Jan Palm

Irradiation Induces a Biphasic Expression of Pro-Inflammatory Cytokines in the Lung

Background and Purpose:The precise pathophysiological mechanisms of radiation-induced lung injury are poorly understood, but have been shown to correlate with dysregulation of different cytokines. The purpose of this study was to evaluate the time course of the pro-inflammatory cytokines tumor necrosis factor-(TNF-)α, interleukin-(IL-)1α and IL-6 after whole-lung irradiation.Material and Methods:The thoraces of C57BL/6J mice were irradiated with 12 Gy. Treated and control mice were sacrificed at 0.5, 1, 3, 6, 12, 24, 48, 72 h, 1, 2, 4, 8, 16, and 24 weeks post irradiation (p. i.). Real-time multiplex RT-PCR (reverse transcriptase polmyerase chain reaction) was established to evaluate the expression of TNF-α, IL-1α and IL-6 in the lung tissue of the mice. For histological analysis, lung tissue sections were stained by hematoxylin and eosin.Results:Multiplex RT-PCR analysis revealed a biphasic expression of these pro-inflammatory cytokines in the lung tissue after irradiation. After an initial increase at 1 h p. i. for TNF-α and at 6 h p. i. for IL-1α and IL-6, the mRNA expression of these pro-inflammatory cytokines returned to basal levels (48 h, 72 h, 1 week, 2 weeks p. i.). During the pneumonic phase, TNF-α, IL-1α and IL-6 were significantly elevated and revealed their maximum at 8 weeks p. i. Histopathologic evaluation of the lung sections obtained within 4 weeks p. i. revealed only minor lung damage in 5–30% of the lung tissue. By contrast, at 8, 16, and 24 weeks p. i., 70–90% of the lung tissue revealed histopathologically detectable organizing alveolitis.Conclusion:Irradiation induces a biphasic expression of pro-inflammatory cytokines in the lung. The initial transitory cytokine response occurred within the first hours after lung irradiation with no detectable histopathologic alterations. The second, more persistent cytokine elevation coincided with the onset of histologically discernible organizing acute pneumonitis.Hintergrund und Ziel:Die genaue Pathophysiologie der strahleninduzierten Lungenschädigung ist bislang nur unvollständig geklärt, scheint aber mit einer Dysregulation verschiedener Zytokine assoziiert zu sein. Das Ziel dieser experimentellen Studie war es, den zeitlichen Expressionsverlauf der proinflammatorischen Zytokine Tumor-Nekrose-Faktor-(TNF-)α, Interleukin-(IL-)1α und IL-6 nach Lungenbestrahlung zu untersuchen.Material und Methodik:Bei C57BL/6J-Mäusen wurde eine Ganzlungenbestrahlung mit 12 Gy durchgeführt. Die Versuchstiere und unbestrahlte Kontrolltiere wurden zu unterschiedlichen Zeitpunkten (0,5, 1, 3, 6, 12, 24, 48, 72 h bzw. 1, 2, 4, 8, 16 und 24 Wochen) nach Bestrahlung getötet. Im Lungengewebe wurde die mRNA-Expression von TNF-α, IL-1α und IL-6 mit Hilfe der Real-Time-multiplex-RT-PCR (Reverse-Transkriptase-Polymerase-Kettenreaktion) quantifiziert. Für die histologische Beurteilung der Lungenpräparate wurde eine Hämatoxylin-Eosin-Färbung durchgeführt.Ergebnisse:Im Rahmen der strahleninduzierten Lungenschädigung konnte mit Hilfe der Multiplex-RT-PCR eine biphasische Expression dieser proinflammatorischen Zytokine nachgewiesen werden. Nach einem initialen Anstieg—bereits 1 h nach Bestrahlung für TNF-α und nach 6 h für IL-1α und IL-6—ging die Zytokinexpression auf Ausgangswerte zurück. Während der Pneumonitisphase war die mRNA-Expression von TNF-α, IL-1α und IL-6 signifikant erhöht und ereichte 8 Wochen nach Bestrahlung ihr Maximum. Während sich bis zu 4 Wochen nach Bestrahlung histopathologisch nur eine geringe Lungenschädigung in 5–30% des Gesamtlungengewebes beobachten ließ, waren nach 8, 16 bzw. 24 Wochen 70–90% des Gesamtlungengewebes im Sinne einer radiogenen Pneumonitis geschädigt.Schlussfolgerung:Im Rahmen der radiogenen Lungenreaktion konnte eine biphasische Expression der proinflammatorischen Zytokine nachgewiesen werden. Die initiale Zytokinerhöhung erfolgte in den ersten Stunden nach Lungenbestrahlung, ohne dass histopathologisch eine Lungenschädigung nachweisbar war. Die zweite, länger persistierende Zytokinerhöhung (einige Wochen nach Bestrahlung) korrelierte mit dem Beginn einer histologisch nachweisbaren radiogenen Pneumonitis.

Cytokine Plasma Levels: Reliable Predictors for Radiation Pneumonitis?

Background Radiotherapy (RT) is the primary treatment modality for inoperable, locally advanced non-small-cell lung cancer (NSCLC), but even with highly conformal treatment planning, radiation pneumonitis (RP) remains the most serious, dose-limiting complication. Previous clinical reports proposed that cytokine plasma levels measured during RT allow to estimate the individual risk of patients to develop RP. The identification of such cytokine risk profiles would facilitate tailoring radiotherapy to maximize treatment efficacy and to minimize radiation toxicity. However, cytokines are produced not only in normal lung tissue after irradiation, but are also over-expressed in tumour cells of NSCLC specimens. This tumour-derived cytokine production may influence circulating plasma levels in NSCLC patients. The aim of the present study was to investigate the prognostic value of TNF-α, IL-1β, IL-6 and TGF-β1 plasma levels to predict radiation pneumonitis and to evaluate the impact of tumour-derived cytokine production on circulating plasma levels in patients irradiated for NSCLC. Methodology/Principal Findings In 52 NSCLC patients (stage I–III) cytokine plasma levels were investigated by ELISA before and weekly during RT, during follow-up (1/3/6/9 months after RT), and at the onset of RP. Tumour biopsies were immunohistochemically stained for IL-6 and TGF-β1, and immunoreactivity was quantified (grade 1–4). RP was evaluated according to LENT-SOMA scale. Tumour response was assessed according to RECIST criteria by chest-CT during follow-up. In our clinical study 21 out of 52 patients developed RP (grade I/II/III/IV: 11/3/6/1 patients). Unexpectedly, cytokine plasma levels measured before and during RT did not correlate with RP incidence. In most patients IL-6 and TGF-β1 plasma levels were already elevated before RT and correlated significantly with the IL-6 and TGF-β1 production in corresponding tumour biopsies. Moreover, IL-6 and TGF-β1 plasma levels measured during follow-up were significantly associated with the individual tumour responses of these patients. Conclusions/Significance The results of this study did not confirm that cytokine plasma levels, neither their absolute nor any relative values, may identify patients at risk for RP. In contrast, the clear correlations of IL-6 and TGF-β1 plasma levels with the cytokine production in corresponding tumour biopsies and with the individual tumour responses suggest that the tumour is the major source of circulating cytokines in patients receiving RT for advanced NSCLC.

Matrix-Metallo-Proteinases and their tissue inhibitors in radiation-induced lung injury

Purpose: Remodeling of extracellular matrix (ECM) after lung damage depends on collagen degrading Matrix-Metallo-Proteinases (MMP) and their endogenous inhibitors (Tissue-Inhibitors of Metallo-Proteinases, TIMP). Transforming growth factor (TGF)-β1 has been implicated in the pathogenesis of radiation-induced lung fibrosis upon its effects on fibroblast proliferation and collagen synthesis. Lung cancer patients have often elevated TGF-β1 plasma levels as a result of increased TGF-β1 expression in their tumours. On this background, we investigated the effect of irradiation on the MMP/TIMP system in the lung tissue of normal and transgenic TGF-β1 mice, in which TGF-β1 is overexpressed in the liver resulting in high TGF-β1 plasma levels. Material and methods: Transgenic (TG) and wild-type (WT) mice underwent thoracic irradiation with 12 Gy or sham-irradiation. For each study group (TG 12 Gy; TG 0 Gy; WT 12 Gy; WT 0 Gy) 8 mice were sacrificed at 4 and 8 weeks after (sham-) irradiation. The TGF-β1, TIMP-1/-2/-3 expression in the lung tissue was quantified by Western blot; the MMP-2 and MMP-9 activity was analysed by zymography. The cellular origin of the MMP and TIMP was localised by immunohistochemistry. Results: Irradiation had no influence on the TIMP-1/-2/-3, but increased significantly the MMP-2 /-9 expression. In the lung tissue of TG mice the TIMP-1/-2/-3 expression was elevated, the MMP-9 activity was decreased. The immunhistochemical study showed that parenchymal and inflammatory cells express these MMP/TIMP. Conclusion: Our results provide evidence that the overexpression of MMP-2 and MMP-9 is involved in the inflammatory response of radiation-induced lung injury. MMP-2 and MMP-9 are known to degrade collagen IV of basement membranes, therefore affecting the structural integrity of lung tissue. In contrast, in lung tissue of TG mice the TIMP-1/-2/-3 expression was up-regulated and the MMP-9 activity was diminished, thereby decreasing possibly the ECM degradation leading to lung fibrosis.

Ewing’s sarcoma and peripheral primitive neuroectodermal tumor cells produce large quantities of bioactive tumor necrosis factor-α (tnf-α) after radiation exposure

Abstract Purpose In the present study, we examined human Ewing’s sarcoma (ES) and peripheral primitive neuroectodermal tumor (pPNET) cell lines that are able to produce TNF-α as part of the response to irradiation. Radiation-induced tumor cell production of TNF-α may enhance irradiation efficacy and improve the effect of local tumor irradiation. On the other hand, radiation-induced tumor cell production of TNF-α may adversely affect the normal tissue. Methods and materials Twelve different ES/pPNET cell lines were investigated in vitro and (after establishment as tumor xenografts in athymic nude mice) in vivo for their TNF-α mRNA expression (real-time quantitative reverse transcriptase polymerase chain reaction) and TNF-α protein production ( in vitro : enhanced amplified sensitivity immunoassay; in vivo : immunohistochemistry) after exposure to different irradiation doses (2, 5, 10, 20, 30, or 40 Gy) and after different time intervals (1, 3, 6, 12, 24, 48, or 72 h after irradiation). The bioactivity of the TNF-α protein was evaluated in chromogenic cytotoxicity and neutralization assays. Results Nine out of 12 ES/pPNET cell lines express constitutively significant quantities of bioactive TNF-α in vitro . ES/pPNET cells originating from primary tumors secreted higher TNF-α levels than cells derived from metastatic lesions. In 5 of the 9 TNF-α–producing cell lines, TNF-α mRNA and protein levels were upregulated after irradiation exposure in a time- and dose-dependent manner. After establishment of the ES/pPNET cell lines in athymic nude mice, the radiation-induced TNF-α release could be demonstrated also in the xenograft tumors in vivo (analogous to the in vitro experiments). Using the same methods for quantitative analysis, it was determined that the TNF-α expression of the radiation-responsive tumor cells was up to 2000-fold higher compared to the maximal radiation-induced TNF-α release in normal lung tissue measured during the pneumonic phase. Conclusion Certain ES/pPNET cell lines produce extremely large quantities of bioactive TNF-α after radiation exposure in a time- and dose-dependent manner. Radiation-induced TNF-α production of tumor cells may be of paramount importance in respect to not only tumor behavior, but also to potential damage to normal tissue and the clinical status of the host.

Set-up errors and planning margins in planar and CBCT image-guided radiotherapy using three different imaging systems: A clinical study for prostate and head-and-neck cancer.

PURPOSE The purpose of this work is to compare the positioning accuracy achieved by three different imaging techniques and planar vs. CBCT imaging for two common IGRT indications. METHODS A collective of prostate cancer and head-and-neck cancer patients treated at our institution during the year 2013 was retrospectively analyzed. For all treatment fractions (3078 in total), the kind of acquired set-up image and the performed couch shift before treatment were assessed. The distribution of couch corrections was compared for three different imaging systems available at our institution: the treatment beam line operating at 6 MV, a dedicated imaging beam line of nominally 1 MV, and the kVision system at 70-121 kV. Shifts were analyzed for planar and cone-beam CT images. Based on the set-up corrections, CTV to PTV expansion margins were calculated. RESULTS The difference in set-up corrections performed for the three energies and both techniques (planar vs. CBCT) was not significant for head-and-neck cancer patients. For prostate cancer all shifts had equal variance. Averages ranged from -0.7 to +0.7 mm. The set-up margins calculated on the basis of the observed shifts are 4.0 mm (AP) and 3.8 mm (SI, LR) for the head-and-neck PTV and 6.6 mm (SI), 6.7 mm (AP) and 7.9 mm (LR) for the prostate cancer patients. CONCLUSIONS For three different linac-based imaging energies and planar/CBCT imaging, no relevant differences in set-up shifts were observed. The suggested set-up margins for these indications are of the order of 4 mm for head-and-neck and 6-8 mm for prostate treatment.

DNA-damage foci to detect and characterize DNA repair alterations in children treated for pediatric malignancies.

Purpose In children diagnosed with cancer, we evaluated the DNA damage foci approach to identify patients with double-strand break (DSB) repair deficiencies, who may overreact to DNA-damaging radio- and chemotherapy. In one patient with Fanconi anemia (FA) suffering relapsing squamous cell carcinomas of the oral cavity we also characterized the repair defect in biopsies of skin, mucosa and tumor. Methods and Materials In children with histologically confirmed tumors or leukemias and healthy control-children DSB repair was investigated by counting γH2AX-, 53BP1- and pATM-foci in blood lymphocytes at defined time points after ex-vivo irradiation. This DSB repair capacity was correlated with treatment-related normal-tissue responses. For the FA patient the defective repair was also characterized in tissue biopsies by analyzing DNA damage response proteins by light and electron microscopy. Results Between tumor-children and healthy control-children we observed significant differences in mean DSB repair capacity, suggesting that childhood cancer is based on genetic alterations affecting DNA repair. Only 1 out of 4 patients with grade-4 normal-tissue toxicities revealed an impaired DSB repair capacity. The defective DNA repair in FA patient was verified in irradiated blood lymphocytes as well as in non-irradiated mucosa and skin biopsies leading to an excessive accumulation of heterochromatin-associated DSBs in rapidly cycling cells. Conclusions Analyzing human tissues we show that DSB repair alterations predispose to cancer formation at younger ages and affect the susceptibility to normal-tissue toxicities. DNA damage foci analysis of blood and tissue samples allows one to detect and characterize DSB repair deficiencies and enables identification of patients at risk for high-grade toxicities. However, not all treatment-associated normal-tissue toxicities can be explained by DSB repair deficiencies.

mARC prostate treatment planning with Varian Eclipse for flat vs. FFF beams

PURPOSE The modulated arc (mARC) technique as an alternative to VMAT is a rotational IMRT irradiation with burst mode delivery. Varian has recently implemented an option for mARC-planning into the Eclipse treatment planning system (TPS) and so far mARC-planning with this TPS has not been evaluated systematically. Therefore, for prostate treatment with Eclipse we compare mARC with IMRT using flat (6MV) and flattening-filter-free (FFF, 7MV) beam energies. METHODS For ten prostate cancer patients standardized re-contouring and re-planning was performed with a prescription of 76Gy to the complete planning-target-volume (PTV). IMRT and mARC plans (6MV vs. FFF 7MV) were compared pairwise considering indices for plan quality. All plans were delivered on an anthromorphic phantom equipped with thermoluminescent dosimeters to measure out-of-field dose and treatment times. RESULTS Regarding PTV coverage, there was no marked preference for either technique or energy. The evaluation of organs at risk showed improved bladder sparing of the mARC plans up to about 75Gy; above this dose the IMRT plans achieved significant better sparing. The use of the FFF-beam-energy and mARC-technique resulted in a significant decrease in out-of-field dose. This combination also led to a drastic reduction of treatment time by factor of three in comparison with 6MV IMRT. CONCLUSION While highly conformal treatment plans could be created by the use of all modalities, the combination of the high dose rate with mARC appears to be the preferable option as it benefits from a marked decrease in treatment time and out-of-field dose.

Planning study and dose measurements of intracranial stereotactic radiation surgery with a flattening filter-free linac

PURPOSE Flattening filter-free (FFF) beams have recently become available for radiation therapy, offering much higher dose rates but complicating treatment owing to the nonflat profile. Stereotactic treatment is one of the most evident scenarios to investigate the use of FFF beams. METHODS AND MATERIALS We present a planning study of a FFF 7-MV beam for the treatment of brain metastases using multiple noncoplanar arcs. Plan differences as compared with flat 6 MV photon fields are estimated using different measures of quality. Absolute dosimetry and fluence distribution are verified and the out-of-field dose is measured. RESULTS The FFF 7-MV plans are slightly better than the flat 6-MV plans as evaluated by a number of quality indices, dose to organs at risk, and out-of-field dose, although differences may not be clinically relevant. Verification does not pose any problems. CONCLUSIONS The FFF 7-MV treatment plans are marginally superior to the flat-beam 6-MV plans in almost all cases, with greatly reduced treatment times (almost 50%).

Increasing genomic instability during cancer therapy in a patient with Li-Fraumeni syndrome

Highlights • Tumor suppressor p53 plays a central role in maintaining genomic stability by promoting cell-cycle checkpoints and apoptosis after genotoxic insults.• Here, we show that a patient with Li-Fraumeni syndrome receiving craniospinal irradiation including large volumes of bone marrow developed progressive genomic instability of the hematopoietic system.