Tomasz Kuszewski

A cross-platform public domain PC image-analysis program for the comet assay.

The single-cell gel electrophoresis, also known as the comet assay, has gained wide-spread popularity as a simple and reliable method to measure genotoxic and cytotoxic effects of physical and chemical agents as well as kinetics of DNA repair. Cells are generally stained with fluorescent dyes. The analysis of comets--damaged cells which form a typical comet-shaped pattern--is greatly facilitated by the use of a computer image-analysis program. Although several image-analysis programs are available commercially, they are expensive and their source codes are not provided. For Macintosh computers a cost-free public domain macro is available on the Internet. No ready for use, cost-free program exists for the PC platform. We have, therefore, developed such a public domain program under the GNU license for PC computers. The program is called CASP and can be run on a variety of hardware and software platforms. Its practical merit was tested on human lymphocytes exposed to gamma-rays and found to yield reproducible results. The binaries for Windows 95 and Linux, together with the source code can be obtained from: http://www.casp.of.pl.

Chromosomal Aberrations and Micronuclei In Lymphocytes of Breast Cancer Patients after an Accident during Radiotherapy with 8 MeV Electrons1

Abstract Wojcik, A., Stephan, G., Sommer, S., Buraczewska, I., Kuszewski, T., Wieczorek, A. and Gózdz, S. Chromosomal Aberrations and Micronuclei In Lymphocytes of Breast Cancer Patients after an Accident during Radiotherapy with 8 MeV Electrons. Radiat. Res.160, 677–683 (2003). In February 2001 a radiation accident occurred in a radiotherapy unit of an oncology hospital in Poland. Five breast cancer patients undergoing radiotherapy received a single high dose of 8 MeV electrons. The exact doses are not known, but they were heterogeneous and may have reached about 100 Gy. To assess whether such exposure would be detectable in peripheral blood lymphocytes, chromosomal aberrations and micronuclei were analyzed in lymphocytes from the accident patients and compared to values for lymphocytes from 10 control patients who were not involved in the accident but who received similar radiotherapy treatments. Lymphocytes were harvested for analysis of chromosomal aberrations at three different culture times to determine whether heavily damaged cells reached mitosis with a delay. There was no effect of harvest time on the frequencies of chromosomal aberrations, indicating that there was no delay of heavily damaged cells in entering mitosis. A good correlation was observed between micronuclei and chromosomal aberrations. In lymphocytes from three of the accident patients, significantly enhanced frequencies of both aberrations and micronuclei were found. The great individual variability observed in the frequency of cytogenetic damage in lymphocytes from both control and accident patients precluded the unambiguous identification of all accident patients.

Individual variations in the micronucleus assay for biological dosimetry after high dose exposure.

The micronucleus assay is widely used as a biological dosimeter. Due to an inhibitory effect of radiation on cell proliferation the assay yields satisfactory results only when the absorbed dose is below about 5Gy. In 2002 Müller and Rode suggested that a modified version of the test, based on the analysis of the ratio of trinucleated to tetranucleated cells and the frequency of micronuclei (Mn) in binucleated cells containing at least one Mn, can be applied to detect a dose reaching 15Gy (Mutat. Res. 502 (2002) 47-51). Their conclusion was based on the results of experiments with lymphocytes from one donor and nothing is known about the possible influence of individual variability on the applicability of the Mn test to detect high doses of radiation. The aim of the present study was to validate the modified micronucleus assay with lymphocytes of 5 donors. Their blood was exposed to 0, 5, 10, 15 and 20Gy of (60)Co gamma rays. The levels of Mn and of cell proliferation were assessed using various approaches. A strong inter-individual variability was observed for all endpoints. The results clearly show that the assessment of cell proliferation is essential for the interpretation of results. Unfortunately, it was not possible to identify one single proliferation marker that gives all necessary information.

Comparative analysis of three functional predictive assays in lymphocytes of patients with breast and gynaecological cancer treated by radiotherapy.

Purpose There is a need to develop predictive tests that would allow identifying cancer patients with a high risk of developing side effects to radiotherapy. We compared the predictive value of three functional assays: the G0 aberration assay, the G2 aberration assay and the alkaline comet assay in lymphocytes of breast cancer and gynaecological cancer patients. Material and methods Peripheral blood was collected from 35 patients with breast cancer and 34 patients with gynaecological cancer before the onset of therapy. Chromosomal aberrations were scored in lymphocytes irradiated in the G0 or G2 phase of the cell cycle. DNA repair kinetics was performed with the alkaline comet assay following irradiation of unstimulated lymphocytes. The results were compared with the severity of early and late side effects to radiotherapy. Results No correlation was observed between the results of the assays and the severity of side effects. Moreover, each assay identified different patients as radiosensitive. Conclusions There is no simple correlation between the in vitro sensitivity of lymphocytes and the risk of developing early and late side effects.

Usage of ICP Algorithm for Initial Alignment in B-Splines FFD Image Registration in Breast Cancer Radiotherapy Planning

Estimation of a resected tumor lodge localization after a breast cancer surgery is a demanding task for the radiotherapy planning. The image registration techniques can be used to improve the radiotherapy. The initial alignment of two volumes is an important aspect of medical image registration procedure. We propose usage of the iterative closest point in two different scenarios: as a initial alignment, replacing intensity based rigid registration and as a initial transform to speed-up traditional rigid registration process. Two versions of the algorithm are presented: a point matching between bone structures and a line matching between volume edges. The correctness and usefulness are evaluated using: a target registration error, comparison of the computation time and convergence ratios, and visual inspection. The results demonstrate that the usage of iterative closest point algorithm significantly improve the initial alignment process in terms of the computation time.

Modulation of radiation-induced cytogenetic damage in human peripheral blood lymphocytes by hypothermia.

PURPOSE Recent studies have shown that low temperature (hypothermia) at exposure can act in a radio-protective manner at the level of cytogenetic damage. The mechanisms of this phenomenon are not understood, but it was suggested to be due to hypothermia-induced perturbations of the cell cycle. The purpose of the present study was to detect whether a reduced frequency of micronuclei is observed in peripheral blood lymphocytes (PBL) irradiated at low temperature and harvested sequentially at 3 time points. Additionally, the level of apoptosis was estimated by microscopic analysis of the MN slides. MATERIALS AND METHODS Experiments were carried out with blood drawn from three donors at the Stockholm University and from three donors at the Jan Kochanowski University. Prior to irradiation, blood samples were incubated for 20min and irradiated at the respective temperature (0°C and 37°C) with gamma rays. Whole blood cultures were set up, cytochalasin B was added after 44h of irradiation and the samples were harvested after 72, 96 and 120h of incubation time. RESULTS AND CONCLUSIONS The frequency of micronuclei was markedly lower in PBL harvested at 72h, 96h and 120h following irradiation at 0°C as compared to 37°C. This indicates that the temperature effect observed in peripheral blood lymphocytes after irradiation is not related to a temporary perturbation of the cell cycle. Also, it is not due to selective elimination of damaged cells by apoptosis.

Improving oncoplastic breast tumor bed localization for radiotherapy planning using image registration algorithms

Knowledge about tumor bed localization and its shape analysis is a crucial factor for preventing irradiation of healthy tissues during supportive radiotherapy and as a result, cancer recurrence. The localization process is especially hard for tumors placed nearby soft tissues, which undergo complex, nonrigid deformations. Among them, breast cancer can be considered as the most representative example. A natural approach to improving tumor bed localization is the use of image registration algorithms. However, this involves two unusual aspects which are not common in typical medical image registration: the real deformation field is discontinuous, and there is no direct correspondence between the cancer and its bed in the source and the target 3D images respectively. The tumor no longer exists during radiotherapy planning. Therefore, a traditional evaluation approach based on known, smooth deformations and target registration error are not directly applicable. In this work, we propose alternative artificial deformations which model the tumor bed creation process. We perform a comprehensive evaluation of the most commonly used deformable registration algorithms: B-Splines free form deformations (B-Splines FFD), different variants of the Demons and TV-L1 optical flow. The evaluation procedure includes quantitative assessment of the dedicated artificial deformations, target registration error calculation, 3D contour propagation and medical experts visual judgment. The results demonstrate that the currently, practically applied image registration (rigid registration and B-Splines FFD) are not able to correctly reconstruct discontinuous deformation fields. We show that the symmetric Demons provide the most accurate soft tissues alignment in terms of the ability to reconstruct the deformation field, target registration error and relative tumor volume change, while B-Splines FFD and TV-L1 optical flow are not an appropriate choice for the breast tumor bed localization problem, even though the visual alignment seems to be better than for the Demons algorithm. However, no algorithm could recover the deformation field with sufficient accuracy in terms of vector length and rotation angle differences.

Application of B-splines FFD image registration in breast cancer radiotherapy planning

Localization of a resected tumor lodge after a breast cancer surgery is a challenging task for the radiotherapy planning. Currently, the problem is handled by creating radiation dose margins, which take into account the lack of information about lodge localization. We propose an alternative approach based on the image registration techniques, that aims to align computed tomography 3D images before and after surgery and to estimate the resected tumor lodge localization. The rigid registration and free-form deformations based on B-Splines are evaluated with additional 3D images preprocessing and analytical gradient calculation to speed-up the registration process. The correctness and usefulness are evaluated using target registration error, visual inspection, and physician opinion. The results demonstrate that the usage of image registration techniques can improve the quality of the radiotherapy planning.

CT segmentation based on MRI images in context of prostate radiotherapy planning

This paper addresses a problem of automatic segmentation of computed tomography (CT) data in context of prostate radiotherapy planning. A new 3D algorithm is proposed in which a prostate is automatically contoured in CT images. The proposed segmentation scenario consists of the following steps: 1) both CT and magnetic resonance (MR) data of a patient are acquired, 2) due to better visibility of soft tissues in MR images, soft organs are segmented in MR data using active contour method (snakes) with additional gradient vector flow enhancement, 3) then obtained 3D contours are mapped from MR to CT images (using mutual information criterion) by means of a flexible registration technique in which global affine transformation is combined with local B-spline free from deformation method. During segmentation of the MR images prior knowledge about a mean ellipsoidal prostate shape, extracted before, plays a role of an addition constraint. Obtained results are compared with manual segmentation done by medical doctors using Dice similarity measure.

Application of demons image registration algorithms in resected breast cancer lodge localization

An estimation of a resected cancer lodge localization after breast tumor surgery is a challenging task during radiotherapy planning. Knowledge about the tumor lodge position and shape could improve the radiation dose distribution. However, the tumor no longer exists after the surgery, but information about its position is available in the 3D image acquired before the surgery. Therefore, image registration algorithms can be used to estimate the tumor lodge localization and potentially improve the radiotherapy planning. In this work, we evaluate different variants of a Demons image registration algorithm. The nonparametric Demons algorithms are compared to a parametric registration procedure, the B-Splines free-form deformations. The results are evaluated using a target registration error and a medical expert visual inspection. The results show that for small deformations, the diffeomorphic, symmetric Demons are the most reliable, but for larger deformations, parametric B-Splines free-form deformations provide better results. Results demonstrate that there is still a place for a specialized algorithm development.