Sonia Tejada

A small noncoding RNA signature found in exosomes of GBM patient serum as a diagnostic tool

BACKGROUND Glioblastoma multiforme (GBM) is the most frequent malignant brain tumor in adults, and its prognosis remains dismal despite intensive research and therapeutic advances. Diagnostic biomarkers would be clinically meaningful to allow for early detection of the tumor and for those cases in which surgery is contraindicated or biopsy results are inconclusive. Recent findings show that GBM cells release microvesicles that contain a select subset of cellular proteins and RNA. The aim of this hypothesis-generating study was to assess the diagnostic potential of miRNAs found in microvesicles isolated from the serum of GBM patients. METHODS To control disease heterogeneity, we used patients with newly diagnosed GBM. In the discovery stage, PCR-based TaqMan Low Density Arrays followed by individual quantitative reverse transcriptase polymerase chain reaction were used to test the differences in the miRNA expression levels of serum microvesicles among 25 GBM patients and healthy controls paired by age and sex. The detected noncoding RNAs were then validated in another 50 GBM patients. RESULTS We found that the expression levels of 1 small noncoding RNA (RNU6-1) and 2 microRNAs (miR-320 and miR-574-3p) were significantly associated with a GBM diagnosis. In addition, RNU6-1 was consistently an independent predictor of a GBM diagnosis. CONCLUSIONS Altogether our results uncovered a small noncoding RNA signature in microvesicles isolated from GBM patient serum that could be used as a fast and reliable differential diagnostic biomarker.

Prognostic value of residual fluorescent tissue in glioblastoma patients after gross total resection in 5-aminolevulinic Acid-guided surgery.

BACKGROUND There is evidence in the literature supporting that fluorescent tissue signal in fluorescence-guided surgery extends farther than tissue highlighted in gadolinium in T1 sequence magnetic resonance imaging (MRI), which is the standard to quantify the extent of resection. OBJECTIVE To study whether the presence of residual fluorescent tissue after surgery carries a different prognosis for glioblastoma (GBM) cases with complete resection confirmed by MRI. METHODS A retrospective review in our center found 118 consecutive patients with high-grade gliomas operated on with the use of fluorescence-guided surgery with 5-aminolevulinic acid. Within that series, the 52 patients with newly diagnosed GBM and complete resection of enhancing tumor (CRET) in early MRI were selected for analysis. We studied the influence of residual fluorescence in the surgical field on overall survival and neurological complication rate. Multivariate analysis included potential relevant factors: age, Karnofsky Performance Scale, O-methylguanine methyltransferase methylation promoter status, tumor eloquent location, preoperative tumor volume, and adjuvant therapy. RESULTS The median overall survival was 27.0 months (confidence interval = 22.4-31.6) in patients with nonresidual fluorescence (n = 25) and 17.5 months (confidence interval = 12.5-22.5) for the group with residual fluorescence (n = 27) (P = .015). The influence of residual fluorescence was maintained in the multivariate analysis with all covariables, hazard ratio = 2.5 (P = .041). The neurological complication rate was 18.5% in patients with nonresidual fluorescence and 8% for the group with residual fluorescence (P = .267). CONCLUSION GBM patients with CRET in early MRI and no fluorescent residual tissue had longer overall survival than patients with CRET and residual fluorescent tissue.

Pathological characterization of the glioblastoma border as shown during surgery using 5-aminolevulinic acid-induced fluorescence

Thirty consecutive surgical patients with glioblastoma, were operated upon using fluorescence induced by 5‐aminolevulinic acid as guidance. The fluorescent quality of the tissue was used to take biopsies from the tumor center, from the invasive area around it and from adjacent normal‐looking tissue. These samples were analyzed with HE, Ki‐67 and nestin. Nestin expression in tissue surrounding glioblastoma cases was compared to tissue surrounding vascular lesions, metastasis and hippocampal sclerosis. The rate of gross total resection assessed by volumetric MRI was 83%. Using HE examination as the gold standard, fluorescence identified solid tumor with 100% positive predictive value, invasive areas with 97%, and normal tissue with 67% negative predictive value. Ki67 stained some cells in 69% of the non‐fluorescent samples around the tumor. There was always strong nestin expression around the tumor but it was similar to control cases in non‐glioma lesions with subacute expansion. 5‐aminolevulinic acid fluorescence guidance is very reliable and can help to study the tumor–brain interface. Nestin expression is strong and constant in the tissue around the tumor, but is mostly an acute glial reaction, not specific of the neoplasm. Nestin staining is not recommended as a tumor stem cell marker.

Quantitative volumetric analysis of gliomas with sequential MRI and 11C-methionine PET assessment: patterns of integration in therapy planning

PurposeThe aim of the study was to evaluate the volumetric integration patterns of standard MRI and 11C-methionine positron emission tomography (PET) images in the surgery planning of gliomas and their relationship to the histological grade.MethodsWe studied 23 patients with suspected or previously treated glioma who underwent preoperative 11C-methionine PET because MRI was imprecise in defining the surgical target contour. Images were transferred to the treatment planning system, coregistered and fused (BrainLAB). Tumour delineation was performed by 11C-methionine PET thresholding (vPET) and manual segmentation over MRI (vMRI). A 3-D volumetric study was conducted to evaluate the contribution of each modality to tumour target volume. All cases were surgically treated and histological classification was performed according to WHO grades. Additionally, several biopsy samples were taken according to the results derived either from PET or from MRI and analysed separately.ResultsFifteen patients had high-grade tumours [ten glioblastoma multiforme (GBM) and five anaplastic), whereas eight patients had low-grade tumours. Biopsies from areas with high 11C-methionine uptake without correspondence in MRI showed tumour proliferation, including infiltrative zones, distinguishing them from dysplasia and radionecrosis. Two main PET/MRI integration patterns emerged after analysis of volumetric data: pattern vMRI-in-vPET (11/23) and pattern vPET-in-vMRI (9/23). Besides, a possible third pattern with differences in both directions (vMRI-diff-vPET) could also be observed (3/23). There was a statistically significant association between the tumour classification and integration patterns described above (p < 0.001, κ = 0.72). GBM was associated with pattern vMRI-in-vPET (9/10), low-grade with pattern vPET-in-vMRI (7/8) and anaplastic with pattern vMRI-diff-vPET (3/5).ConclusionThe metabolically active tumour volume observed in 11C-methionine PET differs from the volume of MRI by showing areas of infiltrative tumour and distinguishing from non-tumour lesions. Differences in 11C-methionine PET/MRI integration patterns can be assigned to tumour grades according to the WHO classification. This finding may improve tumour delineation and therapy planning for gliomas.

Voxel-Based Analysis of Dual-Time-Point 18F-FDG PET Images for Brain Tumor Identification and Delineation

We have investigated dual-time-point 18F-FDG PET for the detection and delineation of high-grade brain tumors using quantitative criteria applied on a voxel basis. Methods: Twenty-five patients with suspected high-grade brain tumors and inconclusive MRI findings underwent 11C-methionine PET and dual-time-point 18F-FDG PET. Images from each subject were registered and spatially normalized. Parametric maps of standardized uptake value (SUV) and tumor–to–normal gray matter (TN) ratio for each PET image were obtained. Tumor diagnosis was evaluated according to 4 criteria comparing standard and delayed 18F-FDG PET images: any SUV increase, SUV increase greater than 10%, any TN increase, and TN increase greater than 10%. Voxel-based analysis sensitivity was assessed using 11C-methionine as a reference and compared with visual and volume-of-interest analysis for dual-time-point PET images. Additionally, volumetric assessment of the tumor extent that fulfills each criterion was compared with the volume defined for 11C-methionine PET. Results: The greatest sensitivity for tumor identification was obtained with any increase of TN ratio (100%), followed by a TN increase greater than 10% (96%), any SUV increase (80%), and an SUV increase greater than 10% (60%). These values were superior to visual analysis of standard 18F-FDG (sensitivity, 40%) and delayed 18F-FDG PET (sensitivity, 52%). Volume-of-interest analysis of dual-time-point PET reached a sensitivity of only 64% using the TN increase criterion. Regarding volumetry, voxel-based analysis with the TN ratio increase as a criterion, compared with 11C-methionine PET, detected 55.4% of the tumor volume, with the other criteria detecting volumes lower than 20%. Nevertheless, volume detection presented great variability, being better for metastasis (78%) and glioblastomas (56%) than for anaplastic tumors (12%). A positive correlation was observed between the volume detected and the time of acquisition of the delayed PET image (r = 0.66, P < 0.001), showing volumes greater than 75% when the delayed image was obtained at least 6 h after 18F-FDG injection. Conclusion: Compared with standard 18F-FDG PET studies, quantitative dual-time-point 18F-FDG PET can improve sensitivity for the identification and volume delineation of high-grade brain tumors.

Phase I Study of DNX-2401 (Delta-24-RGD) Oncolytic Adenovirus: Replication and Immunotherapeutic Effects in Recurrent Malignant Glioma

Purpose DNX-2401 (Delta-24-RGD; tasadenoturev) is a tumor-selective, replication-competent oncolytic adenovirus. Preclinical studies demonstrated antiglioma efficacy, but the effects and mechanisms of action have not been evaluated in patients. Methods A phase I, dose-escalation, biologic-end-point clinical trial of DNX-2401 was conducted in 37 patients with recurrent malignant glioma. Patients received a single intratumoral injection of DNX-2401 into biopsy-confirmed recurrent tumor to evaluate safety and response across eight dose levels (group A). To investigate the mechanism of action, a second group of patients (group B) underwent intratumoral injection through a permanently implanted catheter, followed 14 days later by en bloc resection to acquire post-treatment specimens. Results In group A (n = 25), 20% of patients survived > 3 years from treatment, and three patients had a ≥ 95% reduction in the enhancing tumor (12%), with all three of these dramatic responses resulting in > 3 years of progression-free survival from the time of treatment. Analyses of post-treatment surgical specimens (group B, n = 12) showed that DNX-2401 replicates and spreads within the tumor, documenting direct virus-induced oncolysis in patients. In addition to radiographic signs of inflammation, histopathologic examination of immune markers in post-treatment specimens showed tumor infiltration by CD8+ and T-bet+ cells, and transmembrane immunoglobulin mucin-3 downregulation after treatment. Analyses of patient-derived cell lines for damage-associated molecular patterns revealed induction of immunogenic cell death in tumor cells after DNX-2401 administration. Conclusion Treatment with DNX-2401 resulted in dramatic responses with long-term survival in recurrent high-grade gliomas that are probably due to direct oncolytic effects of the virus followed by elicitation of an immune-mediated antiglioma response.

Dendritic cell vaccination in glioblastoma after fluorescence-guided resection

AIM To assess whether the addition of a customized, active immunotherapy to standard of care including fluorescence-guided surgery, may provide hints of an improved survival for patients with poor-prognosis, incurable glioblastoma multiform. METHODS Preliminary to our ongoing, phase-II clinical trial, we conducted a small pilot study enrolling five consecutive patients with resectable glioblastoma. In terms of Recursive Partitioning Analysis, four patients were class V and one was class IV. In all five cases, fluorescence-guided surgery was employed, followed by rapid steroid discontinuation. Patients were then treated with a combination of standard radio-chemotherapy with temozolomide and tumor lysate-pulsed, mature dendritic cell-based vaccinations. RESULTS Though all five patients ultimately progressed, with any further treatment left to the sole decision of the treating oncologist, active immunotherapy was very well tolerated and induced specific immune responses in all three patients for whom enough material was available for such an assessment. Median progression-free survival was 16.1 mo. Even more important, median and mean overall survival were 27 mo and 26 mo, respectively. Three patients have died with an overall survival of 9 mo, 27 mo and 27.4 mo, while the other two are still alive at 32 mo and 36 mo, the former receiving treatment with bevacizumab, while the latter has now been off therapy for 12 mo. Four of five patients were alive at two years. CONCLUSION Active immunotherapy with tumor lysate-pulsed, autologous dendritic cells is feasible, safe, well tolerated and biologically efficacious. A phase-II study is ongoing to possibly improve further on our very encouraging clinical results.

Phase I Trial of DNX-2401 for Diffuse Intrinsic Pontine Glioma Newly Diagnosed in Pediatric Patients

BACKGROUND There are no effective treatments for diffuse intrinsic pontine gliomas (DIPGs); these tumors cannot be surgical resected, and diagnosis is based on magnetic resonance imaging. As a result, tumor tissues for molecular studies and pathologic diagnosis are infrequent. New clinical trials are investigating novel medications and therapeutic techniques in an effort to improve treatment of patients with DIPGs. OBJECTIVE To determine the safety, tolerability, and toxicity of an oncolytic adenovirus, DNX-2401, injected into the cerebellar peduncle in pediatric subjects with DIPG and to collect tumor samples of this type of tumor. METHODS Phase I, single-center, uncontrolled trial. A tumor biopsy will be performed through the cerebellar peduncle, and DNX-2401 will be injected immediately after the biopsy. Standard therapy consisting of radiotherapy and chemotherapy will follow in 2 to 6 wk. EXPECTED OUTCOMES Improvement of overall survival and quality of life in patients with DIPG and collection of tumor specimens to study the molecular profiling of these tumors. DISCUSSION The aims of this trial are to contribute to the sample collection of DIPG and to offer treatment during the tumor tissue biopsy using the virus. If this virus works as expected, it could kill the tumor cells with no damage to healthy tissue, functioning as a targeted therapy. It is important to note that edema has not been observed with this virus in all trials performed to date. The information obtained through this and other similar studies may be useful for developing or improving new therapies in the battle against DIPG.

Abstract CT027: Oncolytic virus DNX-2401 with a short course of temozolomide for glioblastoma at first recurrence: Clinical data and prognostic biomarkers

DNX-2401 is a replication competent oncolytic adenovirus that has shown a potent antitumor effect in preclinical glioma models and a safe profile when administer as a single agent in glioma patients. Previously, our group showed that combination of DNX-2401 with temozolomide significantly increased the survival of mice bearing gliomas. Even though virotherapy is becoming a real therapeutic option still the precise mechanism of action, the interplay with the tumor microenvironment and biomarkers that predict response remain elusive. The main objective of this clinical trial was to investigate safety, OR, and OS following treatment with DNX-2401 and temozolomide. In addition, we aim to seek biomarkers that predict response to the virus. We included 31 glioma patients at first recurrence after prior surgery and standard treatment with radiotherapy, and temozolomide. Tumor resection or biopsy was performed to confirm recurrent disease. All patients received a single intraparenchymal injection of DNX-2401 (3x1010 vp) followed by 4 cycles of 150 mg/m2 temozolomide 2-4 weeks later. Ninety-four percent of patients completed all 4 planned cycles. The safety objective of the trial was achieved with no severe toxicities related to DNX-2401. Grade 3-4 adverse events were consistent with those associated with temozolomide (e.g., lymphopenia) or underlying disease. Several objective responses were observed with three patients alive 30, 19 and 27 months after treatment. The study is ongoing and results will be updated. Patients with higher titers of neutralizing antibodies prior to virus administration appeared to respond better to treatment. No differences were seen in the levels of serum cytokines amongst patients and treatment times. FGF2 is a growth factor that has been shown to increased cell susceptibility to virus infection and replication through inhibition of the interferon pathway. Interestingly, high expression of FGF2 in tumor samples was associated with significantly longer overall survival. FGF2 expression was inversely correlated with IFN-γ expression in the tumor and in exosomes isolated from liquid biopsies in the same patient. These data also suggest that patients with stronger innate responses did worse than those where the IFN pathway was downregulated. In summary, our data demonstrate that DNX-2401 in combination with temozolomide is well tolerated, can be safely administered and shows therapeutic activity. Moreover, FGF2 as a prognostic biomarker of DNX-2401 treatment response deserves further investigation. Citation Format: Marta M. Alonso, Marc Garcia-Moure, Marisol Gonzalez-Huarriz, Miguel Marigil, Ruben Hernandez-Alcoceba, Maria Bunales, Sandra Hervas, Jaime Gallego, Candelaria Gomez-Manzano, Juan Fueyo, Frederick Lang, Joanna Peterkin, Ricardo Diez-Valle, Sonia Tejada. Oncolytic virus DNX-2401 with a short course of temozolomide for glioblastoma at first recurrence: Clinical data and prognostic biomarkers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT027. doi:10.1158/1538-7445.AM2017-CT027

DNX-2401, an oncolytic virus, for the treatment of newly diagnosed diffuse intrinsic pontine gliomas: A case report

Diffuse intrinsic pontine gliomas (DIPGs) are aggressive glial brain tumors that primarily affect children, for which there is no curative treatment. Median overall survival is only one year. Currently, the scientific focus is on expanding the knowledge base of the molecular biology of DIPG, and identifying effective therapies. Oncolytic adenovirus DNX-2401 is a replication-competent, genetically modified virus capable of infecting and killing glioma cells, and stimulating an anti-tumor immune response. Clinical trials evaluating intratumoral DNX-2401 in adults with recurrent glioblastoma have demonstrated that the virus has a favorable safety profile and can prolong survival. Subsequently, these results have encouraged the transition of this biologically active therapy from adults into the pediatric population. To this aim, we have designed a clinical Phase I trial for newly diagnosed pediatric DIPG to investigate the feasibility, safety, and preliminary efficacy of delivering DNX-2401 into tumors within the pons following biopsy. This case report presents a pediatric patient enrolled in this ongoing Phase I trial for children and adolescents with newly diagnosed DIPG. The case involves an 8-year-old female patient with radiologically diagnosed DIPG who underwent stereotactic tumor biopsy immediately followed by intratumoral DNX-2401 in the same biopsy track. Because there were no safety concerns or new neurological deficits, the patient was discharged 3 days after the procedures. To our knowledge, this is the first report of intratumoral DNX-2401 for a patient with DIPG in a clinical trial. We plan to demonstrate that intratumoral delivery of an oncolytic virus following tumor biopsy for pediatric patients with DIPG is a novel and feasible approach and that DNX-2401 represents an innovative treatment for the disease.