Dušan Milanović

Increased radiosensitivity and radiothermosensitivity of human pancreatic MIA PaCa-2 and U251 glioblastoma cell lines treated with the novel Hsp90 inhibitor NVP-HSP990

Background and purposeHeat shock Protein 90 (Hsp90) is a molecular chaperone that folds,stabilizes, and functionally regulates many cellular proteins involved inoncogenic signaling and in the regulation of radiosensitivity. It isupregulated in response to stress such a heat. Hyperthermia is a potentradiosensitizer, but induction of Hsp90 may potentially limit its efficacy.Our aim was to investigate whether the new Hsp90 inhibitor NVP-HSP990increases radiosensitivity, thermosensitivity and radiothermosensitivity ofhuman tumor cell lines.Material and methodsU251 glioblastoma and MIA PaCa-2 pancreatic carcinoma cells were used. Todetermine clonogenic survival, colony forming assays were performed. Cellviability and proliferation were assesed by Trypan blue staining. Cell cycleand apoptosis analyses were performed by flow cytometry. DAPI staining wasused to detect mitotic catastrophe.ResultsNVP- HSP990 increased the thermosensitivity, radiosensitivity andradio-thermosensitivity of both cell lines in clonogenic assays.72 hours after irradiation with 4 Gy, a significant reduction incell number associated with considerable G2/M acumulation and mitoticcatastrophe as well as cell death by apoptosis/necrosis was observed.ConclusionsTreatment with NVP- HSP990 strongly sensitized U251 and MIA PaCa-2cells to hyperthermia and ionizing radiation or combination thereof throughaugmentation of G2/M arrest, mitotic catastrophe and associatedapoptosis.

Stereotactic fractionated radiotherapy of the resection cavity in patients with one to three brain metastases

OBJECTIVES The goal of this study is to evaluate the role of stereotactic fractionated radiotherapy (SFRT) in patients with one to three brain metastases after surgical resection. METHODS AND MATERIALS We performed a retrospective single-institutional study in patients undergoing SFRT of surgical cavity after resection of ≤3 brain metastases. 60 patients with newly diagnosed brain metastases treated with SFRT following resection were included. The total irradiation dose was 30 Gy (5 Gy/d, BED 45 Gy) after complete macroscopical resection and 35 Gy (5 Gy/d, BED 52.5 Gy) in patients with macroscopic residual tumour after surgery. Macroscopic residual tumour was defined as contrast enhancement next to the resection cavity on the postoperative T1-MRI. The gross tumour volume (GTV) encompassing the residual tumour was delineated on the T1-MRI, the clinical target volume (CTV) encompassed the surgical cavity plus 1mm and the planning target volume (PTV) the CTV plus 2mm. RESULTS Eight of 60 patients had no imaging follow-up due to morbidity/mortality. Two of 52 (3.8%) patients experienced local failures only, 25 of 52 (48.1%) patients experienced distant intracranial failures only and 4 (7.7%) patients experienced both local and distant intracranial failures. In summary, there were 6 (11.5%) local failures and 29 (55.8%) distant failures. Age was significant for local control in the Cox regression test (p=0.046). Thirty-seven of 60 (61.7%) patients died during follow-up. Median follow-up was 8 months. Median overall survival was 15 months. Cox regression for survival was significant for KPS score ≤70% and size of PTV. No severe side effects were seen. Patients undergoing whole brain radiation therapy (WBRT) as salvage therapy in case of progression had no severe side effects either. CONCLUSION In the light of encouraging local control rates, SFRT could be an alternative to WBRT after surgical resection of ≤3 brain metastases. Due to the high rate of distant intracranial failure regular follow-up with MRI is mandatory.

The influence of the combined treatment with Vadimezan (ASA404) and taxol on the growth of U251 glioblastoma xenografts

BackgroundOne of the most important biological characteristics of Glioblastoma multiforme (GBM) is high vascular density. Vadimezan (ASA404, DMXAA) belongs to the class of small molecule vascular disrupting agents (VDA) that cause disruption of established tumor vessels and subsequent tumor hemorrhagic necrosis. Its selective antivascular effect is mediated by intratumoral induction of several cytokines including tumor necrosis factor-α (TNF-α), granulocyte-colony-stimulating factor (G-CSF), interleukin 6 (IL-6) and macrophage inflammatory protein 1α (MIP-1α). Preclinical studies have demonstrated that ASA404 acts synergistically with taxanes. In this study, we investigated if treatment of mice bearing U251 human glioblastoma xenografts with ASA404 and taxol may be synergistic. Therapy response was evaluated by measuring changes in tumor size and metabolic activity using 18F-FDG PET (Fluorodeoxyglucose - positron emision tomography) imaging.MethodsU251 cells were inoculated s.c. in the right hind limb of NMRI-Foxn1nu athymic female nude mice. Animals were randomly assigned into 4 groups (7–9 animals/group) for treatment: control, taxol, ASA404, and ASA404 plus taxol. The animals received either a single dose of taxol (10 mg/kg), ASA404 (27.5 mg/kg), or taxol (10 mg/kg) plus ASA404 (27.5 mg/kg) administered i.p.; ASA404 was administred 24 h after the treatment with taxol. 4 and 24 h after treatment with ASA404 (28 and 48 h hours after treatment with taxol) 18 F-FDG PET scans were performed.ResultsThe treatment with taxol did not affect the tumor growth in comparison to untreated controls. The treatment of animals with single dose ASA404 alone or in combination with taxol caused a significant delay in tumor growth. The combined treatment did not decrease the growth of the xenografts significantly more than ASA404 alone, but early changes in tumor 18 F-FDG uptake preceded subsequent growth inhibition. The tumor weights, which were determined at the end of treatment, were lower in case of combined treatment.ConclusionsThe treatment with ASA404 alone or in combination with taxol showed antitumoral effects in our glioblastoma model probably through destruction of blood vessels. The implications for the anticancer effect of this compound warrant further preclinical studies. 18F-FDG PET appears to be a promising tool to monitor treatment with ASA404 early in the course of therapy.

Inhibition of 13-cis retinoic acid-induced gene expression of reactive-resistance genes by thalidomide in glioblastoma tumours in vivo

The cell differentiation potential of 13-cis retinoic acid (RA) has not succeeded in the clinical treatment of glioblastoma (GBM) so far. However, RA may also induce the expression of resistance genes such as HOXB7 which can be suppressed by Thalidomide (THAL). Therefore, we tested if combined treatment with RA+THAL may inhibit growth of glioblastoma in vivo. Treatment with RA+THAL but not RA or THAL alone significantly inhibited tumour growth. The synergistic effect of RA and THAL was corroborated by the effect on proliferation of glioblastoma cell lines in vitro. HOXB7 was not upregulated but microarray analysis validated by real-time PCR identified four potential resistance genes (IL-8, HILDPA, IGFBPA, and ANGPTL4) whose upregulation by RA was suppressed by THAL. Furthermore, genes coding for small nucleolar RNAs (snoRNA) were identified as a target for RA for the first time, and their upregulation was maintained after combined treatment. Pathway analysis showed upregulation of the Ribosome pathway and downregulation of pathways associated with proliferation and inflammation. In conclusion, combined treatment with RA + THAL delayed growth of GBM xenografts and suppressed putative resistance genes associated with hypoxia and angiogenesis. This encourages further pre-clinical and clinical studies of this drug combination in GBM.

Local control and overall survival after frameless radiosurgery: A single center experience

Highlights • LINAC-based frameless radiosurgery shows favorable local control.• 18–20 Gy were delivered as single fraction.• No treatment related side effects ≥grade 2 were observed.• SRS and deferred WBRT remain salvage therapies for distant intracranial relapse.• Extracranial stable disease and GTV ≤ 2.5 cm3 were significant predictors of OS.