Annette Raabe

Association of single nucleotide polymorphisms in ATM, GSTP1, SOD2, TGFB1, XPD and XRCC1 with clinical and cellular radiosensitivity

PURPOSE To examine the association of polymorphisms in ATM (codon 158), GSTP1 (codon 105), SOD2 (codon 16), TGFB1 (position -509), XPD (codon 751), and XRCC1 (codon 399) with fibrosis and also individual radiosensitivity. METHODS AND MATERIALS Retrospective analysis with 69 breast cancer patients treated with breast-conserving radiotherapy; total dose delivered was restricted to vary between 54 and 55Gy. Fibrosis was evaluated according to LENT/SOMA score. DNA was extracted from blood samples; cellular radiosensitivity was measured using the G0 assay and polymorphisms by PCR-RFLP and MALDI-TOF, respectively. RESULTS Twenty-five percent of all patients developed fibrosis of grade 2 or 3. This proportion tends to be higher in patients being polymorphic in TGFB1 or XRCC1 when compared to patients with wildtype genotype, whereas for ATM, GSTP1, SOD2 and XPD the polymorphic genotype appears to be associated with a lower risk of fibrosis. However, none of these associations are significant. In contrast, when a risk score is calculated based on all risk alleles, there was significant association with an increased risk of fibrosis (per risk allele odds ratio (ORs)=2.09, 95% confidence interval (CI): 1.32-3.55, p=0.0005). All six polymorphisms were found to have no significant effect on cellular radiosensitivity. CONCLUSIONS It is most likely that risk for radiation-induced fibrosis can be assessed by a combination of risk alleles. This finding needs to be replicated in further studies.

Social stress induces structural and functional alterations of phagocytes in rainbow trout (Oncorhynchus mykiss)

Stress induced by social conflict in rainbow trout is known to result in physiological and haematological alterations as well as immune suppression. Based on these findings we sought to determine whether structure and function of the phagocytes in subordinate fish respond to the stressful situation. Phagocytes are essentially involved in the immune defence of teleosts. Histological and ultrastructural studies of haemopoietic pronephros tissue have shown that under stress, potential phagocytes (histiocytes, reticulum cells, endothelial cells, and neutrophils) become activated. They show marked hypertrophy, increased pseudopod formation and enhanced autophagocytosis. Erythrophagocytosis is particularly conspicuous suggesting some impairment of the red blood cell vitality. In vivo and in vitro tests using yeast cells also indicate an average increase in the phagocytosis rate in the subordinate fish. However, a considerable percentage of the phagocytes show signs of degeneration. They disintegrate, thereby forming necrotic tissue areas.

Association of single nucleotide polymorphisms in the genes ATM, GSTP1, SOD2, TGFB1, XPD and XRCC1 with risk of severe erythema after breast conserving radiotherapy

PurposeTo examine the association of polymorphisms in ATM (codon 158), GSTP1 (codon 105), SOD2 (codon 16), TGFB1 (position −509), XPD (codon 751), and XRCC1 (codon 399) with the risk of severe erythema after breast conserving radiotherapy.Methods and materialsRetrospective analysis of 83 breast cancer patients treated with breast conserving radiotherapy. A total dose of 50.4 Gy was administered, applying 1.8 Gy/fraction within 42 days. Erythema was evaluated according to the Radiation Therapy Oncology Group (RTOG) score. DNA was extracted from blood samples and polymorphisms were determined using either the Polymerase Chain Reaction based Restriction-Fragment-Length-Polymorphism (PCR-RFL) technique or Matrix-Assisted-Laser-Desorption/Ionization –Time-Of-Flight-Mass-Spectrometry (MALDI-TOF). Relative excess heterozygosity (REH) was investigated to check compatibility of genotype frequencies with Hardy-Weinberg equilibrium (HWE). In addition, p-values from the standard exact HWE lack of fit test were calculated using 100,000 permutations. HWE analyses were performed using R.ResultsFifty-six percent (46/83) of all patients developed erythema of grade 2 or 3, with this risk being higher for patients with large breast volume (odds ratio, OR = 2.55, 95% confidence interval, CI: 1.03–6.31, p = 0.041). No significant association between SNPs and risk of erythema was found when all patients were considered. However, in patients with small breast volume the TGFB1 SNP was associated with erythema (p = 0.028), whereas the SNP in XPD showed an association in patients with large breast volume (p = 0.046). A risk score based on all risk alleles was neither significant in all patients nor in patients with small or large breast volume. Risk alleles of most SNPs were different compared to a previously identified risk profile for fibrosis.ConclusionsThe genetic risk profile for erythema appears to be different for patients with small and larger breast volume. This risk profile seems to be specific for erythema as compared to a risk profile for fibrosis.

Treatment of solitary brain metastasis: Resection followed by Whole brain radiation therapy (WBRT) and a radiation boost to the metastatic site

Background:Whole brain radiation therapy (WBRT) is reported to improve local control after resection of brain metastases. Improvement of survival was only observed in patients with controlled extracranial disease. The optimum radiation schedule has yet to be defined. The authors’ experience with a postoperative approach including WBRT and a radiation boost to the metastatic site is presented.Patients and Methods:Criteria for inclusion into this retrospective analysis were solitary brain metastasis, Karnofsky performance status ≥ 70%, and controlled extracranial disease. Two therapies were compared for local control and survival: surgery followed by 40 Gy WBRT (group A) versus surgery followed by 40 Gy WBRT and a 10 Gy boost (group B). Statistical analysis was performed using the Kaplan-Meier method and log-rank test.Results:33 patients were included (17 group A, 16 group B). The results suggested better local control (p = 0.0087) and survival (p = 0.0023) for group B. 17/17 patients (100%) of group A and 13/16 patients (81%) of group B showed progression of brain metastasis, 8/17 and 3/16 patients in the area of metastatic surgery. Median time to progression was 7 (1–22) months in group A and 12 (3–42) months in group B. The number of cancer-related deaths amounted to 17/17 (100%) in group A after a median interval of 9 (3–26) months, and to 9/16 (56%) in group B after 14 (4–46) months.Conclusion:After resection of solitary brain metastasis, a radiation boost in addition to WBRT seems to improve local control and survival when compared to postoperative WBRT alone. The results should be confirmed in a larger prospective trial.Hintergrund:Gemäß Literatur führt eine Ganzhirnbestrahlung nach Resektion von Hirnmetastasen zu einer besseren lokalen Kontrolle. Ein besseres Gesamtüberleben wurde nur für Patienten mit kontrollierten extrazerebralen Manifestationen beschrieben. Das optimale Strahlentherapieregime ist bisher nicht geklärt. Erfahrungen mit einem postoperativen Regime, bestehend aus Ganzhirnbestrahlung und Boost der Metastasenregion, werden präsentiert.Patienten und Methodik:Einschlusskriterien für diese retrospektive Analyse waren: Vorliegen einer solitären Hirnmetastase, Karnofsky-Index ≥ 70% und Kontrolle extrazerebraler Manifestationen (Tabelle 1). Zwei Therapien wurden für die lokale Kontrolle und das Gesamtüberleben verglichen: Metastasenresektion mit anschließender Ganzhirnbestrahlung bis 40 Gy (Gruppe A) versus Resektion mit anschließender Ganzhirnbestrahlung bis 40 Gy und einem Boost von 10 Gy (Gruppe B). Die statistische Analyse erfolgte mittels Kaplan-Meier-Methode und Log-Rank-Test.Ergebnisse:33 Patienten wurden eingeschlossen (17 Gruppe A, 16 Gruppe B). Der Vergleich beider Gruppen ergab eine bessere lokale Kontrolle (p = 0,0087) und ein besseres Gesamtüberleben (p = 0,0023) für Gruppe B (Abbildung 1). Bei 17/17 Patienten (100%) in Gruppe A und 13/16 Patienten (81%) in Gruppe B zeigte sich ein intrazerebraler Progress, bei 8/17 und 3/16 Patienten im Bereich der resezierten Metastase. Das mediane progressionsfreie Intervall betrug 7 (1–22) Monate in Gruppe A und 12 (3–42) Monate in Gruppe B. In Gruppe A verstarben 17/17 Patienten (100%) nach median 9 (3–26) Monaten tumorbedingt, in Gruppe B 9/16 Patienten (56%) nach median 14 (4–46) Monaten.Schlussfolgerung:Nach Resektion solitärer Hirnmetastasen scheint ein zusätzlich zur Ganzhirnbestrahlung applizierter Boost der Metastasenregion die lokale Kontrolle und das Überleben zu verbessern. Die Ergebnisse müssen an einem größeren Kollektiv in Form einer prospektiven Studie überprüft werden.

The potential role of G2- but not of G0-radiosensitivity for predisposition of prostate cancer

PURPOSE Comparing the chromosomal radiosensitivity of prostate cancer patients with that of healthy donors. MATERIALS AND METHODS The study was performed on 81 prostate cancer patients characterised by a clinical stage of predominantly pT2c or pT3a and a median age of 67 years. As healthy donors 60 male monozygotic twin pairs were recruited with a median age of 28 years. Chromosomal radiosensitivity was measured using both G0- and G2-assay. RESULTS No difference between healthy donors and prostate cancer patients was detected concerning G0-radiosensitivity, since medians were similar (Hodges-Lehmann estimate: -0.05, 95% CI: -0.18-0.08, p=0.4167). However, a pronounced difference was determined for G2-radiosensitivity with prostate cancer patients showing a significantly higher sensitivity compared to healthy donors (Hodges-Lehmann estimate: -0.41, 95% CI: -0.53 to -0.30, p=1.75(-9)). Using the 90% quantile of G2-radiosensitivity in healthy donors as a threshold for discrimination the fraction of prostate cancer patients with elevated radiosensitivity increased to 49%. CONCLUSION G2-, but not G0-radiosensitivity is a promising marker for predisposition of prostate cancer.

No Effect of the Transforming Growth Factor β1 Promoter Polymorphism C-509T on TGFB1 Gene Expression, Protein Secretion, or Cellular Radiosensitivity

PURPOSE To study whether the promoter polymorphism (C-509T) affects transforming growth factor β1 gene (TGFB1) expression, protein secretion, and/or cellular radiosensitivity for both human lymphocytes and fibroblasts. METHODS AND MATERIALS Experiments were performed with lymphocytes taken either from 124 breast cancer patients or 59 pairs of normal monozygotic twins. We used 15 normal human primary fibroblast strains as controls. The C-509T genotype was determined by polymerase chain reaction-restriction fragment length polymorphism or TaqMan single nucleotide polymorphism (SNP) genotyping assay. The cellular radiosensitivity of lymphocytes was measured by G0/1 assay and that of fibroblasts by colony assay. The amount of extracellular TGFB1 protein was determined by enzyme-linked immunosorbent assay, and TGFB1 expression was assessed via microarray analysis or reverse transcription-polymerase chain reaction. RESULTS The C-509T genotype was found not to be associated with cellular radiosensitivity, neither for lymphocytes (breast cancer patients, P=.811; healthy donors, P=.181) nor for fibroblasts (P=.589). Both TGFB1 expression and TGFB1 protein secretion showed considerable variation, which, however, did not depend on the C-509T genotype (protein secretion: P=.879; gene expression: lymphocytes, P=.134, fibroblasts, P=.605). There was also no general correlation between TGFB1 expression and cellular radiosensitivity (lymphocytes, P=.632; fibroblasts, P=.573). CONCLUSION Our data indicate that any association between the SNP C-509T of TGFB1 and risk of normal tissue toxicity cannot be ascribed to a functional consequence of this SNP, either on the level of gene expression, protein secretion, or cellular radiosensitivity.

Association between SNPs in defined functional pathways and risk of early or late toxicity as well as individual radiosensitivity

Background and purposeThe aim of this study was to determine the impact of functional single nucleotide polymorphism (SNP) pathways involved in the ROS pathway, DNA repair, or TGFB1 signaling on acute or late normal toxicity as well as individual radiosensitivity.Materials and methodsPatients receiving breast-conserving surgery and radiotherapy were examined either for erythema (n = 83), fibrosis (n = 123), or individual radiosensitivity (n = 123). The 17 SNPs analyzed are involved in the ROS pathway (GSTP1, SOD2, NQO1, NOS3, XDH), DNA repair (XRCC1, XRCC3, XRCC6, ERCC2, LIG4, ATM) or TGFB signaling (SKIL, EP300, APC, AXIN1, TGFB1). Associations with biological and clinical endpoints were studied for single SNPs but especially for combinations of SNPs assuming that a SNP is either beneficial or deleterious and needs to be weighted.ResultsWith one exception, no significant association was seen between a single SNP and the three endpoints studied. No significant associations were also observed when applying a multi-SNP model assuming that each SNP was deleterious. In contrast, significant associations were obtained when SNPs were suggested to be either beneficial or deleterious. These associations increased, when each SNP was weighted individually. Detailed analysis revealed that both erythema and individual radiosensitivity especially depend on SNPs affecting DNA repair and TGFB1 signaling, while SNPs in ROS pathway were of minor importance.ConclusionFunctional pathways of SNPs may be used to form a risk score allowing to predict acute and late radiation-induced toxicity but also to unravel the underlying biological mechanisms.ZusammenfassungHintergrund und ZielFür ein SNP-Netzwerk („single nucleotide polymorphism“, Einzelnukleotidpolymorphismus), welches im ROS-Signalweg, an der DNA-Reparatur und im TGFB1-Signalweg involviert ist, sollen die Bedeutung für die akute und späte Toxizität sowie die individuelle Strahlenempfindlichkeit bestimmt werden.Material und MethodenNach Strahlentherapie wurden Brustkrebspatientinnen entweder hinsichtlich des Erythems (n = 83), einer Fibrose (n = 123) oder der individuellen Strahlenempfindlichkeit (n = 123) untersucht. Die 17 untersuchten SNPs sind entweder am ROS-Pathway (GSTP1, SOD2, NQO1, NOS3, XDH), bei der DNA-Reparatur (XRCC1, XRCC3, XRCC6, ERCC2, LIG4, ATM) oder dem TGFB Signalling (SKIL, EP300, APC, AXIN1, TGFB1) beteiligt. Die Assoziation mit biologischen und klinischen Endpunkten wurde für einzelne, aber insbesondere für Kombinationen von SNPs untersucht, wobei angenommen wurde, dass ein SNPs sowohl von Vorteil als auch von Nachteil sein kann und auch gewichtet werden sollte.ErgebnisseMit einer Ausnahme wurde für einen einzelnen SNP keine signifikante Assoziation identifiziert. Ebenfalls keine signifikante Assoziation wurde gefunden, wenn alle SNPs in einem Wert zusammengefasst werden, unter der Annahme, dass ein SNP immer nachteilig ist. Im Gegensatz dazu ergeben sich signifikante Assoziationen, wenn davon ausgegangen wird, dass ein SNP entweder nachteil- oder vorteilhaft sein kann. Diese Assoziationen werden noch stärker, wenn die SNPs individuell gewichtet werden. Eine detaillierte Analyse des Netzwerks ergibt, dass das Erythem und die individuelle Strahlenempfindlichkeit insbesondere durch SNPs in der DNA-Reparatur und dem TGFB1-Signalweg bestimmt werden, während SNPs im ROS-Signalweg ohne große Bedeutung sind.SchlussfolgerungFunktionale SNP-Netzwerke können genutzt werden, um einen Risikoscore zu bilden, der es erlaubt das Risiko für akute und späte Toxizität vorherzusagen und die zugrundeliegenden Mechanismen aufzuklären.

No effect of the hemoglobin solution HBOC-201 on the response of the rat R1H tumor to fractionated irradiation.

Background and Purpose:Tumor hypoxia is regarded as one important underlying feature of radioresistance. The authors report on an experimental approach to improve tumor response to radiation by combining fractionated irradiation with HBOC-201, an ultrapurified polymerized hemoglobin solution, which is currently used in clinical phase II/III trials as alternative oxygen carrier and proved to be highly effective in tissue oxygenation (tpO2).Material and Methods:: Subcutaneously growing rhabdomyosarcoma R1H tumors of the rat were treated with either 40 Gy (2 Gy/fraction, 20 fractions in 2 weeks, ambient) followed by graded top-up doses (clamped) alone, or in combination with HBOC-201, or with HBOC-201 plus carbogen (95% O2 + 5% CO2). Local tumor control (TCD50%) and growth delay were used as endpoints. In addition, the effect of HBOC-201 alone or in combination with carbogen on the tpO2 of tumor and muscle was determined using a flexible stationary probe (Licox, GMS).Results:TCD50% values of 119 Gy (95% confidence interval 103;135), 111 Gy (84;138), and 102 Gy (83;120) were determined for tumors irradiated alone, in combination with HBOC-201, and with HBOC-201 plus carbogen, respectively. Although the dose-response curves showed a slight shift to lower doses when HBOC-201 or HBOC-201 plus carbogen was added, the differences in TCD50% were not statistically significant. No effect was seen on the growth delay of recurrent tumors. HBOC-201 alone did not effect tumor or muscle tpO2. In combination with carbogen the mean tpO2 of muscle raised from 23.9 mmHg to 59.3 mmHg (p < 0.05), but this effect was less pronounced than the increase in tpO2 by carbogen alone.Conclusion:Low-dose application of HBOC-201 does not improve the response of the rhabdomyosarcoma R1H of the rat to fractionated irradiation.Hintergrund und Ziel:Strategien zur Überwindung der hypoxievermittelten Radioresistenz von Tumoren sind gegenwärtig von großem klinischen und wissenschaftlichen Interesse. Eine Verbesserung der Sauerstoffverfügbarkeit im Tumor kann als Folge der gesteigerten O2-Transportkapazität durch eine Erhöhung der Hämoglobinkonzentration erreicht werden. Die zellfreie Hämoglobinlösung HBOC-201 wird erfolgreich in klinischen Phase-II/III-Studien eingesetzt und vermag die Sauerstoffversorgung von Gewebe effektiv zu erhöhen. Die vorliegende Arbeit sollte klären, ob HBOC-201 im Rahmen einer fraktionierten Strahlentherapie die Tumorhypoxie verringern und das Therapieergebnis verbessern kann.Material und Methodik:Eine fraktionierte Bestrahlung von subkutan wachsenden R1H-Tumoren der Ratte wurde mit täglicher, niedrigdosierter i. v. Gabe von HBOC-201 bzw. von HBOC-201 plus Carbogen (95% O2 + 5% CO2) kombiniert. Es wurden Gesamtdosen von 40 Gy fraktioniert verabreicht (2 Gy/Fraktion, 20 Fraktionen in 2 Wochen) und mit Top-up-Dosen auf Gesamtdosen aufgesättigt, die eine Bestimmung der lokalen Tumorkontrolle ermöglichten. Neben der lokalen Kontrolldosis (TCD50%) wurde die Wachstumsverzögerung der Rezidive bestimmt. Zusätzlich wurde der Effekt von HBOC-201 allein und in Kombination mit Carbogen auf die Gewebsoxygenierung (tpO2) polarometrisch bestimmt.Ergebnisse:Die Kombination einer fraktionierten Bestrahlung mit HBOC-201 bzw. mit HBOC-201 plus Carbogen hatte keinen statistisch signifikanten Einfluss auf die lokale Tumorkontrolle. Es wurde eine nur geringfügige Verringerung der TCD50%-Werte von 119 Gy (95%-Konfidenzintervall: 103;135) auf 111 Gy (84;138) bzw. auf 102 Gy (83;120) beobachtet. Die Wachstumsverzögerung der Rezidive wies ebenfalls keine Veränderung auf. Die Oxygenierung von Tumor- und Muskelgewebe konnte durch Carbogenatmung, nicht aber durch HBOC-201 verbessert werden.Schlussfolgerung:Die Wirksamkeit einer fraktionierten Tumorbestrahlung wird durch niedrigdosierte Hämoglobinlösung HBOC-201 nicht verbessert.

Influence of the Hemoglobin Solution HBOC-201 on Tissue Oxygenation in the Rat R1H-Tumor

Background: HBOC-201 is an ultra purified bovine hemoglobin solution. It has already been used in clinical phase II/III trials for emergency treatments. Animal experiments have shown that HBOC-201 is highly effective in tissue oxygenation. The study was performed in order to assess the potential of low dose HBOC-201 to improve tumor oxygenation. Methods: 30 rats with a subcutaneously growing rhabdomyosarcoma R1H tumor were randomly assigned either to be ventilated with carbogen (n = 10), or to receive an IV injection of 0.3 g/kg HBOC-201 (n = 10) or a combination of 0.3 g/kg HBOC-201 and carbogen breathing (n = 10). Under general anesthesia the effects of the respective treatment on the tissue oxygen tension (tpO2) of the tumor were determined using a flexible stationary probe at baseline (b) and 15 and 60 min after application of the respective medication. Results: HBOC-201 alone failed to improve tumor tpO2 (b: 1.3 ± 1.2 mmHg; 15 min: 1.4 ± 1 mmHg; 60 min: 1 ± 1 mmHg). In combination with carbogen the mean tpO2 of the tumor raised in comparison to baseline values (b: 3.1 ± 4.6 mmHg; 15 min: 8.5 ± 11* mmHg; 60 min: 4.8 ± 5 mmHg; *p < 0.05 vs. b), but this effect was less pronounced than the increase in tpO2 by carbogen alone (b: 3.4 ± 3.4 mmHg; 15 min: 9 ± 10* mmHg; 60 min: 13 ± 19* mmHg; *p < 0.05 vs. b). Conclusion: The application of low dose hemoglobin solution HBOC-201 does not result in improvement of tissue oxygenation in the rat rhabdomyosarcoma R1H.

Radiobiological determination of growth kinetics and radiosensitivity of pulmonary micrometastases of the R1H tumour

The aim of the study was to determine the radiosensitivity and the growth kinetics of pulmonary micrometastases of the R1H tumour of the rat. Lung metastases were induced by intravenous injection of viable tumour cells. At different time intervals (3-32 days) after injection, lungs were locally irradiated with 200 kVp X-rays, using 1.5 Gy/fraction. Total doses of 6-33 Gy were administered within 11 days. Endpoints used were survival time, local control rate, and number of metastases in the lungs at autopsy. The data were evaluated using the multi-target model. Beginning in the fifth week after tumour cell inoculation the animals started to exhibit a pronounced dyspnoea and were sacrificed. Sections revealed an extensive metastatic infiltration of the lungs. With increasing total dose a prolongation of survival time as well as an increase in cure rate was observed. The number of metastases found in the lungs decreased with increasing total dose. It is concluded that metastatic growth does not start earlier than 3 days after tumour cell inoculation and accelerates continuously. The doubling time of the tumour cells in the micrometastases decreases continuously and from 5.2 to 1.2 days between day 3 and 40, whereas larger metastases containing more than 10(6) cells show gompertzian growth kinetics. The cell doubling time in this stage of metastatic growth is longer than 5 days. During the first 4 weeks of metastatic growth the radiosensitivity of metastatic R1H cells in the lungs is the same as in vitro.