suitable for use in biological dosimetry.

Low-Dose Hyper-Radiosensitivity Is Not a Common Effect in Normal Asynchronous and G2-Phase Fibroblasts of Cancer Patients

PURPOSE In our previous study, using the micronucleus assay, a low-dose hyper-radiosensitivity (HRS)-like phenomenon was observed for normal fibroblasts of 2 of the 40 cancer patients investigated. In this article we report, for the first time, the survival response of primary fibroblasts from 25 of these patients to low-dose irradiation and answer the question regarding the effect of G2-phase enrichment on HRS elicitation. METHODS AND MATERIALS The clonogenic survival of asynchronous as well as G2-phase enriched fibroblast populations was measured. Separation of G2-phase cells and precise cell counting was performed using a fluorescence-activated cell sorter. Sorted and plated cells were irradiated with single doses (0.1-4 Gy) of 6-MV x-rays. For each patient, at least 4 independent experiments were performed, and the induced-repair model was fitted over the whole data set to confirm the presence of HRS effect. RESULTS The HRS response was demonstrated for the asynchronous and G2-phase enriched cell populations of 4 patients. For the rest of patients, HRS was not defined in either of the 2 fibroblast populations. Thus, G2-phase enrichment had no effect on HRS elicitation. CONCLUSIONS The fact that low-dose hyper-radiosensitivity is not a common effect in normal human fibroblasts implies that HRS may be of little consequence in late-responding connective tissues with regard to radiation fibrosis.

The contribution of women to radiobiology: Marie Curie and beyond.

Marie Sklodowska-Curie, an extraordinary woman, a Polish scientist who lived and worked in France, led to the development of nuclear energy and the treatment of cancer. She was the laureate of two Nobel Prizes, the first woman in Europe who obtained the degree of Doctor of Science and opened the way for women to enter fields which had been previously reserved for men only. As a result of her determination and her love of freedom, she has become an icon for many female scientists active in radiation sciences. They are successors of Maria Curie and without the results of their work, improvement in radiation oncology will not be possible. Many of them shared some elements of Maria Curies biography, like high ethical and moral standards, passionate dedication to work, strong family values, and scientific collaboration with their husbands. The significance of Tikvah Alper, Alma Howard, Shirley Hornsey, Juliana Denekamp, Helen Evans, Eleanor Blakely, Elizabeth L. Travis, Fiona Stewart, Andree Dutreix, Catharine West, Peggy Olive, Ingela Turesson, Penny Jeggo, Irena Szumiel, Eleonor Blakely, Sara Rockwell and Carmel Mothersill contribution to radiation oncology is presented. All the above mentioned ladies made significant contribution to the development of radiotherapy (RT) and more efficient cancer treatment. Due to their studies, new schedules of RT and new types of ionizing radiation have been applied, lowering the incidence of normal tissue toxicity. Their achievements herald a future of personalized medicine.

An association between low-dose hyper-radiosensitivity and the early G2-phase checkpoint in normal fibroblasts of cancer patients.

In our previous study, low-dose hyper-radiosensitivity (HRS) effect was demonstrated for normal fibroblasts (asynchronous and G2-phase enriched) of 4 of the 25 cancer patients investigated. For the rest of patients, HRS was not defined in either of the 2 fibroblast populations. Thus, the study indicated that G2-phase enrichment had no influence on HRS identification. The conclusion contradicts that reported for human tumor cells, and suggests different mechanism of HRS in normal human cells. In the present paper we report, for the first time, the activity of early G2-phase checkpoint after low-dose irradiation in normal fibroblasts of these 4 HRS-positive patients and 4 HRS-negative patients and answer the question regarding the role of this checkpoint in normal human cells. The response of the early G2-phase checkpoint was determined by assessment of the progression of irradiated cells into mitosis using the mitotic marker, phosphorylated histone H3. We found evident differences in the activity of the early G2-phase checkpoint between HRS-positive and HRS-negative fibroblasts. In HRS-positive fibroblasts the checkpoint was not triggered and DNA damage was not recognized after doses lower than 0.2Gy resulting in HRS response. On the contrary, in HRS-negative fibroblasts the early G2-phase checkpoint was activated regardless of the dose in the range 0.1-2Gy. In conclusion, although cell cycle phase has no effect on the presence of HRS effect in normal human fibroblasts, the data reported here indicate that HRS response in these cells is associated with the functioning of early G2-phase checkpoint in a threshold-dose dependent manner, similarly as it takes place in most of human tumor and other cells.

Prognostic significance of proliferation rate and DNA ploidy in astrocytic gliomas treated with radiotherapy

Summary Aim The proliferative potential, and DNA ploidy in 50 brain tumours (15 grade I & II, and 35 grade III & IV astrocytomas) were investigated using bromodeoxyuridine (BrdUrd) incorporation and flow cytometry. Materials/Methods Tumour samples taken from each patient during surgery were incubated in vitro for one hour at 37°C with bromodeoxyuridine (BrdUrd), using the high pressure oxygen method. The percentage of BrdUrd-labelled cells (BrdUrd Labelling index, BrdUrd LI), and the total DNA content were evaluated. After surgery, 21 patients received conventionally fractionated radiotherapy (RT), 11 patients received accelerated RT, and 18 patients underwent hypofractionated RT. Results The tumours showed variability in BrdUrd LI values, which ranged from 0.3 to 15.8%. A significantly higher mean value for BrdUrd LI was shown in grades AIII & IV (3.5%), than in astrocytomas of grades AI & II (1.5%, p=0.005). A lower though not statistically significant percentage of DNA aneuploidy was observed in low-grade (40.2%) glioma than was seen in high-grade (65.7%) glioma. Univariate analysis showed that younger (≤50 years) patients (p=0.001), those with AI & II glioma (p=0.000), low tumour proliferation rate (BrdUrd LI ≤2.1%, p=0.006) and conventional or hypofractionated RT (p=0.000) had a significantly higher 5-year survival rate. Tumour ploidy had no influence on patients’ survival (p=0.261). However, a Cox multivariate analysis showed that only the patients’ age (>50 years), high grade tumours (AIII & IV) and accelerated RT were significantly unfavourable prognostic factors in terms of survival. Conclusions To improve RT results, younger patients (≤50 years) with fast proliferating tumours should receive more aggressive treatment.

Positive effect of single nucleotide RAD51 135G>C polymorphism and low Ku70 protein expression on female rectal cancer patients survival after preoperative radiotherapy.

BACKGROUND/AIMS This is a retrospective analysis of 103 patients having locally advanced rectal cancer who received short-course radiotherapy (SCRT). The objective of the study was to check whether a polymorphism in the RAD51 gene (135 G>C), Ku70 protein expression, and tumor microenvironment: proliferation rate measured by BrdUrdLI and Ki-67LI, hypoxia (glucose transporter-1 expression), P53 protein expression, and DNA ploidy can influence DNA repair capacity, the factors contributing to patient overall survival (OS) and the incidence of recurrences and metastases. MATERIALS AND METHODS RAD51 (135 G>C) polymorphism was evaluated using restriction fragment length polymorphism polymerase chain reaction, and proteins were identified using immunohistochemistry. RESULTS There were 3 (2.9%) tumors with RAD51 CC, 75 (72.8%) with GG, and 25 (24.3%) with GC genotypes. The median follow-up time was 63.1 months (range 2-120). Patients with CC genotype survived significantly longer than those with GG and GC genotypes and did not develop any recurrences or distant metastases. Female patients with Ku70 expression (<75.1) or RAD51CC genotype (impaired DNA damage repair and radiosensitive) had significantly longer OS (p=0.013) than those with Ku70>75.1 % or RAD51GG,GC (radioresistant phenotype) and male patients in the log-rank test. In multivariate analysis, positive prognostic factors for OS in the male patients were grade=1 and <17 days break in the treatment, whereas in the female subgroup, only radiosensitive phenotype (Ku70 <75.1% or RAD51CC genotype). CONCLUSION To the best of our knowledge, this is the first study to provide evidence for the positive effect of CC genotype of RAD51 or low Ku70 expression on OS in females with rectal cancer after SCRT.