Publisher Correction: Actively personalized vaccination trial for newly diagnosed glioblastoma.

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Actively personalized vaccination trial for newly diagnosed glioblastoma.

Patients with glioblastoma currently do not sufficiently benefit from recent breakthroughs in cancer treatment that use checkpoint inhibitors1,2. For treatments using checkpoint inhibitors to be successful, a high mutational load and responses to neoepitopes are thought to be essential3. There is limited intratumoural infiltration of immune cells4 in glioblastoma and these tumours contain only 30-50 non-synonymous mutations5. Exploitation of the full repertoire of tumour antigens-that is, both unmutated antigens and neoepitopes-may offer more effective immunotherapies, especially for tumours with a low mutational load. Here, in the phase I trial GAPVAC-101 of the Glioma Actively Personalized Vaccine Consortium (GAPVAC), we integrated highly individualized vaccinations with both types of tumour antigens into standard care to optimally exploit the limited target space for patients with newly diagnosed glioblastoma. Fifteen patients with glioblastomas positive for human leukocyte antigen (HLA)-A*02:01 or HLA-A*24:02 were treated with a vaccine (APVAC1) derived from a premanufactured library of unmutated antigens followed by treatment with APVAC2, which preferentially targeted neoepitopes. Personalization was based on mutations and analyses of the transcriptomes and immunopeptidomes of the individual tumours. The GAPVAC approach was feasible and vaccines that had poly-ICLC (polyriboinosinic-polyribocytidylic acid-poly-L-lysine carboxymethylcellulose) and granulocyte-macrophage colony-stimulating factor as adjuvants displayed favourable safety and strong immunogenicity. Unmutated APVAC1 antigens elicited sustained responses of central memory CD8+ T cells. APVAC2 induced predominantly CD4+ T cell responses of T helper 1 type against predicted neoepitopes.

Diagnostic yield of simultaneous dynamic contrast-enhanced magnetic resonance perfusion measurements and [18F]FET PET in patients with suspected recurrent anaplastic astrocytoma and glioblastoma.

PURPOSE: Both amino acid positron emission tomography (PET) and magnetic resonance imaging (MRI) blood volume (BV) measurements are used in suspected recurrent high-grade gliomas. We compared the separate and combined diagnostic yield of simultaneously acquired dynamic contrast-enhanced (DCE) perfusion MRI and O-(2-[18F]-fluoroethyl)-L-tyrosine ([18F]FET) PET in patients with anaplastic astrocytoma and glioblastoma following standard therapy. METHODS: A total of 76 lesions in 60 hybrid [18F]FET PET/MRI scans with DCE MRI from patients with suspected recurrence of anaplastic astrocytoma and glioblastoma were included retrospectively. BV was measured from DCE MRI employing a 2-compartment exchange model (2CXM). Diagnostic performances of maximal tumour-to-background [18F]FET uptake (TBRmax), maximal BV (BVmax) and normalised BVmax (nBVmax) were determined by ROC analysis using 6-month histopathological (n = 28) or clinical/radiographical follow-up (n = 48) as reference. Sensitivity and specificity at optimal cut-offs were determined separately for enhancing and non-enhancing lesions. RESULTS: In progressive lesions, all BV and [18F]FET metrics were higher than in non-progressive lesions. ROC analyses showed higher overall ROC AUCs for TBRmax than both BVmax and nBVmax in both lesion-wise (all lesions, p = 0.04) and in patient-wise analysis (p < 0.01). Combining TBRmax with BV metrics did not increase ROC AUC. Lesion-wise positive fraction/sensitivity/specificity at optimal cut-offs were 55%/91%/84% for TBRmax, 45%/77%/84% for BVmax and 59%/84%/72% for nBVmax. Combining TBRmax and best-performing BV cut-offs yielded lesion-wise sensitivity/specificity of 75/97%. The fraction of progressive lesions was 11% in concordant negative lesions, 33% in lesions only BV positive, 64% in lesions only [18F]FET positive and 97% in concordant positive lesions. CONCLUSION: The overall diagnostic accuracy of DCE BV imaging is good, but lower than that of [18F]FET PET. Adding DCE BV imaging did not improve the overall diagnostic accuracy of [18F]FET PET, but may improve specificity and allow better lesion-wise risk stratification than [18F]FET PET alone.

Feasibility of multi-parametric PET and MRI for prediction of tumour recurrence in patients with glioblastoma.

BACKGROUND: Recurrence in glioblastoma patients often occur close to the original tumour and indicates that the current treatment is inadequate for local tumour control. In this study, we explored the feasibility of using multi-modality imaging at the time of radiotherapy planning. Specifically, we aimed to identify parameters from pre-treatment PET and MRI with potential to predict tumour recurrence. MATERIALS AND METHODS: Sixteen patients were prospectively recruited and treated according to established guidelines. Multi-parametric imaging with 18F-FET PET/CT and 18F-FDG PET/MR including diffusion and dynamic contrast enhanced perfusion MRI were performed before radiotherapy. Correlations between imaging parameters were calculated. Imaging was related to the voxel-wise outcome at the time of tumour recurrence. Within the radiotherapy target, median differences of imaging parameters in recurring and non-recurring voxels were calculated for contrast-enhancing lesion (CEL), non-enhancing lesion (NEL), and normal appearing grey and white matter. Logistic regression models were created to predict the patient-specific probability of recurrence. The most important parameters were identified using standardized model coefficients. RESULTS: Significant median differences between recurring and non-recurring voxels were observed for FDG, FET, fractional anisotropy, mean diffusivity, mean transit time, extra-vascular, extra-cellular blood volume and permeability derived from scans prior to chemo-radiotherapy. Tissue-specific patterns of voxel-wise correlations were observed. The most pronounced correlations were observed for 18F-FDG- and 18F-FET-uptake in CEL and NEL. Voxel-wise modelling of recurrence probability resulted in area under the receiver operating characteristic curve of 0.77 from scans prior to therapy. Overall, FET proved to be the most important parameter for recurrence prediction. CONCLUSION: Multi-parametric imaging before radiotherapy is feasible and significant differences in imaging parameters between recurring and non-recurring voxels were observed. Combining parameters in a logistic regression model enabled patient-specific maps of recurrence probability, where 18F-FET proved to be most important. This strategy could enable risk-adapted radiotherapy planning.

Early treatment response evaluation using FET PET compared to MRI in glioblastoma patients at first progression treated with bevacizumab plus lomustine.

BACKGROUND: The goal of this prospective study was to compare the value of both conventional MRI and O-(2-18F-fluoroethyl)-L-tyrosine (FET) PET for response evaluation in glioblastoma patients treated with bevacizumab plus lomustine (BEV/LOM) at first progression. METHODS: After chemoradiation with concomitant and adjuvant temozolomide, 21 IDH wild-type glioblastoma patients at first progression (age range, 33-75 years; MGMT promoter unmethylated, 81%) were treated with BEV/LOM. Contrast-enhanced MRI and FET-PET scans were performed at baseline and after 8-10 weeks. We obtained FET metabolic tumor volumes (MTV) and tumor/brain ratios. Threshold values of FET-PET parameters for treatment response were established by ROC analyses using the post-progression overall survival (OS) ≤/>9 months as the reference. MRI response assessment was based on RANO criteria. The predictive ability of FET-PET thresholds and MRI changes on early response assessment was evaluated subsequently concerning OS using uni- and multivariate survival estimates. RESULTS: Early treatment response as assessed by RANO criteria was not predictive for an OS>9 months (P = 0.203), whereas relative reductions of all FET-PET parameters significantly predicted an OS>9 months (P < 0.05). The absolute MTV at follow-up enabled the most significant OS prediction (sensitivity, 85%; specificity, 88%; P = 0.001). Patients with an absolute MTV below 5 ml at follow-up survived significantly longer (12 vs. 6 months, P < 0.001), whereas early responders defined by RANO criteria lived only insignificantly longer (9 vs. 6 months; P = 0.072). The absolute MTV at follow-up remained significant in the multivariate survival analysis (P = 0.006). CONCLUSIONS: FET-PET appears to be useful for identifying responders to BEV/LOM early after treatment initiation.

Simultaneous evaluation of brain tumour metabolism, structure and blood volume using [(18)F]-fluoroethyltyrosine (FET) PET/MRI: feasibility, agreement and initial experience.

PURPOSE: Both [(18)F]-fluoroethyltyrosine (FET) PET and blood volume (BV) MRI supplement routine T1-weighted contrast-enhanced MRI in gliomas, but whether the two modalities provide identical or complementary information is unresolved. The aims of the study were to investigate the feasibility of simultaneous structural MRI, BV MRI and FET PET of gliomas using an integrated PET/MRI scanner and to assess the spatial and quantitative agreement in tumour imaging between BV MRI and FET PET. METHODS: A total of 32 glioma patients underwent a 20-min static simultaneous PET/MRI acquisition on a Siemens mMR system 20 min after injection of 200 MBq FET. The MRI protocol included standard structural MRI and dynamic susceptibility contrast (DSC) imaging for BV measurements. Maximal relative tumour FET uptake (TBRmax) and BV (rBVmax), and Dice coefficients were calculated to assess the quantitative and spatial congruence in the tumour volumes determined by FET PET, BV MRI and contrast-enhanced MRI. RESULTS: FET volume and TBRmax were higher in BV-positive than in BV-negative scans, and both VOLBV and rBVmax were higher in FET-positive than in FET-negative scans. TBRmax and rBVmax were positively correlated (R (2) = 0.59, p < 0.001). FET and BV positivity were in agreement in only 26 of the 32 patients and in 42 of 63 lesions, and spatial congruence in the tumour volumes as assessed by the Dice coefficients was generally poor with median Dice coefficients exceeding 0.1 in less than half the patients positive on at least one modality for any pair of modalities. In 56 % of the patients susceptibility artefacts in DSC BV maps overlapped the tumour on MRI. CONCLUSION: The study demonstrated that although tumour volumes determined by BV MRI and FET PET were quantitatively correlated, their spatial congruence in a mixed population of treated glioma patients was generally poor, and the modalities did not provide the same information in this population of patients. Combined imaging of brain tumour metabolism and perfusion using hybrid PET/MR systems may provide complementary information on tumour biology, but the potential clinical value remains to be determined in future trials.

Early changes in perfusion of glioblastoma during radio- and chemotherapy evaluated by T1-dynamic contrast enhanced magnetic resonance imaging.

BACKGROUND: The survival times of patients with glioblastoma differ widely and biomarkers that would enable individualized treatment are needed. The objective of this study was to measure changes in the vascular physiology of tumor using T1-dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) in patients with glioblastoma during early stages of radio- and chemotherapy (Tx) and explore possible correlations with treatment outcomes. MATERIAL AND METHODS: An exploratory prospective study was planned. Patients underwent DCE-MRI at baseline, after approximately one and six weeks of Tx and three and six months post-Tx. DCE-MRI at three Tesla generated maps of blood flow (BF), blood volume (BV), permeability (Ki) and volume of distribution (Vd) using a combination of model-free deconvolution and Patlak plots. Regions of interest in contrast enhancing tumor and in normal appearing white matter were contoured. Progression-free survival (PFS) was the primary clinical outcome. Patients with PFS > 6 months were compared with those with PFS < 6 months. Parameters of vascular physiology and changes in these during Tx were compared for these two groups at all time points using non-parametric statistics. RESULTS: Eleven eligible patients were included and 46 DCE-MRI examinations were carried out. BF in tumor increased for all patients early during Tx (p = 0.005) and then fell to a level below baseline at post-Tx examinations (p = 0.016). A similar but non-significant trend was seen for tumor BV. There was no detectable difference between patients with PFS > 6 months versus PFS < 6 months with regards to baseline values or changes during and after Tx. CONCLUSIONS: Although no correlations to outcomes were found, the results of this exploratory study may be hypothesis generating and will be examined in a larger patient group.

Immunotherapy drives mesenchymal tumour cell state shift and TME immune response in glioblastoma patients.

BACKGROUND: Glioblastoma is a highly aggressive type of brain tumour for which there is no curative treatment available. Immunotherapies have shown limited responses in unselected patients, and there is an urgent need to identify mechanisms of treatment resistance to design novel therapy strategies. METHODS: Here we investigated the phenotypic and transcriptional dynamics at single-cell resolution during nivolumab immune checkpoint treatment of glioblastoma patients. RESULTS: We present the integrative paired single-cell RNA-seq analysis of 76 tumour samples from patients in a clinical trial of the PD-1 inhibitor nivolumab and untreated patients. We identify a distinct aggressive phenotypic signature in both tumour cells and the tumour microenvironment in response to nivolumab. Moreover, nivolumab-treatment was associated with an increased transition to mesenchymal stem-like tumour cells, and an increase in TAMs and exhausted and proliferative T cells. We verify and extend our findings in large external glioblastoma dataset (n = 298), develop a latent immune signature and find 18% of primary glioblastoma samples to be latent immune, associated with mesenchymal tumour cell state and TME immune response. Finally, we show that latent immune glioblastoma patients are associated with shorter overall survival following immune checkpoint treatment (p = 0.0041). CONCLUSIONS: We find a resistance mechanism signature in a quarter of glioblastoma patients associated with a tumour-cell transition to a more aggressive mesenchymal-like state, increase in TAMs and proliferative and exhausted T cells in response to immunotherapy. These patients may instead benefit from neuro-oncology therapies targeting mesenchymal tumour cells.

Single agent- and combination treatment with two targeted suicide gene therapy systems is effective in chemoresistant small cell lung cancer cells.

BACKGROUND: Transcriptional targeted suicide gene (SG) therapy driven by the insulinoma-associated 1 (INSM1) promoter makes it possible to target suicide toxin production and cytotoxicity exclusively to small cell lung cancer (SCLC) cells and tumors. It remains to be determined whether acquired chemoresistance, as observed in the majority of SCLC patients, desensitizes SCLC cells to INSM1 promoter-driven SG therapy. METHODS: A panel of SCLC cell lines resistant to clinically relevant chemotherapeutics was characterized regarding the expression of proteins involved in response to chemotherapy and regarding INSM1 promoter activity. Sensitivity towards INSM1 promoter-driven SG therapy was tested using different systems: Yeast cytosine deaminase-uracil phosphoribosyl transferase (YCD-YUPRT) in combination with the prodrug 5-fluorocytosine (5-FC) or Escherichia coli nitroreductase (NTR) together with the bromomustard prodrug SN27686. RESULTS: The chemoresistant cell lines displayed heterogeneous expression profiles of molecules involved in multidrug resistance, apoptosis and survival pathways. Despite this, the INSM1 promoter activity was found to be unchanged or increased in SCLC chemoresistant cells and xenografts compared to chemosensitive variants. INSM1 promoter-driven SG therapy with YCD-YUPRT/5-FC or NTR/SN27686, was found to induce high levels of cytotoxicity in both chemosensitive and chemoresistant SCLC cells. Moreover, the combination of INSM1 promoter-driven YCD-YUPRT/5-FC therapy and chemotherapy, as well as the combination of INSM1 promoter-driven YCD-YUPRT/5-FC and NTR/SN27686 therapy, was observed to be superior to single agent therapy in chemoresistant SCLC cells. CONCLUSIONS: Collectively, the present study demonstrates that targeted SG therapy is a potent therapeutic approach for chemoresistant SCLC patients, with the highest efficacy achieved when applied as combination SG therapy or in combination with standard chemotherapy.

A phase II trial with bevacizumab and irinotecan for patients with primary brain tumors and progression after standard therapy.

UNLABELLED: The combination of irinotecan and bevacizumab has shown efficacy in the treatment of recurrent glioblastoma multiforme (GBM). A prospective, phase II study of 85 patients with various recurrent brain tumors was carried out. Primary endpoints were progression free survival (PFS) and response rate. MATERIAL AND METHODS: Patients with recurrent primary brain tumors with performance status 0-2 were eligible. Intravenous bevacizumab 10 mg/kg and irinotecan 125/340 mg/m(2) were administered every 14 days. Evaluation was carried out every eight weeks using MRI and Macdonald response criteria. Treatment was continued until progression. RESULTS: In total 85 patients were included with the following histologies: GBM (n = 32), glioma WHO gr. III (n = 33), glioma WHO gr. II (n = 12) and others (n = 8). Patients received a median of four cycles. ORR (overall response rate) for glioblastoma was 25% and 59% achieved stable disease (SD). Median PFS was 5.2 months. For grade III gliomas ORR was 21% and 45% had SD. Median PFS was 3.7 months. No objective responses occurred in grade II gliomas. In the non-glioma population, one PR as well as several long PFS times were observed. DISCUSSION AND CONCLUSION: The combination of bevacizumab and irinotecan is well tolerated and moderately efficacious in glioblastoma and glioma WHO gr. III. A majority of patients achieve at least disease stabilization. Prolonged progression-free survival in non-glioma patients warrants further research.

The effect of adenovirus-mediated gene expression of FHIT in small cell lung cancer cells.

The candidate tumor suppressor fragile histidine traid (FHIT) is frequently inactivated in small cell lung cancer (SCLC). Mutations in the p53 gene also occur in the majority of SCLC leading to the accumulation of the mutant protein. Here we evaluated the effect of FHIT gene therapy alone or in combination with the mutant p53-reactivating molecule, PRIMA-1(Met)/APR-246, in SCLC. Overexpression of FHIT by recombinant adenoviral vector (Ad-FHIT)-mediated gene transfer in SCLC cells inhibited their growth by inducing apoptosis and when combined with PRIMA-1(Met)/APR-246, a synergistic cell growth inhibition was achieved.

Cetuximab insufficiently inhibits glioma cell growth due to persistent EGFR downstream signaling.

Overexpression and/or amplification of the epidermal growth factor receptor (EGFR) is present in 35-45% of primary glioblastoma multiforme tumors and has been correlated with a poor prognosis. In this study, we investigated the effect of cetuximab and intracellular signaling pathways downstream of EGFR, important for cell survival and proliferation. We show insufficient EGFR downregulation and competition with endogenous EGFR ligands upon cetuximab treatment. Dose-response experiments showed inhibition of EGFR phosphorylation without affecting two of the prominent downstream signaling pathways. Our results indicate that amplification and/or overexpression of EGFR is an unsatisfactory predictor for response to cetuximab.

Genomic profiling of newly diagnosed glioblastoma patients and its potential for clinical utility - a prospective, translational study.

Glioblastoma (GBM) is an incurable brain tumor for which new treatment strategies are urgently needed. Next-generation sequencing of GBM has most often been performed retrospectively and on archival tissue from both diagnostic and relapse surgeries with limited knowledge of clinical information, including treatment given. We sought to investigate the genomic composition prospectively in treatment-naïve patients, searched for possible targetable aberrations, and investigated for prognostic and/or predictive factors. A total of 108 newly diagnosed GBM patients were included. Clinical information, progression-free survival, and overall survival (OS) were noted. Tissues were analyzed by whole-exome sequencing, single nucleotide polymorphism (SNP) and transcriptome arrays, and RNA sequencing; assessed for mutations, fusions, tumor mutational burden (TMB), and chromosomal instability (CI); and classified into GBM subgroups. Each genomic report was discussed at a multidisciplinary tumor board meeting to evaluate for matching trials. From 111 consecutive patients, 97.3% accepted inclusion in this study. Eighty-six (77%) were treated with radiation therapy/temozolomide (TMZ) and adjuvant TMZ. One NTRK2 and three FGFR3-TACC3 fusions were identified. Copy number alterations in GRB2 and SMYD4 were significantly correlated with worse median OS together with known clinical variables like age, performance status, steroid dose, and O6-methyl-guanine-DNA-methyl-transferase status. Patients with CI-median or TMB-high had significantly worse median OS compared to CI-low/high or TMB-low/median. In conclusion, performing genomic profiling at diagnosis enables evaluation of genomic-driven therapy at the first progression. Furthermore, TMB-high or CI-median patients had worse median OS, which can support the possibility of offering experimental treatment already at the first line for this group.

Acute damage by naphthalene triggers expression of the neuroendocrine marker PGP9.5 in airway epithelial cells.

Protein Gene Product 9.5 (PGP9.5) is highly expressed in nervous tissue. Recently PGP9.5 expression has been found to be upregulated in the pulmonary epithelium of smokers and in non-small cell lung cancer, suggesting that it also plays a role in carcinogen-inflicted lung epithelial injury and carcinogenesis. We investigated the expression of PGP9.5 in mice in response to two prominent carcinogens found in tobacco smoke: Naphthalene and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). By immunostaining, we found that PGP9.5 protein was highly expressed throughout the airway epithelium in the days immediately following a single injection of naphthalene. In contrast, PGP9.5 was exclusively confined to neurons and neuroendocrine cells in the control and NNK-exposed lungs. Furthermore, we investigated the expression of PGP9.5 mRNA in the lungs by quantitative RT-PCR (qPCR). PGP9.5 mRNA expression was highly upregulated in the days immediately following naphthalene injection and gradually returning to that of control mice 5 days after naphthalene injection. In contrast, exposure to NNK did not result in a significant increase in PGP9.5 mRNA 10 weeks after exposure. No increased expression of two other neuroendocrine markers was found in the non-neuroendocrine epithelial cells after naphthalene exposure. In contrast, immunostaining for the cell cycle regulator p27(Kip1), which has previously been associated with PGP9.5 in lung cancer cells, revealed transient downregulation of p27(Kip1) in naphthalene exposed airways compared to controls, indicating that the rise in PGP9.5 in the airway epithelium is related to downregulation of p27(Kip1). This study is the first to specifically identify the carcinogen naphthalene as an inducer of PGP9.5 expression in non-neuroendocrine epithelium after acute lung injury and further strengthens the accumulating evidence of PGP9.5 as a central player in lung epithelial damage and early carcinogenesis.

VEGF-C sustains VEGFR2 activation under bevacizumab therapy and promotes glioblastoma maintenance.

Background: Glioblastoma ranks among the most lethal cancers, with current therapies offering only palliation. Paracrine vascular endothelial growth factor (VEGF) signaling has been targeted using anti-angiogenic agents, whereas autocrine VEGF/VEGF receptor 2 (VEGFR2) signaling is poorly understood. Bevacizumab resistance of VEGFR2-expressing glioblastoma cells prompted interrogation of autocrine VEGF-C/VEGFR2 signaling in glioblastoma. Methods: Autocrine VEGF-C/VEGFR2 signaling was functionally investigated using RNA interference and exogenous ligands in patient-derived xenograft lines and primary glioblastoma cell cultures in vitro and in vivo. VEGF-C expression and interaction with VEGFR2 in a matched pre- and post-bevacizumab treatment cohort were analyzed by immunohistochemistry and proximity ligation assay. Results: VEGF-C was expressed by patient-derived xenograft glioblastoma lines, primary cells, and matched surgical specimens before and after bevacizumab treatment. VEGF-C activated autocrine VEGFR2 signaling to promote cell survival, whereas targeting VEGF-C expression reprogrammed cellular transcription to attenuate survival and cell cycle progression. Supporting potential translational significance, targeting VEGF-C impaired tumor growth in vivo, with superiority to bevacizumab treatment. Conclusions: Our results demonstrate VEGF-C serves as both a paracrine and an autocrine pro-survival cytokine in glioblastoma, promoting tumor cell survival and tumorigenesis. VEGF-C permits sustained VEGFR2 activation and tumor growth, where its inhibition appears superior to bevacizumab therapy in improving tumor control.

Solid lipid nanoparticles can effectively bind DNA, streptavidin and biotinylated ligands.

Cationic solid lipid nanoparticles (SLN) have recently been suggested for non-viral gene delivery, as these particles consist of well tolerated substances, can bind DNA directly via electrostatic interactions and mediate gene transfer in vitro. We here report the development of SLN complexes, which can be targeted to specific surface receptors. A formulation of SLN was prepared by the microemulsion technique comprising of stearylamine and the matrix lipid Compritol ATO 888 with a size of approximately 100 nm and a zeta-potential of +15. These SLN are able to condense DNA in complexes, which are very stable under physiological conditions, and they display low cytotoxicity in cell culture. In addition to binding of DNA, the SLN can simultaneously bind substantial amounts of streptavidin directly via electrostatic interactions. The SLN:DNA: streptavidin complexes are stable and are capable of binding biotinylated ligands, which can interact with surface receptors. This method allows for development of a fast and simple method of preparing a targeted non-viral gene therapy vector.

Urokinase-Type Plasminogen Activator Receptor as a Potential PET Biomarker in Glioblastoma.

UNLABELLED: Glioblastoma is one of the most malignant types of human cancer, and the prognosis is poor. The development and validation of novel molecular imaging biomarkers has the potential to improve tumor detection, grading, risk stratification, and treatment monitoring of gliomas. The aim of this study was to explore the potential of PET imaging of the urokinase-type plasminogen activator receptor (uPAR) in glioblastoma. METHODS: The uPAR messenger RNA expression of tumors from 19 glioblastoma patients was analyzed, and a cell culture derived from one of these patients was used to establish an orthotopic xenograft model of glioblastoma. Tumor growth was monitored using bioluminescence imaging. Five to six weeks after inoculation, all mice were scanned with small-animal PET/CT using two new uPAR PET ligands ((64)Cu-NOTA-AE105 and (68)Ga-NOTA-AE105) and, for comparison, O-(2-(18)F-fluoroethyl)-l-tyrosine ((18)F-FET). One MRI scan was obtained for each mouse to confirm tumor location. The uPAR specificity of (64)Cu-NOTA-AE105 was confirmed by alignment of hematoxylin- and eosin-stained and uPAR immunohistochemistry-stained slides of the brain with the activity distribution as determined using autoradiography. RESULTS: uPAR expression was found in all 19 glioblastoma patient tumors, and high expression of uPAR correlated with decreased overall survival (P = 0.04). Radiolabeling of NOTA-AE105 with (64)Cu and (68)Ga was straightforward, resulting in a specific activity of approximately 20 GBq/µmol and a radiochemical purity of more than 98% for (64)Cu-NOTA-AE105 and more than 97% for (68)Ga-NOTA-AE105. High image contrast resulting in clear tumor delineation was found for both (68)Ga-NOTA-AE105 and (64)Cu-NOTA-AE105. Absolute uptake in tumor was higher for (18)F-FET (3.5 ± 0.8 percentage injected dose [%ID]/g) than for (64)Cu-NOTA-AE105 (1.2 ± 0.4 %ID/g) or (68)Ga-NOTA-AE105 (0.4 ± 0.1 %ID/g). A similar pattern was observed in background brain tissue, where uptake was 1.9 ± 0.1 %ID/g for (18)F-fluorothymidine, compared with 0.05 ± 0.01 %ID/g for (68)Ga-NOTA-AE105 and 0.11 ± 0.02 %ID/g for (64)Cu-NOTA-AE105. The result was a significantly higher tumor-to-background ratio for both (68)Ga-NOTA-AE105 (7.6 ± 2.1, P < 0.05) and (64)Cu-NOTA-AE105 (10.6 ± 2.3, P < 0.01) than for (18)F-FET PET (1.8 ± 0.3). Autoradiography of brain slides confirmed that the accumulation of (64)Cu-NOTA-AE105 corresponded well with uPAR-positive cancer cells. CONCLUSION: On the basis of our translational study, uPAR PET may be a highly promising imaging biomarker for glioblastoma. Further clinical exploration of uPAR PET in glioblastoma is therefore justified.

Assessment of Quantitative and Allelic MGMT Methylation Patterns as a Prognostic Marker in Glioblastoma.

Methylation of the O(6)-methylguanine-DNA methyltransferase (MGMT) gene is a predictive and prognostic marker in newly diagnosed glioblastoma patients treated with temozolomide but how MGMT methylation should be assessed to ensure optimal detection accuracy is debated. We developed a novel quantitative methylation-specific PCR (qMSP) MGMT assay capable of providing allelic methylation data and analyzed 151 glioblastomas from patients receiving standard of care treatment (Stupp protocol). The samples were also analyzed by immunohistochemistry (IHC), standard bisulfite pyrosequencing, and genotyped for the rs1690252 MGMT promoter single nucleotide polymorphism. Monoallelic methylation was observed more frequently than biallelic methylation, and some cases with monoallelic methylation expressed the MGMT protein whereas others did not. The presence of MGMT methylation was associated with better overall survival (p = 0.006; qMSP and p = 0.002; standard pyrosequencing), and the presence of the protein was associated with worse overall survival (p = 0.009). Combined analyses of qMSP and standard pyrosequencing or IHC identified additional patients who benefited from temozolomide treatment. Finally, low methylation levels were also associated with better overall survival (p = 0.061; qMSP and p = 0.02; standard pyrosequencing). These data support the use of both MGMT methylation and MGMT IHC but not allelic methylation data as prognostic markers in patients with temozolomide-treated glioblastoma.

Specific sensitivity of small cell lung cancer cell lines to the snake venom toxin taipoxin.

Small cell lung cancer (SCLC) is a malignant disease, for which no satisfactory treatment is presently available and consequently, new specific therapeutic targets are in high demand. A global gene expression analysis previously performed, identified the neuronal pentraxin receptor (NPR) as highly and relatively specifically expressed in SCLC, consistent with the neuroendocrine features of this cancer. Normally, NPR is exclusively expressed in neurons, where it associates with the homologous proteins neuronal pentraxins 1 and 2 (NP1 and NP2) in complexes capable of binding the snake venom neurotoxin taipoxin. The purpose of the present study was to assess the toxic effect of taipoxin in SCLC-cell lines and to determine if toxicity correlates to NPR and NP1 and NP2 expression levels. NPR was detected by Western blot analysis in all the tested SCLC and in control cell lines of different origin. The receptor co-purified with cell membrane in SCLC, indicating that NPR is surface associated. Microarray signals for NP1 and NP2mRNA was detected in a subset of SCLC-cell lines and validated by Northern blot analysis. Furthermore, NP1 protein was detected by Western blot analysis in a few SCLC-cell lines, but not in the control cell lines. A number of SCLC-cell lines showed marked sensitivity to taipoxin (IC50: 3-130 nM) at toxin concentrations leaving the control cell lines unaffected. The sensitivity to taipoxin did not correlate with the expression levels of NP1 protein and NP2-mRNA, suggesting that expression of these proteins may not be required for taipoxin induced toxicity in SCLC. The demonstrated toxic effect of taipoxin in SCLC may prove to be of importance for designing novel specific treatment modalities for this disease.

Replacement and suicide gene therapy for targeted treatment of lung cancer.

Lung cancer is the leading cause of cancer-related death in the developed world; consequently, novel therapeutic strategies are in high demand. A major problem with the present treatment modalities is the lack of tumor specificity giving rise to dose-limiting toxicity and side effects. Gene therapy constitutes an experimental approach gaining increased attention as a putative future cancer therapeutic strategy. Using this strategy, cancer cytotoxicity can be obtained by replacing mutated genes with functional analogues or introducing a suicide gene into the malignant cells. Insight into the molecular biology of cancer cells has identified a number of regulatory gene sequences, which can be used to selectively activate the therapeutic gene specifically in cancer cells, thereby reducing nonspecific toxicity. Although further improvements are necessary, recent encouraging results have shown promise for future clinical application of gene therapy. This article presents an update on the experimental and clinical results obtained within the field of lung cancer gene therapy, concentrating on strategies to specifically activate expression of the therapeutic gene in cancer cells. Furthermore, status of the development of delivery vector constructs for lung cancer gene therapy will be presented.