Brain Tumor Progression Research Articles

Heterogeneous Nuclear Ribonucleoprotein C1/C2 Controls the Metastatic Potential of Glioblastoma by Regulating PDCD4

MicroRNAs (miRNAs) have been implicated in the pathogenesis and progression of brain tumors. miR-21 is one of the most highly overexpressed miRNAs in glioblastoma multiforme (GBM), and its level of expression correlates with the tumor grade. Programmed cell death 4 (PDCD4) is a well-known miR-21 target and is frequently downregulated in glioblastomas in accordance with increased miR-21 expression. Downregulation of miR-21 or overexpression of PDCD4 can inhibit metastasis. Here, we investigate the role of heterogeneous nuclear ribonucleoprotein C1/C2 (hnRNPC) in the metastatic potential of the glioblastoma cell line T98G. hnRNPC bound directly to primary miR-21 (pri-miR-21) and promoted miR-21 expression in T98G cells. Silencing of hnRNPC lowered miR-21 levels, in turn increasing the expression of PDCD4, suppressing Akt and p70S6K activation, and inhibiting migratory and invasive activities. Silencing of hnRNPC reduced cell proliferation and enhanced etoposide-induced apoptosis. In support of a role for hnRNPC in the invasiveness of GBM, highly aggressive U87MG cells showed higher hnRNPC expression levels and hnRNPC abundance in tissue arrays and also showed elevated levels as a function of brain tumor grade. Taken together, our data indicate that hnRNPC controls the aggressiveness of GBM cells through the regulation of PDCD4, underscoring the potential usefulness of hnRNPC as a prognostic and therapeutic marker of GBM.

Elevating SOX2 Levels Deleteriously Affects the Growth of Medulloblastoma and Glioblastoma Cells

Medulloblastomas and glioblastomas are devastating tumors that respond poorly to treatment. These tumors have been shown to express SOX2 and overexpression of SOX2 has been correlated with poor prognosis. Although knockdown of SOX2 impairs the growth and tumorigenicity of brain tumor cells, it was unclear how elevating SOX2 levels would affect their fate. Interestingly, studies conducted with neural stem cells have shown that small increases or decreases in the level of this transcription factor significantly alter their fate. Here, we report that elevating SOX2 3-fold above endogenous levels in U87 and U118 glioblastoma, and DAOY medulloblastoma cells significantly impairs their ability to proliferate. We extended these findings and determined that elevating SOX2 in DAOY cells remodels their cell-cycle profile by increasing the proportion of cells in the G1-compartment, and induces the expression of genes associated with differentiation. Furthermore, we show that elevating SOX2 leads to a dramatic induction of CD133 expression in DAOY cells, yet inhibits the ability of both CD133+ and CD133− cells to form neurospheres. Together, these findings argue that SOX2 levels must be carefully controlled in glioblastomas and medulloblastomas to maintain their fate. Equally important, our data suggests that increases in the expression of SOX2 during brain tumor progression are likely to be linked closely with changes in other critical genes that work in concert with SOX2 to enhance the tumorigenicity of brain tumors. Importantly, we demonstrate that this is also likely to be true for other cancers that express SOX2. Moreover, these studies demonstrate the advantage of using inducible promoters to study the effects of SOX2 elevation, as compared to gene expression systems that rely on constitutive expression.

Biophysical modeling of brain tumor progression: From unconditionally stable explicit time integration to an inverse problem with parabolic PDE constraints for model calibration

A novel unconditionally stable, explicit numerical method is introduced to the field of modeling brain cancer progression on a tissue level together with an inverse problem (IP) based on optimal control theory that allows for automated model calibration with respect to observations in clinical imaging data. Biophysical models of cancer progression on a tissue level are in general based on the assumption that the spatiotemporal spread of cancerous cells is determined by cell division and net migration. These processes are typically described in terms of a parabolic partial differential equation (PDE). In the present work a parallelized implementation of an unconditionally stable, explicit Euler (EE(⋆)) time integration method for the solution of this PDE is detailed. The key idea of the discussed EE(⋆) method is to relax the strong stability requirement on the spectral radius of the coefficient matrix by introducing a subdivision regime for a given outer time step. The performance is related to common implicit numerical methods. To quantify the numerical error, a simplified model that has a closed form solution is considered. To allow for a systematic, phenomenological validation a novel approach for automated model calibration on the basis of observations in medical imaging data is developed. The resulting IP is based on optimal control theory and manifests as a large scale, PDE constrained optimization problem. The numerical error of the EE(⋆) method is at the order of standard implicit numerical methods. The computing times are well below those obtained for implicit methods and by that demonstrate efficiency. Qualitative and quantitative analysis in 12 patients demonstrates that the obtained results are in strong agreement with observations in medical imaging data. Rating simulation success in terms of the mean overlap between model predictions and manual expert segmentations yields a success rate of 75% (9 out of 12 patients). The discussed EE(⋆) method provides desirable features for image-based model calibration or hybrid image registration algorithms in which the model serves as a biophysical prior. This is due to (i) ease of implementation, (ii) low memory requirements, (iii) efficiency, (iv) a straightforward interface for parameter updates, and (v) the fact that the method is inherently matrix-free. The explicit time integration method is confirmed via experiments for automated model calibration. Qualitative and quantitative analysis demonstrates that the proposed framework allows for recovering observations in medical imaging data and by that phenomenological model validity.

Cognitive function testing in adult brain tumor trials: lessons from a comprehensive review

Neurocognitive function (NCF) impairment is an important component of primary and metastatic brain tumors and their therapeutic interventions. As a result, modern clinical trials of cranial irradiation for adult cancer patients have incorporated NCF testing as a primary or secondary end point. In doing so, these clinical trials have provided a novel insight into our understanding of the NCF effects of cranial irradiation and brain tumor progression. In this article, we review these clinical trials both in terms of the trial findings and in terms of the types of NCF tests used in these trials. We also provide an introduction to the strengths and limitations of these NCF tests, as well as expert commentary on the current status and future directions of NCF testing in brain tumor trials.

Abstract 3982: Cancer genome sequencing analysis, storage, discovery and delivery

Abstract Genome instability and structural rearrangement is a distinctive hallmark of the cancer genome. With next-gen sequencing technologies, our ability to measure structural rearrangements that occur throughout tumorigenesis and progression has improved significantly, while creating an urgent need for rearrangement discovery, analysis, and visualization methods to aid in our understanding of these events. We have developed a sequencing analysis pipeline that streamlines the discovery of an individual tumor's mutations, small indels, copy number alterations, allele-specific amplifications and deletions, and genomic rearrangements. Rearrangements are refined to base-pair precision using unmapped, putative split reads found in the vicinity of the breakpoint. Results are presented in an interactive, web-based genome browser that provides analysis and visualization of both high-level, processed results as well as the raw data from which they were derived. From the web-based interface, results from multiple samples can be aggregated into user-defined sample cohorts to help identify features that are shared among a significant number of the samples. Using this sequencing analysis pipeline, we discovered high-confident, small- and large-scale somatic events in 17 whole genome glioblastoma multiforme (GBM) tumor samples from The Cancer Genome Atlas (TCGA) project, using their matched normal sequences to filter out germline variants. Among many interesting structural aberrations identified in these samples, we find four samples exhibiting EGFR amplifications and the presence of the EGFRvIII mutant, characterized by the in-frame deletion of exons 2-7 to produce a constitutively active form of the receptor. Comparing the read support of the EGFRvIII-associated breakpoints to the number of normally-mapped reads in the neighborhood suggest that, in all cases, the EGFRvIII mutant emerges after the amplification of wild-type EGFR, existing as a small fraction of total number of EGFR copies. In other samples, evidence of high copy number amplicons containing both MDM2 and EGFR are discovered at detectable levels in the blood sequencing data, raising the possibility that patient-specific PCR-based assays could be developed to quantitate the presence of somatic rearrangements as a proxy to monitor the progression of brain tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3982. doi:1538-7445.AM2012-3982

Abstract 452: Novel MRI-based biomarker for early assessment of glioma recurrence

Abstract Gliomas continue to be the most common form of brain malignancy in adult patients. Even with advancements in the clinical management of these patients, assessment of tumor recurrence continues to be based on late or serial changes in tumor volume as measured by CT or MRI [1; 2]. The purpose of this study was to develop and evaluate a new voxel-based method, referred as parametric response mapping (PRM), for early detection of brain tumor progression using standard MR images (FLAIR and contrast-enhanced T1-weighted). PRM results were evaluated relative to standard MRI-based criteria of clinical progression assessment in patients with Grade III/IV gliomas. Fourteen patients with grades III/IV glioma underwent MRI before and during treatment (every 2 months). MRI scans, acquired on a 1.5T or a 3T clinical scanner, consisted of fluid-attenuated inversion recovery imaging (FLAIR) and contrast-enhanced T1-weighted (CE-T1w) images. All images were spatially registered to CE-T1w pre-treatment scans and tumors were manually contoured on the T1w-Gd images. Subsequent to PRM analysis, FLAIR images were normalized to the signal using the white matter (rFLAIR). PRM, applied to the whole brain volume on rFLAIR, was performed by first generating voxel-based difference maps at each longitudinal follow-up scan using the baseline as the subtrahend. Baseline was defined as either the pre-treatment or subsequent rFLAIR image. Individual voxels were classified based on the extent of change observed in the rFLAIR difference maps. Three classifications were used: voxels with a significant increase in rFLAIR values (PRMrFLAIR+), significant decrease (PRMrFLAIR-) or statistically unchanged (PRMrFLAIR0). Disease recurrence was defined by PRM as the sum of red voxels that exceed 20% of the tumor volume as delineated on T1w-Gd. PRMrFLAIR detected tumor recurrence on average 18±3 weeks earlier than the MacDonald criteria. It was also observed that PRMrFLAIR+ found outside the tumor CE-T1w regions revealed the spatial location at which the tumor would progress on subsequent scans based on current standard progression criteria. These results show that the PRM approach provides for the early detection and spatial depiction of brain tumor progression prior to detection by currently available conventional MRI-based criteria. While further validation of PRM for detection of tumor recurrence is currently underway, the ability to detect tumor progression significantly earlier than currently possible is anticipated to have a major impact on patient care. [1] Macdonald, D. R., et al. (1990). J Clin Oncol 8(7): 1277-1280. [2] Wen, P. Y., et al. (2010). J Clin Oncol 28(11): 1963-1972. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 452. doi:1538-7445.AM2012-452

Interferon regulatory factor 7 regulates glioma stem cells via interleukin-6 and Notch signalling

Inflammatory microenvironment signalling plays a crucial role in tumour progression (i.e. cancer cell proliferation, survival, angiogenesis and metastasis) in many types of human malignancies. However, the role of inflammation in brain tumour pathology remains poorly understood. Here, we report that interferon regulatory factor 7 is a crucial regulator of brain tumour progression and heterogeneity. Ectopic expression of interferon regulatory factor 7 in glioma cells promotes tumorigenicity, angiogenesis, microglia recruitment and cancer stemness in vivo and in vitro through induction of interleukin 6, C-X-C motif chemokine 1 and C-C motif chemokine 2. In particular, interferon regulatory factor 7-driven interleukin 6 plays a pivotal role in maintaining glioma stem cell properties via Janus kinase/signal transducer and activator of transcription-mediated activation of Jagged-Notch signalling in glioma cells and glioma stem cells derived from glioma patients. Accordingly, the short hairpin RNA-mediated depletion of interferon regulatory factor 7 in glioma stem cells markedly suppressed interleukin 6-Janus kinase/signal transducer and activator of transcription-mediated Jagged-Notch-signalling pathway, leading to decreases in glioma stem cell marker expression, tumoursphere-forming ability, and tumorigenicity. Furthermore, in a mouse model of wound healing, depletion of interferon regulatory factor 7 suppressed tumour progression and decreased cellular heterogeneity. Finally, interferon regulatory factor 7 was overexpressed in patients with high-grade gliomas, suggesting its potential as an independent prognostic marker for glioma progression. Taken together, our findings indicate that interferon regulatory factor 7-mediated inflammatory signalling acts as a major driver of brain tumour progression and cellular heterogeneity via induction of glioma stem cell genesis and angiogenesis.

Assessment of intra-observer variability in measurement of high-grade brain tumors

A 25% increase in bidimensional products (BPs) of tumor diameter has been used as a criterion for brain tumor progression. We studied intra-observer variability in measurements of BPs. Ten patients with contrast-enhancing glioblastoma multiforme underwent baseline and follow-up MR imaging. Seven observers measured BPs in various planes. Differences in BPs between scans were expressed as a percentage of baseline. This calculation was performed for both readings of the baseline and follow-up scans. Differences between change from baseline to follow-up on each reading (termed D values) were calculated for each reader (total of 196 D values). Median D value in each plane was calculated for each reader. Range of D values was 12.36-33.64% in axial plane (average 10.63%), 12.18-38.62% in coronal plane (average 26.84%) and 15.12-35.48% in sagittal plane (average 26.11%). Across all planes, 88 (45%) D values were >25%. When all imaging planes for any single observation were combined, in 76% of cases, at least one D value of >25% was seen. Based on the high degree of intra-observer variability, tumor measurements producing an increase in BP of >25% can routinely be obtained solely by chance.

Loss of Brain-enriched miR-124 MicroRNA Enhances Stem-like Traits and Invasiveness of Glioma Cells

miR-124 is a brain-enriched microRNA that plays a crucial role in neural development and has been shown to be down-regulated in glioma and medulloblastoma, suggesting its possible involvement in brain tumor progression. Here, we show that miR-124 is down-regulated in a panel of different grades of glioma tissues and in all of the human glioma cell lines we examined. By integrated bioinformatics analysis and experimental confirmation, we identified SNAI2, which is often up-regulated in glioma, as a direct functional target of miR-124. Because SNAI2 has been shown to regulate stem cell functions, we examined the roles of miR-124 and SNAI2 in glioma cell stem-like traits. The results showed that overexpression of miR-124 and knockdown of SNAI2 reduced neurosphere formation, CD133(+) cell subpopulation, and stem cell marker (BMI1, Nanog, and Nestin) expression, and these effects could be rescued by re-expression of SNAI2. Furthermore, enhanced miR-124 expression significantly inhibited glioma cell invasion in vitro. Finally, stable overexpression of miR-124 and knockdown of SNAI2 inhibited the tumorigenicity and invasion of glioma cells in vivo. These findings reveal, for the first time, that the tumor suppressor activity of miR-124 could be partly due to its inhibitory effects on glioma stem-like traits and invasiveness through SNAI2.

Spurious progression in pediatric brain tumors

In this study, we sought to characterize post-therapy MRI changes mimicking progression, which we refer to as "spurious progression" (SP) in children with brain tumors. We analyzed whether SP is associated with particular tumor types or therapeutic modalities. Between 2000 and 2009, we identified 181 consecutive children <21 years of age at our center who were treated for brain tumors and had at least three MRI scans within a year after completing therapy. SP was defined as MRI abnormalities characterized by increase in size, enhancement, edema, or cystic changes within 12 months following therapy, and stabilization or improvement on subsequent imaging. One-hundred forty-one patients with brain tumors were evaluable. Fifty-six (40%) had imaging abnormalities initially suggestive of disease progression; of these, 34 (24%) had true disease progression (TP). The remaining 22 (16%) had SP based on either stability, decrease in enhancement, edema, size, or disappearance of these cystic or non-cystic abnormalities. SP occurred in patients with low grade (n = 20) and high grade lesions (n = 2). Median time to SP was 2.4 months (range, 0.7-8.3 months), with time to stability, decrease, or disappearance at a median of 4 months (range 1.4-7.7 months). Five patients were clinically symptomatic from SP and were treated with steroids, cyst drainage, and/or surgery. Therefore, SP occurs more commonly in children with low grade tumors, but can also occur with high grade brain tumors, regardless of therapeutic approach.

Overexpression of S6 Kinase 1 in Brain Tumours Is Associated with Induction of Hypoxia-Responsive Genes and Predicts Patients' Survival

mTOR/S6K pathway is a crucial regulator of cell growth and metabolism. Deregulated signalling via S6K has been linked to various human pathologies, including metabolic disorders and cancer. Many of the molecules signalling upstream of S6K have been shown to be either mutated or overexpressed in tumours, leading to S6K activation. The role of S6K1 in brain tumours is not fully investigated. In this study, we investigated the gene expression profile of S6 kinases in brain and CNS tumours using the publically available Cancer Microarray Database. We found that S6K1 but not S6K2 gene is overexpressed in brain tumours and this upregulation is associated with patients' poor survival. Furthermore, we interrogated Oncomine database for the expression profile of hypoxia-induced genes using a literature-defined concept. This gene list included HIF1A, VEGFA, SOX4, SOX9, MMP2, and NEDD9. We show that those genes are upregulated in all brain tumour studies investigated. Additionally, we analysed the coexpression profile of S6K1 and hypoxia responsive genes. The analysis was done across 4 different brain studies and showed that S6K1 is co-overexpressed with several hypoxia responsive genes. This study highlights the possible role of S6K1 in brain tumour progression and prediction of patients' survival. However, new epidemiological studies should be conducted in order to confirm these associations and to refine the role of S6K1 in brain tumours as a useful marker for patients' survival.

Paramagnetic Ions Affect Relaxation Rate Dispersion of Blood: Implications for Magnetic Resonance Relaxation Dispersion Imaging

The proton relaxation rate dispersion of paramagnetic ions in blood samples was studied in the frequency interval from 10 kHz to 10 MHz. With the Field cycling method we applied, the samples were first magnetized by a relatively high magnetic field (0.5 T). By electronic means this field was rapidly (1 ms) reduced to a lower value in the interval 0.1 mT - 0.5 T, where the excited proton spin may relax during a time interval of about 3·T1max. Then the magnetic field is again quickly raised up to the higher level for the detection of the NMR-signal. The relaxation characteristics were analyzed by applying a model with three compartments of water proton-spin exchange. For each compartment we estimated a correlation frequency by fitting the dispersion curves to a sum of Lorentz distributions. We found that low concentrations of paramagnetic ions have a large influence on the relaxation rate dispersion in the low frequency region 100 mT) and low (<10 mT) relaxation fields. Such sequences of images could be used to study the oxygen status and metabolism of the brain as well as the generation and distribution of reactive oxygen species (ROS). This technique called “Magnetic resonance Relaxation Dispersion Imaging” (MARDI) would be particularly suitable to analyze various neurological diseases such as stroke, MS, Alzheimer´s disease as well as brain tumor progression. It might also be suitable for imaging of tumour oxygenation and in vivo ROS distributions in radiation therapy, which could be used in dose planning and for analyzing and optimizing the effect of various radiation therapy regimes.

In-silico Modelling of Tumour-Immune System Interactions for Glioblastomas

AbstractIn the present work, a new mathematical approach for modelling the influence of the immune system, more precisely of microglial cells, on the progression of malignant primary brain tumours is presented. A hybrid approach is used to model the cellular tumour progression, the development of the local nutrient concentration and of the density of the extracellular matrix (ECM). The resting microglia in primary brain tumours are activated and attracted by signals emitted by tumour cells, which are described by a partial differential equation. The secretion of matrix degrading enzymes from amoeboid immune cells can be modelled with the help of an additional term for the degradation of the ECM. This supports a more invasive migration of tumour cells. To our knowledge, we present for the first time a model of microglial cells in the context of tumour growth. The qualitative results are identical to the cell arrangements described in the literature. In addition, the comparison with in-vitro data matches in a qualitative manner. The proposed model, thus, represents a promising approach for modelling brain tumour growth at the cellular level in the light of the innate immune system.

A Generic Framework for Modeling Brain Deformation as a Constrained Parametric Optimization Problem to Aid Non-diffeomorphic Image Registration in Brain Tumor Imaging

In the present paper a novel computational framework for modeling tumor induced brain deformation as a biophysical prior for non-rigid image registration is described. More precisely, we aim at providing a generic building block for non-rigid image registration that can be used to resolve inherent irregularities in non-diffeomorphic registration problems that naturally arise in serial and cross-population brain tumor imaging studies due to the presence (or progression) of pathology. The model for the description of brain cancer dynamics on a tissue level is based on an initial boundary value problem (IBVP). The IBVP follows the accepted assumption that the progression of primary brain tumors on a tissue level is governed by proliferation and migration of cancerous cells into surrounding healthy tissue. The model of tumor induced brain deformation is phrased as a parametric, constrained optimization problem. As a basis of comparison and to demonstrate generalizability additional soft constraints (penalties) are considered. A back-tracking line search is implemented in conjunction with a limited memory Broyden-Fletcher-Goldfarb-Shanno (LBFGS) method in order to handle the numerically delicate log-barrier strategy for confining volume change. Numerical experiments are performed to test the flexible control of the computed deformation patterns in terms of varying model parameters. The results are qualitatively and quantitatively related to patterns in patient individual magnetic resonance imaging data. Numerical experiments demonstrate the flexible control of the computed deformation patterns. This in turn strongly suggests that the model can be adapted to patient individual imaging patterns of brain tumors. Qualitative and quantitative comparison of the computed cancer profiles to patterns in medical imaging data of an exemplary patient demonstrates plausibility. The designed optimization problem is based on computational tools widely used in non-rigid image registration, which in turn makes the model generally applicable for integration into non-rigid image registration algorithms.

Collagen VI Ablation Retards Brain Tumor Progression Due to Deficits in Assembly of the Vascular Basal Lamina

To investigate the importance of the vascular basal lamina in tumor blood vessel morphogenesis and function, we compared vessel development, vessel function, and progression of B16F10 melanoma tumors in the brains of wild-type and collagen VI-null mice. In 7-day tumors in the absence of collagen VI, the width of the vascular basal lamina was reduced twofold. Although the ablation of collagen VI did not alter the abundance of blood vessels, a detailed analysis of the number of either pericytes or endothelial cells (or pericyte coverage of endothelial cells) showed that collagen VI-dependent defects during the assembly of the basal lamina have negative effects on both pericyte maturation and the sprouting and survival of endothelial cells. As a result of these deficits, vessel patency was reduced by 25%, and vessel leakiness was increased threefold, resulting in a 10-fold increase in tumor hypoxia along with a fourfold increase in hypoxia-inducible factor-1α expression. In 12-day collagen VI-null tumors, vascular endothelial growth factor expression was increased throughout the tumor stroma, in contrast to the predominantly vascular pattern of vascular endothelial growth factor expression in wild-type tumors. Vessel size was correspondingly reduced in 12-day collagen VI-null tumors. Overall, these vascular deficits produced a twofold decrease in tumor volume in collagen VI-null mice, confirming that collagen VI-dependent basal lamina assembly is a critical aspect of vessel development.

A Novel Sequential Treatment Utilizing CPX-351 As Salvage Chemotherapy Followed by a Reduced Intensity Conditioning Allogeneic Stem-Cell Transplantation for Patients with Refractory Leukemia

Abstract 3030 Introduction:Allogeneic Hematopoeitic Stem cell Transplantation (HSCT) using standard ablative or reduced intensity conditioning regimens is often ineffective in patients with primary refractory and relapsed acute leukemia. Sequential administration of cytoreductive chemotherapy followed by a Reduced Intensity Conditioning (RIC) regimen may lead to improved results (Schmid et al Blood 2006). CPX-351 is a novel liposomal formulation that encapsulates the combination of cytarabine and daunorubicin in a fixed 5: 1 ratio. In vitro, it selectively concentrates in the marrow compared to other organs. Clinically, CPX-351 is well tolerated, with a favorable extramedullary toxicity profile, making it an appropriate cytoreductive agent prior to conditioning for HSCT.Patients and Methods: In a 3+3 phase I trial, patients with relapsed or primary refractory acute leukemia were treated with escalating doses of CPX-351 starting at 60 mg/m2 on days - 28, -26 and -24 followed by RIC with IV busulfan 3.2 mg/kg/day on days -6 to -3 and fludarabine 30 mg /m2/day on days -6 to -3 (Bu/Flu). GVHD prophylaxis consisted of tacrolimus starting on day -3, at a starting dose of 0.03 mg/kg/24 hours as a continuous infusion and adjusted to achieve a trough level between 10 and 15 ng/ml. and methotrexate 10 mg/m2 IV on days 1, 3, 6 and 11 (Methotrexate dose was reduced to 5 mg/m2 after observing grade 3 mucositis in the first 3 patients). The protocol was amended to include a phase 1B in addition to the above mentioned phase 1A. In phase 1B, patients were treated with escalating doses of CPX-351 starting at 60 mg/m2 on days - 21, -19 and -17 followed by IV Bu/Flu conditioning. Thirty two patients (AML-27, ALL-2, CML in blast crisis-1, high risk MDS-2) have been enrolled to date. Nine patients are in-evaluable due to short follow up (2), sepsis resulting in aborted transplant plans (4), appendicitis (1), early death at day +12 due to sepsis prior to engraftment (1) and donor’s unavailability after receiving one dose of CPX-351 (1). Twenty three patients who underwent HSCT are evaluable (AML-19, ALL-2, high risk MDS-2). We calculated the transplant co-morbidity index as well as the prognostic score identified by the CIBMTR in patients with refractory leukemia undergoing HSCT (Duval et al. JCO 2010). The twenty three evaluable patients have a median age of 58 (range 33–72), co- morbidity index 2 (range 0–6) and CIBMTR score 3 (range (2–5). All patients received HLA compatible grafts (MUD-15, MRD-8). Results:Nineteen patients achieved complete hematologic remission by day 30 post transplant. All nineteen patients had adequate donor’s engraftment for neutrophils and platelets at a median time of 15 days (range 12–35) and 16.5 days (range 10–90) respectively. Four patients continued to have active disease by day 30. Five more patients had a disease relapse before or shortly after day 100 and one patient had a relapse 22 months post transplant for a total of 10 relapsed patients (43%). Three patients died of acute GI GVHD 2, 6 and 9 months after transplant (all were in remission). One patient died of a pre existing brain tumor progression and one patient died of liver cirrhosis due to iron overload one year after transplant for a total non relapse mortality of 21%. At a median follow up of 6 months, eight patients are alive without evidence of leukemia (35%). Acute GVHD grade II-IV occurred in 8 patients (35%) and was the cause of death in 3 patients. Chronic GVHD occurred in 3 patients (13%). Grade 2 mucosal injury as defined by Bearman toxicity criteria was the most common toxicity developing in 15 patients (65%). Conclusion:The maximum tolerated (MTD) dose of CPX-351 followed by RIC HSCT was not found after a series of 4 treatment cohorts on Arm A and 2 treatment cohorts on arm B. Further dose escalation to define MTD is ongoing in both arms. Remission status is not a prerequisite for a successful outcome in selected patients who otherwise are candidates for transplantation.TablePatient characteristicsCharacteristicEvaluable-23AgeMedian/range56 (33–72)Male sex. %8 (35%)Co-Morbidity Index, median (range)2 (0–6)CIBMTR prognostic score, median (range)3 (2–5)DiseaseAML, %19 (82%)ALL2High risk MDS2DonorMRD, %8 (35%)MUD, %15 (65%)Cohort A, 3 wks between CPX and RIC15Cohort B, 2 wks between CPX and RIC8Table 2:Patient Outcomes at a median follow up of 6 monthsRelapse Rate10 (43%)Non-Relapse Mortality5 (21%)Leukemia Free Survival6 (36%) Disclosures:Off Label Use: CPX is not FDA approved. Feldman:celator: Consultancy.

Hypothesis: Are Neoplastic Macrophages/Microglia Present in Glioblastoma Multiforme?

Most malignant brain tumours contain various numbers of cells with characteristics of activated or dysmorphic macrophages/microglia. These cells are generally considered part of the tumour stroma and are often described as TAM (tumour-associated macrophages). These types of cells are thought to either enhance or inhibit brain tumour progression. Recent evidence indicates that neoplastic cells with macrophage characteristics are found in numerous metastatic cancers of non-CNS (central nervous system) origin. Evidence is presented here suggesting that subpopulations of cells within human gliomas, specifically GBM (glioblastoma multiforme), are neoplastic macrophages/microglia. These cells are thought to arise following mitochondrial damage in fusion hybrids between neoplastic stem cells and macrophages/microglia.

Identification of a peptide that interacts with Nestin protein expressed in brain cancer stem cells

Glioma stem cells (GSCs) are presumably major culprits for brain tumor initiation, progression, and recurrence after conventional therapies. Thus, selective targeting and eradication of GSCs may provide a promising and effective therapeutic approach. Here, we isolated a GSC-targeting (GSCT) peptide that demonstrated selective binding affinity for many undifferentiated GSCs using in vitro phage display technology. This GSCT peptide binds to isotypes of Nestin proteins specifically expressed in GSCs, enabling it to target Nestin-positive cells in human glioblastoma tissues. In human glioblastoma tissue specimens, the fluorescence-conjugated GSCT peptide could visualize putative GSC populations, showing its possible use as a diagnostic agent. GSCT peptide is also internalized into undifferentiated GSCs specifically in vitro, and moreover, intravenously injected GSCT peptide effectively penetrated into tissues, specifically accumulated in gliomas that arise from subcutaneous and orthotopic implantation, and predominantly targeted Nestin-positive cells in these tumors. Thus, our GSCT peptide may be useful for the development of more promising therapeutic and diagnostic modalities that target GSCs in brain tumors.

Optimal Presentation Modes for Detecting Brain Tumor Progression

A common task in radiology interpretation is visual comparison of images. The purpose of this study was to compare traditional side-by-side and in-place (flicker) image presentation modes with advanced methods for detecting primary brain tumors on MR imaging. We identified 66 patients with gliomas and 3 consecutive brain MR imaging examinations (a "triplet"). A display application that presented images in side-by-side mode with or without flicker display as well as display of image subtraction or automated change detection information (also with and without flicker display) was used by 3 board-certified neuroradiologists. They identified regions of brain tumor progression by using this display application. Each case was reviewed using all modes (side-by-side presentation with and without flicker, subtraction with and without flicker, and change detection with and without flicker), with results compared via a panel rating. Automated change detection with or without flicker (P < .0027) as well as subtraction with or without flicker (P < .0027) were more sensitive to tumor progression than side-by-side presentation in cases where all 3 raters agreed. Change detection afforded the highest interrater agreement, followed by subtraction. Clinically determined time to progression was longer for cases rated as nonprogressing by using subtraction images and change-detection images both with and without flicker display mode compared with side-by-side presentation. Automated change detection and image subtraction, with and without flicker display mode, are superior to side-by-side image comparison.

Elucidating the mechanobiology of malignant brain tumors using a brain matrix-mimetic hyaluronic acid hydrogel platform

Glioblastoma multiforme (GBM) is a malignant brain tumor characterized by diffuse infiltration of single cells into the brain parenchyma, which is a process that relies in part on aberrant biochemical and biophysical interactions between tumor cells and the brain extracellular matrix (ECM). A major obstacle to understanding ECM regulation of GBM invasion is the absence of model matrix systems that recapitulate the distinct composition and physical structure of brain ECM while allowing independent control of adhesive ligand density, mechanics, and microstructure. To address this need, we synthesized brain-mimetic ECMs based on hyaluronic acid (HA) with a range of stiffnesses that encompasses normal and tumorigenic brain tissue and functionalized these materials with short Arg-Gly-Asp (RGD) peptides to facilitate cell adhesion. Scanning electron micrographs of the hydrogels revealed a dense, sheet-like microstructure with apparent nanoscale porosity similar to brain extracellular space. On flat hydrogel substrates, glioma cell spreading area and actin stress fiber assembly increased strongly with increasing density of RGD peptide. Increasing HA stiffness under constant RGD density produced similar trends and increased the speed of random motility. In a three-dimensional (3D) spheroid paradigm, glioma cells invaded HA hydrogels with morphological patterns distinct from those observed on flat surfaces or in 3D collagen-based ECMs but highly reminiscent of those seen in brain slices. This material system represents a brain-mimetic model ECM with tunable ligand density and stiffness amenable to investigations of the mechanobiological regulation of brain tumor progression.