Roger Nadrowitz

The hypoxia-inducible factor-1α is a negative factor for tumor therapy

Tumor hypoxia negatively regulates cell growth and causes a more malignant phenotype by increasing the expression of genes encoding angiogenic, metabolic and metastatic factors. Of clinical importance, insufficient tumor oxygenation affects the efficiency of chemotherapy and radiotherapy by poorly understood mechanisms. The hypoxia-inducible factor (HIF)-1 is a master transcriptional activator of oxygen-regulated genes and HIF-1 is constitutively upregulated in several tumor types. HIF-1 might thus be implicated in tumor therapy resistance. We found that transformed mouse embryonic fibroblasts deficient for HIF-1α are more susceptible to the treatment with carboplatin, etoposide and ionizing radiation than wild-type cells. Increased cell death in HIF-1α-deficient cells was because of apoptosis and did not involve p53 induction. Tumor chemotherapy of experimental fibrosarcoma in immunocompromised mice with carboplatin and etoposide confirmed the enhanced susceptibility of HIF-1α-deficient cells. Agents that did not cause DNA double-strand breaks, such as DNA-synthesis inhibitors or a DNA single-strand break-causing agent equally impaired cell growth, independent of the HIF-1α genotype. Functional repair of a fragmented reporter gene was decreased in HIF-1α-deficient cells. Thus, hypoxia-independent basal HIF-1α expression in tumor cells, as known from untransformed embryonic stem cells, is sufficient to induce target gene expression, probably including DNA double-strand break repair enzymes.

Overexpression of NPM-ALK induces different types of malignant lymphomas in IL-9 transgenic mice

Anaplastic large-cell lymphoma (ALCL) comprises approximately 25% of all non-Hodgkin lymphomas (NHL) in children and young adults, and up to 15% of high-grade NHL in older patients. Over 50% of these tumours carry the translocation t(2;5)(p23;q35). The result of this translocation is the fusion of the nucleophosmin (NPM) gene to the anaplastic lymphoma kinase (ALK) gene. The resulting hybrid protein contains the ALK catalytic domain that consequently confers transforming potential, which contributes to the pathogenesis of ALCL. To further analyse the transforming activity in an animal model, a cDNA encoding the protein product, NPM–ALK, was inserted into the retrovirus vector pLXSN and transduced into mouse bone marrow progenitors. These cells were subsequently used in a bone marrow transplant with the aim of reconstituting the haematopoietic compartments of lethally irradiated recipients. IL-9 transgenic mice were chosen as the animal model system, because dysregulated expression of the IL-9 gene in transgenic mice results in the sporadic development of spontaneous thymic lymphomas. Moreover, IL-9 is known to be expressed in cases of human ALCL. We used 15 IL-9 transgenic mice and eight corresponding wild-type mice (FVB/N) and transplanted them with NPM/ALK infected bone marrow cells. Eight IL-9 transgenic mice, serving as a control group, received pLXSN (vector only)-infected marrow. Reconstituted mice developed NPM–ALK-positive lymphomas, including lymphoblastic lymphomas of T-cell type (T-LB), mature and immature plasmacytoma (PC), and plasmoblastic/anaplastic diffuse large-B-cell lymphoma after about 19–20 weeks. The combined overexpression of NPM–ALK and IL-9 led to the transformation of murine lymphoid cells with accelerated and enhanced development of T-LB in 46% of the mice, which only very rarely occurs in IL-9 transgenic mice only. Of the 15 animals, five (33%) developed plasmacytic/plasmoblastic neoplasms, of which the most aggressive tumours share many features with anaplastic/plasmoblastic diffuse large-B-cell lymphoma on the basis of morphology, a characteristic growth pattern and ALK expression.

Evaluation of prognostic factors and two radiation techniques in patients treated with surgery followed by radio(chemo)therapy or definitive radio(chemo)therapy for locally advanced head-and-neck cancer

Background and Purpose:Conventional radiotherapy (RT) still is the standard technique for head-and-neck cancer in many centers worldwide, whereas other centers replaced this technique by 3-D conformal RT, which is associated with more appropriate dose distributions. Comparative studies regarding outcome and toxicity are lacking. This study compared both techniques for overall survival (OS), metastases-free survival (MFS), loco-regional control (LC), and toxicity in stage III/IV head-and-neck cancer.Patients and Methods:Data of 345 patients irradiated for stage III/IV squamous cell head-and-neck cancer were retrospectively analyzed. Patients received conventional RT (group A, n = 166) or 3-D conformal RT (group B, n = 179). Both techniques were compared for outcomes and toxicity. Eleven further potential prognostic factors were investigated: age, gender, performance status, tumor site, grading, T-stage, N-stage, AJCC-stage, chemotherapy, surgery, pre-RT hemoglobin.Results:3-year-OS was 62% in group A and 57% in group B (p = 0.15). 3-year-MFS was 67% and 76% (p = 0.46), 3-year-LC was 65% and 68%, respectively (p = 0.71). On multivariate analysis, gender (p = 0.005), performance status (p < 0.001), T-stage (p = 0.002), and N-stage (p < 0.001) were associated with OS. MFS was influenced by performance status (p < 0.001) and N-stage (p < 0.001), LC by gender (p = 0.021), T-stage (p < 0.001), and pre-RT hemoglobin level (≥ 12 better than < 12 g/dl, p = 0.004). Grade 2–3 xerostomia was less frequent with 3-D conformal RT (43% vs. 58%, p = 0.06). Otherwise, toxicities were similar.Conclusion:Both RT techniques resulted in similar treatment outcomes. Because xerostomia was less with 3-D conformal RT, this technique appeared beneficial for patients, in whom one parotid gland can be spared. Outcome was associated with gender, performance status, tumor stage, and pre-RT hemoglobin.Hintergrund und Ziel:Die konventionelle Strahlentherapie ist in vielen Institutionen weltweit noch die Standardtechnik bei Kopf-Hals-Tumoren. Andere Institutionen haben diese Technik durch die 3D-konformale Strahlentherapie ersetzt. Vergleichende Studien fehlen. Diese Studie vergleicht beide Techniken hinsichtlich Gesamtüberleben (OS), metastasenfreien Überlebens (MFS), lokoregionaler Kontrolle (LC) und Toxizität bei Patienten mit einem Plattenepithelkarzinom der Kopf-Hals-Region im Stadium III/IV.Patienten und Methodik:Daten von 345 Patienten wurden retrospektiv analysiert (Tabelle 1). Die Patienten erhielten eine konventionelle (Gruppe A, n = 166) oder 3D-konformale (Gruppe B, n = 179) Strahlentherapie (Abbildungen 1 und 2). Elf weitere potentielle Prognosefaktoren wurden untersucht: Alter, Geschlecht, Allgemeinzustand, Tumorsitz, Grading, T-Stadium, N-Stadium, AJCC-Stadium, Chemotherapie, Operation, Hämoglobin vor Strahlentherapie (Tabellen 2 bis 4).Ergebnisse:Die 3-Jahres-Überlebensraten waren 62% in Gruppe A und 57% in Gruppe B (p = 0,15, Abbildung 3). Die MFS-Raten waren 67% und 76% (p = 0,46, Abbildung 4), die LC-Raten 65% und 68% (p = 0,71, Abbildung 5). In der Multivarianzanalyse waren Geschlecht (p = 0,005), Allgemeinzustand (p < 0,001), T-Kategorie (p = 0,002) und N-Kategorie (p < 0,001) mit dem OS assoziiert. Allgemeinzustand (p < 0,001) und N-Stadium (p < 0,001) waren mit dem MFS assoziiert, Geschlecht (p = 0,021), T-Stadium (p < 0,001) und Hämoglobin vor Strahlentherapie (≥ 12 g/dl besser als < 12 g/dl, p = 0,004) mit der LC. Xerostomie Grad 2–3 war seltener nach 3D-konformaler Strahlentherapie (43% vs. 58%, p = 0,06). Ansonsten waren Akut- und Spättoxizität in beiden Gruppen ähnlich (Abbildungen 6 und 7).Schlussfolgerung:Beide Bestrahlungstechniken führten zu ähnlichen Behandlungsergebnissen. Da die Xerostomie nach 3Dkonformaler Strahlentherapie geringer ausgeprägt war, scheinen Patienten, bei denen eine Parotis geschont werden kann, von dieser Technik zu profitieren. Die Behandlungsergebnisse wurden durch das Geschlecht, den Allgemeinzustand, das Tumorstadium und den prä-strahlentherapeutischen Hämoglobinwert beeinflusst.

Radiolabeled Cetuximab plus Whole-Brain Irradiation (WBI) for the Treatment of Brain Metastases from Non-Small Cell Lung Cancer (NSCLC)

Background and Purpose:The addition of systemic drugs to whole-brain irradiation has not improved the survival of patients with multiple brain metastases, most likely because the agents did not readily cross the blood-brain barrier (BBB). Radiolabeling of cetuximab was performed to investigate whether this antibody crosses the BBB.Case Report:A patient with multiple brain lesions from non-small cell lung cancer was investigated. The largest metastasis (40 × 33 × 27 mm) was selected the reference lesion. On day 1, 200 mg/m2 cetuximab (0.25% hot and 99.75% cold antibody) were given. On day 3, 200 mg/m2 cetuximab (cold antibody) were given. Weekly doses of 250 mg/m2 cetuximab were administered for 3 months.Results:The reference lesion showed enhancement of radiolabeled cetuximab (123I-Erbi) on scintigraphy; 123I-Erbi crossed the BBB and accumulated in the lesion. The reference lesion measured 31 × 22 × 21 mm at 4 months. Enhancement of contrast medium was less pronounced.Conclusion:This is the first demonstration of cetuximab crossing the BBB and accumulating in brain metastasis.ZusammenfassungHintergrund und Ziel:Durch die systemische Gabe von Medikamenten zusätzlich zur Ganzhirnbestrahlung wurde bislang keine Verbesserung des Überlebens von Patienten mit multiplen Hirnmetastasen erreicht, höchstwahrscheinlich, weil die Substanzen die Blut-Hirn-Schranke (BBB) nicht adäquat überwinden. Cetuximab wurde radioaktiv markiert, um zu untersuchen, ob dieser Antikörper die BBB passieren kann.Fallbericht:Ein Patient mit multiplen Hirnmetastasen eines nichtkleinzelligen Bronchialkarzinoms wurde untersucht. Die größte Metastase (40 × 33 × 27 mm, Abbildung 1) wurde als Referenzmetastase definiert. An Tag 1 wurden 200 mg/m2 Cetuximab (0,25% heißer und 99,75% kalter Antikörper) appliziert, an Tag 3 erneut 200 mg/m2 Cetuximab (kalter Antikörper). Anschließend wurden wöchentlich 250 mg/m2 Cetuximab für 3 Monate verabreicht.Ergebnisse:In der Szintigraphie zeigte die Referenzmetastase eine Anreicherung des radioaktiv markierten Cetuximabs (123I-Erbi, Abbildung 2). 123I-Erbi passierte die BBB und akkumulierte in der Metastase. Nach 4 Monaten maß die Referenzmetastase noch 31 × 22 × 21 mm (Abbildung 3). Die Kontrastmittelanreicherung war geringer ausgeprägt.Schlussfolgerung:Erstmals wird gezeigt, dass Cetuximab die BBB passiert und sich in einer Hirnmetastase anreichert.

Backscatter dose from metallic materials due to obliquely incident high-energy photon beams.

If metallic material is exposed to ionizing radiation of sufficient high energy, an increase in dose due to backscatter radiation occurs in front of this material. Our purpose in this study was to quantify these doses at variable distances between scattering materials and the detector at axial beam angles between 0 degree (zero angle in beams eye view) and 90 degrees. Copper, silver and lead sheets embedded in a phantom of perspex were exposed to 10 MV-bremsstrahlung. The detector we developed is based on the fluorescence property of pyromellitic acid (1,2,4,5 benzenetetracarboxylic acid) after exposure to ionizing radiation. Our results show that the additional doses and the corresponding dose distribution in front of the scattering materials depend quantitatively and qualitatively on the beam angle. The backscatter dose increases with varying beam angle from 0 degree to 90 degrees up to a maximum at 55 degrees for copper and silver. At angles of 0 degree and 55 degrees the integral backscatter doses over a tissue-equivalent depth of 2 mm are 11.2% and 21.6% for copper and 24% and 28% for silver, respectively. In contrast, in front of lead there are no obvious differences of the measured backscatter doses at angles between 0 degree and 55 degrees. With a further increase of the beam angle from 55 degrees to 90 degrees the backscatter dose decreases steeply for all three materials. In front of copper a markedly lower penetrating depth of the backscattered electrons was found for an angle of 0 degree compared to 55 degrees. This dependence from the beam angle was less pronounced in front of silver and not detectable in front of lead. In conclusion, the dependence of the backscatter dose from the angle between axial beam and scattering material must be considered, as higher scattering doses have to be considered than previously expected. This may have a clinical impact since the surface of metallic implants is usually curved.

A Comparative Study on the Protection Profile of Lidocaine, Amifostine, and Pilocarpin on the Parotid Gland during Radiotherapy

The aim of this study was to evaluate the individual and the synergetic radioprotective effect of lidocaine, amifostine, and pilocarpin on the parotid gland. Forty-nine rabbits were randomized into seven groups (n = 7)--control, irradiated sham-treated, irradiated/lidocaine-pretreated, irradiated/amifostine-pretreated, irradiated/pilocarpin-pretreated, irradiated/lidocaine + pilocarpin-pretreated, and irradiated/amifostine + pilocarpin-pretreated groups. One week before irradiation (15 Gy) and 72 hours as well as 1 month afterward, the parotid gland was investigated morphologically, sialoscintigraphically, and immunohistochemically with the use of tenascin-C and alpha smooth muscle actin. Compared with control animals, there was a significant reduction of the salivary ejection fraction in the irradiated untreated group 72 hours following radiation. Only animals pretreated with lidocaine or amifostine (alone or combined with pilocarpin) showed a slight nonsignificant reduction of salivary ejection fraction. Immunohistochemically, we observed a significant loss of alpha smooth muscle actin and an up-regulation of tenascin-C expression in irradiated/untreated glands. These changes were less evident in animals pretreated with lidocaine or lidocaine + pilocarpin. Amifostine and pilocarpin did not show any influence on tenascin-C or alpha smooth muscle actin expression. Ultrastructural damage was observed in irradiated untreated and pilocarpin-pretreated glands. In contrast, lidocaine and amifostine could largely preserve the glandular ultrastructure. One month postradiation, all changes were regressive regardless of treatment protocol. Potential radioprotective agents show different effects on both morphology and function of the parotid gland. Associated immunohistochemical and ultrastructural findings could prove the prevailed protection profile of lidocaine. This may provide a prophylactic approach in the field of radioprotection of salivary glands.

Radioactive EGFR Antibody Cetuximab in Multimodal Cancer Treatment: Stability and Synergistic Effects With Radiotherapy

PURPOSE Systemic therapies when added to whole brain radiotherapy have failed to improve the survival of patients with multiple brain metastases. The epidermal growth factor receptor antibody cetuximab is an attractive option, if it is able to cross the blood-brain barrier. This might be proven with molecular imaging if the radiolabeled antibody is stable long enough to be effective. This study investigated the stability of radiolabeled cetuximab (Erbitux) ((131)I-Erbi) and potential synergistic effects with radiotherapy in vitro. METHODS AND MATERIALS Two cell lines were investigated, A431 with numerous epidermal growth factor receptors, and JIMT without epidermal growth factor receptors. We labeled 0.4 mg cetuximab with 50 MBq of [(131)I] iodide. Stability was determined for 72 h. The cell cultures were incubated with (131)I-Erbi or cold cetuximab for 72 h. Uptake and cell proliferation were measured every 24 h after no radiotherapy or irradiation with 2, 4, or 10 Gy. RESULTS The radiolabeling yield of (131)I-Erbi was always >80%. The radiochemical purity was still 93.6% after 72 h. A431 cells showed a (131)I-Erbi uptake about 100-fold greater than the JIMT controls. After 48 h, the A431 cultures showed significantly decreased proliferation. At 72 h after irradiation, (131)I-Erbi resulted in more pronounced inhibition of cell proliferation than the cold antibody in all radiation dose groups. CONCLUSION (131)I-Erbi was stable for <or=72 h. Radiotherapy led to increased tumor cell uptake of (131)I-Erbi. Radiotherapy and (131)I-Erbi synergistically inhibited tumor cell proliferation. These results provide the prerequisite data for a planned in vivo study of whole brain radiotherapy plus cetuximab for brain metastases.

Increased Radioiodine Uptake of Thyroid Cell Cultures after External Irradiation

Background:Radioiodine uptake (RIU) is one of the main prognostic factors for curative results of radioiodine therapy in patients with differentiated thyroid cancer. Some days after application of 131I, the uptake of a subsequent administration of radioiodine was found to be reduced. In contrast, early after irradiation with high-energy photons glucose and amino acid uptake were observed to be increased. Effects of external irradiation on RIU of thyrocytes using high-energy photons have not been investigated so far.Material and Methods:Two different cell lines (FRTL-5 and ML-1 cells) derived from thyroid tissue were studied in vitro. Cell lines were either incubated with 131I only (controls) or additionally irradiated with single doses of 6 or 10 Gy of high-energy photons using a linear accelerator. Cell number and RIU were determined 24–96 h after 131I application. RIU measurements were repeated after application of sodium perchlorate in excess to investigate specificity of the uptake. Statistical analyses were performed using non-parametric tests.Results:Incubation with radioiodine as well as irradiation with high-energy photons slowed down proliferation in investigated cell lines significantly. Irradiation with solely 131I resulted in stable or slightly decreased iodide uptake. Compared to those cells, the RIU increased significantly in externally irradiated cells, i. e., additional irradiation with 10 Gy resulted in an almost threefold increase of RIU in FRTL-5 after 72 h. The increase of RIU after irradiation was dose-dependent in both cell lines and could be blocked by perchlorate excess.Conclusion:It could be demonstrated that external irradiation increases RIU in thyroid cell cultures early after irradiation. The increase was dose-dependent and specific, as it could be blocked by perchlorate. This effect appears to be similar to the increase of other actively transported substances after irradiation with high-energy photons. Therefore, the results of this study may contribute to the knowledge of a generalized irradiation-induced mechanism which causes the activation of different cellular transporters. The clinical impact of these findings on combined therapy concepts has to be investigated in further experiments.Hintergrund:Der Radioiod-Uptake (RIU) ist ein wesentlicher prognostischer Faktor für den kurativen Erfolg der Radioiodtherapie bei Patienten mit differenziertem Schilddrüsenkarzinom. Es konnte gezeigt werden, dass einige Tage nach 131I-Gabe der Uptake bei einer erneuten Radioiodapplikation deutlich vermindert war. Im Gegensatz dazu wurde eine Zunahme des Glukose- und Aminosäure-Uptakes kurz nach der Bestrahlung mit hochenergetischen Photonen beschrieben. Effekte einer externen Bestrahlung mit hochenergetischen Photonen auf den RIU von Thyreozyten sind bisher noch nicht untersucht worden.Material und Methodik:Die Untersuchungen wurden in vitro an zwei unterschiedlichen Zelllinien (FRTL-5- und ML-1-Zellen) thyreoidalen Ursprungs durchgeführt. Die Zellkulturen wurden entweder nur mit Radioiod inkubiert (Kontrollen) oder zusätzlich einzeitig mit Hilfe eines Linearbeschleunigers mit 6 oder 10 Gy bestrahlt. Die Bestimmung der Zellzahl und des RIU erfolgte 24–96 h nach der 131I-Applikation. Die Messungen wurden nach Zugabe von Natriumperchlorat im Überschuss wiederholt. Statistische Analysen erfolgten mittels nichtparametrischer Tests.Ergebnisse:Die Proliferation der untersuchten Zelllinien wurde sowohl durch die Inkubation mit Radioiod als auch durch die Bestrahlung signifikant vermindert. Die Bestrahlung mit 131I allein führte zu gleichbleibendem oder leicht vermindertem Iodid-Uptake. Verglichen mit diesen Zellen erhöhte die externe Bestrahlung den RIU signifikant. So konnte bei FRTL-5-Zellen durch die zusätzliche Bestrahlung mit 10 Gy der RIU nach 72 h beinahe verdreifacht werden. Der Anstieg des RIU nach Bestrahlung war bei beiden Zelllinien dosisabhängig und konnte durch einen Überschuss an Perchlorat blockiert werden.Schlussfolgerung:In dieser Studie konnte gezeigt werden, dass eine externe Bestrahlung mit hochenergetischen Photonen zu einem Anstieg des RIU von Schilddrüsen-Zellkulturen führt. Dieser Anstieg war dosisabhängig und spezifisch, da er durch Perchlorat geblockt werden konnte. Dieser Effekt weist Ähnlichkeiten zum Anstieg anderer aktiv transportierter Substanzen nach Bestrahlung auf. Die Ergebnisse dieser Studie liefern einen Beitrag zur Kenntnis eines generalisierten Mechanismus, welcher auf eine Aktivierung unterschiedlicher zellulärer Transporter nach Bestrahlung mit hochenergetischen Photonen hinweist. Die klinische Relevanz dieser Ergebnisse für kombinierte Therapieverfahren sollte in weiteren Untersuchungen geklärt werden.

Radioprotective Effect of Lidocaine on Function and Ultrastructure of Salivary Glands Receiving Fractionated Radiation

PURPOSE Radiation-induced xerostomia still represents a common side effect after radiotherapy for head-and-neck malignancies. The aim of the present study was to examine the radioprotective effect of lidocaine hydrochloride during fractionated radiation in an experimental animal model. METHODS AND MATERIALS To evaluate the influence of different radiation doses on salivary gland function and the radioprotective effect of lidocaine, rabbits were irradiated with 15, 25, 30, and 35 Gy (equivalent doses in 2-Gy fractions equivalent to 24, 40, 48, and 56 Gy, respectively). Lidocaine hydrochloride (10 and 12 mg/kg) was administered before every radiation fraction in the treatment groups. Salivary gland function was assessed by flow sialometry and sialoscintigraphy, and the morphologic changes were evaluated using transmission electron microscopy. RESULTS Functional impairment was first observed after 35 Gy and pretreatment with lidocaine improved radiation tolerance of both parotid and submandibular glands. The use of 12 mg/kg lidocaine was superior and displayed significant radioprotection with regard to flow sialometry and sialoscintigraphy. The ultrastructure was largely preserved after pretreatment with both lidocaine doses. CONCLUSIONS Lidocaine represents an effective radioprotective agent and a promising approach for clinical application to avoid radiation-induced functional impairment of salivary glands.

A simple assay for the study of human hair follicle damage induced by ionizing irradiation.

Please cite this paper as: A simple assay for the study of human hair follicle damage induced by ionizing irradiation. Experimental Dermatology 2010; 19: e306–e309.