Martin Simonsson

Low-dose hypersensitive γH2AX response and infrequent apoptosis in epidermis from radiotherapy patients

BACKGROUND AND PURPOSE A low-dose hypersensitivity to radiation can be observed in vitro for many human cell types in terms of increased cell kill per dose unit for doses below 0.5Gy. Quantification of the double-strand break marker gammaH2AX in samples taken in clinical radiotherapy practice has the potential to provide important information about how induction and repair of severe DNA damage and apoptosis are linked to low-dose hypersensitivity. MATERIAL AND METHODS The effects of exposure to low doses (0.05-1.1Gy) were investigated in skin biopsies taken from prostate cancer patients undergoing the first week of radiotherapy. gammaH2AX foci and apoptotic cells were visualised by immunohistochemistry and quantified by image analysis. RESULTS The gammaH2AX foci pattern in biopsies taken 30min after a single fraction revealed a low-dose hypersensitivity below 0.3Gy (p=0.0009). The result was consistent for repeated fractions (p=0.00001). No decrease in foci numbers could be detected when comparing biopsies taken 30min and 2h after single fractions of 0.4 and 1.2Gy. The result was consistent for repeated fractions. Only 43 of 168,000 cells in total were identified as apoptotic, yet a dose dependency could be detected after 1week of radiotherapy (p=0.003). CONCLUSIONS We describe a method based on gammaH2AX to study DNA damage response and apoptosis in a clinical setting. A gammaH2AX hypersensitive response to low doses can be observed in epidermal skin, already 30min following delivered fraction. A very low frequency of apoptosis in normal epithelium suggests that this effect is not an important part of the in vivo response to low doses.

A low-dose hypersensitive keratinocyte loss in response to fractionated radiotherapy is associated with growth arrest and apoptosis

BACKGROUND AND PURPOSE The existence of a hypersensitive radiation response to doses below 0.5Gy is well established for many normal and tumour cell lines. There is also evidence for hypersensitive tissue responses in acute skin damage and kidney function in mice. Recently, we have identified that a hypersensitive gammaH2AX response exists in human epidermis. The aim of this study was to investigate the dose-response of basal clonogenic keratinocytes in normal skin to fractionated radiotherapy with low dose fractions. MATERIALS Skin punch biopsies were taken before and during radiotherapy from prostate cancer patients undergoing radiotherapy with a curative intent. Areas of epidermis receiving daily fractions of approximately 0.1, 0.2, 0.45 and 1.1Gy were biopsied on the same occasion to determine dose-response for each individual patient. In total, 89 cases were assessed either at 1, 2.5, 3, 4, 5 or 6.5 weeks in the treatment course. Biopsy sampling of another 25 patients was performed from areas receiving 0.45 and 1.1Gy per fraction at regular intervals throughout the 7-week treatment period. The number of basal keratinocytes per mm of the interfollicular epidermis was determined. The DNA damage response of the basal keratinocytes was investigated by immunohistochemical staining for molecular markers of growth arrest, mitosis and cell death, using p21, phospho-H3 and gammaH2AX, respectively. The number of stained keratinocytes in the basal layer was counted manually. The p21 staining was also quantified by digital image analysis. RESULTS The individual dose-response relationships revealed a low-dose hypersensitivity for reduction of basal keratinocytes throughout 7 weeks of radiotherapy (p<0.01). Growth arrest and cell proliferation assessed at 1 week and 6.5 weeks showed, in both cases, hypersensitive increase of p21 (p<0.01) and hypersensitive depression of mitosis (p<0.01). Manual counting and digital image analysis of p21 showed good agreement. Cell death was infrequent but increased significantly between 1 and 6.5 weeks and displayed a hypersensitive dose-response at the end of the treatment period. CONCLUSIONS A low-dose hypersensitivity in basal skin keratinocyte reduction is present throughout 7 weeks of radiotherapy. A persistent hypersensitive growth arrest response and cell killing mediate this effect.

Multicellular Tumour Spheroid as a model for evaluation of [18F]FDG as biomarker for breast cancer treatment monitoring

BackgroundIn order to explore a pre-clinical method to evaluate if [18F]FDG is valid for monitoring early response, we investigated the uptake of FDG in Multicellular tumour spheroids (MTS) without and with treatment with five routinely used chemotherapy agents in breast cancer.MethodsThe response to each anticancer treatment was evaluated by measurement of the [18F]FDG uptake and viable volume of the MTSs after 2 and 3 days of treatment.ResultsThe effect of Paclitaxel and Docetaxel on [18F]FDG uptake per viable volume was more evident in BT474 (up to 55% decrease) than in MCF-7 (up to 25% decrease).Doxorubicin reduced the [18F]FDG uptake per viable volume more noticeable in MCF-7 (25%) than in BT474 MTSs.Tamoxifen reduced the [18F]FDG uptake per viable volume only in MCF-7 at the highest dose of 1 μM.No effect of Imatinib was observed.ConclusionMTS was shown to be appropriate to investigate the potential of FDG-PET for early breast cancer treatment monitoring; the treatment effect can be observed before any tumour size changes occur.The combination of PET radiotracers and image analysis in MTS provides a good model to evaluate the relationship between tumour volume and the uptake of metabolic tracer before and after chemotherapy. This feature could be used for screening and selecting PET-tracers for early assessment of treatment response.In addition, this new method gives a possibility to assess quickly, and in vitro, a good preclinical profile of existing and newly developed anti-cancer drugs.

One kilometer (1 km) electric solar wind sail tether produced automatically

We produced a 1 km continuous piece of multifilament electric solar wind sail tether of μm-diameter aluminum wires using a custom made automatic tether factory. The tether comprising 90,704 bonds between 25 and 50 μm diameter wires is reeled onto a metal reel. The total mass of 1 km tether is 10 g. We reached a production rate of 70 m/24 h and a quality level of 1‰ loose bonds and 2‰ rebonded ones. We thus demonstrated that production of long electric solar wind sail tethers is possible and practical.

Predictors of histology, tissue eosinophilia and mast cell infiltration in Hodgkin's lymphoma--a population-based study.

Objective:  Classical Hodgkin’s lymphoma (HL) lesions comprise few tumour cells, surrounded by numerous inflammatory cells. Like in other malignancies, the microenvironment is presumed to be clinically important in HL; however, microenvironment predictors remain poorly characterised. The aim of this study was to investigate how selected patient characteristics and genetic factors affect HL phenotype, in particular tissue eosinophilia, mast cell counts and HL histological subtype.

Automated classification of immunostaining patterns in breast tissue from the Human Protein Atlas

Background: The Human Protein Atlas (HPA) is an effort to map the location of all human proteins (http://www.proteinatlas.org/). It contains a large number of histological images of sections from human tissue. Tissue micro arrays (TMA) are imaged by a slide scanning microscope, and each image represents a thin slice of a tissue core with a dark brown antibody specific stain and a blue counter stain. When generating antibodies for protein profiling of the human proteome, an important step in the quality control is to compare staining patterns of different antibodies directed towards the same protein. This comparison is an ultimate control that the antibody recognizes the right protein. In this paper, we propose and evaluate different approaches for classifying sub-cellular antibody staining patterns in breast tissue samples. Materials and Methods: The proposed methods include the computation of various features including gray level co-occurrence matrix (GLCM) features, complex wavelet co-occurrence matrix (CWCM) features, and weighted neighbor distance using compound hierarchy of algorithms representing morphology (WND-CHARM)-inspired features. The extracted features are used into two different multivariate classifiers (support vector machine (SVM) and linear discriminant analysis (LDA) classifier). Before extracting features, we use color deconvolution to separate different tissue components, such as the brownly stained positive regions and the blue cellular regions, in the immuno-stained TMA images of breast tissue. Results: We present classification results based on combinations of feature measurements. The proposed complex wavelet features and the WND-CHARM features have accuracy similar to that of a human expert. Conclusions: Both human experts and the proposed automated methods have difficulties discriminating between nuclear and cytoplasmic staining patterns. This is to a large extent due to mixed staining of nucleus and cytoplasm. Methods for quantification of staining patterns in histopathology have many applications, ranging from antibody quality control to tumor grading.

Telomerase antagonist imetelstat inhibits esophageal cancer cell growth and increases radiation-induced DNA breaks.

Telomerase is mainly active in human tumor cells, which provides an opportunity for a therapeutic window on telomerase targeting. We sought to evaluate the potential of the thio-phosphoramidate oligonucleotide inhibitor of telomerase, imetelstat, as a drug candidate for treatment of esophageal cancer. Our results showed that imetelstat inhibited telomerase activity in a dose-dependent manner in esophageal cancer cells. After only 1 week of imetelstat treatment, a reduction of colony formation ability of esophageal cancer cells was observed. Furthermore, long-term treatment with imetelstat decreased cell growth of esophageal cancer cells with different kinetics regarding telomere lengths. Short-term imetelstat treatment also increased γ-H2AX and 53BP1 foci staining in the esophageal cancer cell lines indicating a possible induction of DNA double strand breaks (DSBs). We also found that pre-treatment with imetelstat led to increased number and size of 53BP1 foci after ionizing radiation. The increase of 53BP1 foci number was especially pronounced during the first 1h of repair whereas the increase of foci size was prominent later on. This study supports the potential of imetelstat as a therapeutic agent for the treatment of esophageal cancer.

Automated classification of glandular tissue by statistical proximity sampling

Due to the complexity of biological tissue and variations in staining procedures, features that are based on the explicit extraction of properties from subglandular structures in tissue images may have difficulty generalizing well over an unrestricted set of images and staining variations. We circumvent this problem by an implicit representation that is both robust and highly descriptive, especially when combined with a multiple instance learning approach to image classification. The new feature method is able to describe tissue architecture based on glandular structure. It is based on statistically representing the relative distribution of tissue components around lumen regions, while preserving spatial and quantitative information, as a basis for diagnosing and analyzing different areas within an image. We demonstrate the efficacy of the method in extracting discriminative features for obtaining high classification rates for tubular formation in both healthy and cancerous tissue, which is an important component in Gleason and tubule-based Elston grading. The proposed method may be used for glandular classification, also in other tissue types, in addition to general applicability as a region-based feature descriptor in image analysis where the image represents a bag with a certain label (or grade) and the region-based feature vectors represent instances.

Tissue microarray and digital image analysis: a methodological study with special reference to the microenvironment in Hodgkin lymphoma

Glimelius I, Qvarnström F, Simonsson M, Ekwall A, Smedby K E, Molin D & Amini R‐M 
(2012) Histopathology 61, 26–32

Double strand break induction and kinetics indicate preserved hypersensitivity in keratinocytes to subtherapeutic doses for 7 weeks of radiotherapy

BACKGROUND AND PURPOSE Previously we reported that hyper-radiosensitivity (HRS) was evidenced by quantifying DNA double strand break (DSB) foci in epidermis biopsies collected after delivering radiotherapeutic one and five dose fractions. The aim of this study was to determine whether HRS was preserved throughout a 7-week radiotherapy treatment, and also to examine the rate of foci decline and foci persistence between dose fractions. MATERIALS AND METHODS 42 patients with prostate cancer received 7-week fractionated radiotherapy treatment (RT) with daily dose fractions of 0.05-1.10Gy to the skin. Before RT, and at several times throughout treatment, skin biopsies (n=452) were collected at 30min, and 2, 3, 24, and 72h after dose fractions. DSB-foci markers, γH2AX and 53BP1, were labelled in epidermal keratinocytes with immunofluorescence and immunohistochemical staining. Foci were counted both with digital image analysis and manually. RESULTS HRS in keratinocytes was evidenced by the dose-response relationships of DSB foci, observed throughout the treatment course, independent of sampling time and quantification method. Foci observed at 24h after dose fractions indicated considerable DSB persistence. Accordingly, foci significantly accumulated after 5 consecutive dose fractions. For doses below 0.3Gy, persistent foci could be observed even at 72h after damage induction. A comparison of γH2AX and 53BP1 quantifications in double-stained biopsies showed similar HRS dose-response relationships. CONCLUSIONS These results represented the first evidence of preserved HRS, assessed by γH2AX- and 53BP1-labelled DSB foci, throughout a 7-week treatment course with daily repeated subtherapeutic dose fractions.