Publications by authors named "Serena Pellegatta"

49 Publications

Modifications to the Framework Regions Eliminate Chimeric Antigen Receptor Tonic Signaling.

Cancer Immunol Res 2021 Feb 5. Epub 2021 Feb 5.

Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina.

Chimeric antigen receptor (CAR) tonic signaling, defined as spontaneous activation and release of proinflammatory cytokines by CAR-T cells, is considered a negative attribute because it leads to impaired antitumor effects. Here, we report that CAR tonic signaling is caused by the intrinsic instability of the mAb single-chain variable fragment (scFv) to promote self-aggregation and signaling via the CD3ζ chain incorporated into the CAR construct. This phenomenon was detected in a CAR encoding either CD28 or 4-1BB costimulatory endodomains. Instability of the scFv was caused by specific amino acids within the framework regions (FWR) that can be identified by computational modeling. Substitutions of the amino acids causing instability, or humanization of the FWRs, corrected tonic signaling of the CAR, without modifying antigen specificity, and enhanced the antitumor effects of CAR-T cells. Overall, we demonstrated that tonic signaling of CAR-T cells is determined by the molecular instability of the scFv and that computational analyses of the scFv can be implemented to correct the scFv instability in CAR-T cells with either CD28 or 4-1BB costimulation.
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http://dx.doi.org/10.1158/2326-6066.CIR-20-0451DOI Listing
February 2021

High tumor mutational burden and T-cell activation are associated with long-term response to anti-PD1 therapy in Lynch syndrome recurrent glioblastoma patient.

Cancer Immunol Immunother 2021 Mar 3;70(3):831-842. Epub 2020 Nov 3.

Unit of Molecular Neuro-Oncology, Fondazione IRCCS Istituto Neurologico Carlo Besta, Via Celoria 11, 20133, Milan, Italy.

Background: Glioblastomas (GBMs) in patients harboring somatic or germinal mutations of mismatch-repair (MMR) genes exhibit a hypermutable phenotype. Here, we describe a GBM patient with increased tumor mutational burden and germline MMR mutations, treated using anti-PD1 therapy.

Methods: A woman with newly diagnosed GBM (nGBM) was treated by surgery, radiotherapy, and temozolomide. The tumor recurred after 13 months leading to a second surgery and treatment with nivolumab. Whole-exome sequencing was performed on the nGBM, recurrent GBM (rGBM), and blood. Immune infiltration was investigated by immunohistochemistry and the immune response in the blood during treatment was analyzed by flow cytometry.

Results: High density of infiltrating CD163 + cells was found in both GBM specimens. Large numbers of CD3 + and CD8 + T cells were homogeneously distributed in the nGBM. The infiltration of CD4 + T cells and a different CD8 + T cell density were observed in the rGBM. Both GBM shared 12,431 somatic mutations, with 113 substitutions specific to the nGBM and 1,683 specific to the rGBM. Germline variants included pathogenic mutation in the MSH2 (R359S) gene, suggesting the diagnosis of Lynch syndrome. Systemic immunophenotyping revealed the generation of CD8 + T memory cells and persistent activation of CD4 + T cells. The patient is still receiving nivolumab 68 months after the second surgery.

Conclusions: Our observations indicate that the hypermutator phenotype associated with germinal mutations of MMR genes and abundant T-cell infiltration contributes to a durable clinical benefit sustained by a persistent and robust immune response during anti-PD1 therapy.
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http://dx.doi.org/10.1007/s00262-020-02769-4DOI Listing
March 2021

MR-Spectroscopy and Survival in Mice with High Grade Glioma Undergoing Unrestricted Ketogenic Diet.

Nutr Cancer 2020 Sep 21:1-8. Epub 2020 Sep 21.

Neuroscience Department-Neurology/Stroke Unit, Ospedale A. Manzoni, ASST Lecco, Lecco, Italy.

Glioblastoma multiforme (GBM) is considered the most malignant form of primary brain tumor. Despite multimodal treatment, prognosis remains poor. Ketogenic diet (KD) has been suggested for the treatment of GBM. In this study, the syngenic, orthotopic GL261 mouse glioma model was used to evaluate the effects of KD on the metabolic responses of the tumor using 7T magnetic resonance imaging/spectroscopy. GL261 cells were injected into the caudate nucleus of mice. Following implantation, animals were fed with standard chow or underwent a KD. 18 days after initiating the diet, mice fed with KD displayed significantly higher plasmatic levels of ketone bodies and survived longer than those fed with the standard diet. Decreased concentrations of gamma-aminobutyric acid, N-Acetyl-Aspartate and N-acetylaspartylglutamate were found in tumor tissue after 9 days into the KD, while a huge increase in beta-hydroxybutyrate (bHB) was detected in tumor tissue as compared to normal brain. The accumulation of bHB in the tumor tissue in mice undergoing the KD, may suggest either elevated uptake/release of bHB by tumor cells, or the inability of tumor cells in this context to use it for mitochondrial metabolism.
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http://dx.doi.org/10.1080/01635581.2020.1822423DOI Listing
September 2020

Expansion of effector and memory T cells is associated with increased survival in recurrent glioblastomas treated with dendritic cell immunotherapy.

Neurooncol Adv 2019 May-Dec;1(1):vdz022. Epub 2019 Aug 20.

Laboratory of Brain Tumor Immunotherapy, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan, Italy.

Background: The efficacy of dendritic cell (DC) immunotherapy as a single therapeutic modality for the treatment of glioblastoma (GBM) patients remains limited. In this study, we evaluated in patients with GBM recurrence the immune-mediated effects of DC loaded with autologous tumor lysate combined with temozolomide (TMZ) or tetanus toxoid (TT).

Methods: In the phase I-II clinical study DENDR2, 12 patients were treated with 5 DC vaccinations combined with dose-dense TMZ. Subsequently, in eight patients, here defined as Variant (V)-DENDR2, the vaccine site was preconditioned with TT 24 hours before DC vaccination and TMZ was avoided. As a survival endpoint for these studies, we considered overall survival 9 months (OS9) after second surgery. Patients were analyzed for the generation of effector, memory, and T helper immune response.

Results: Four of 12 DENDR2 patients reached OS9, but all failed to show an immunological response. Five of eight V-DENDR2 patients (62%) reached OS9, and one patient is still alive (OS >30 months). A robust CD8 T-cell activation and memory T-cell formation were observed in V-DENDR2 OS>9. Only in these patients, the vaccine-specific CD4 T-cell activation (CD38/HLA-DR) was paralleled by an increase in TT-induced CD4/CD38/CD127 memory T cells. Only V-DENDR2 patients showed the formation of a nodule at the DC injection site infiltrated by CCL3-expressing CD4 T cells.

Conclusions: TT preconditioning of the vaccine site and lack of TMZ could contribute to the efficacy of DC immunotherapy by inducing an effector response, memory, and helper T-cell generation.
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http://dx.doi.org/10.1093/noajnl/vdz022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7212883PMC
August 2019

PGE Is Crucial for the Generation of FAST Whole- Tumor-Antigens Loaded Dendritic Cells Suitable for Immunotherapy in Glioblastoma.

Pharmaceutics 2020 Mar 2;12(3). Epub 2020 Mar 2.

Cell Therapy Production Unit-UPTC and Cerebrovascular Unit, Fondazione IRCCS Istituto Neurologico Carlo Besta, 20133 Milan, Italy.

Dendritic cells (DC) are the most potent antigen-presenting cells, strongly inducers of T cell-mediated immune responses and, as such, broadly used as vaccine adjuvant in experimental clinical settings. DC are widely generated from human monocytes following in vitro protocols which require 5-7 days of differentiation with GM-CSF and IL-4 followed by 2-3 days of activation/maturation. In attempts to shorten the vaccine's production, Fast-DC protocols have been developed. Here we reported a Fast-DC method in compliance with good manufacturing practices for the production of autologous mature dendritic cells loaded with antigens derived from whole tumor lysate, suitable for the immunotherapy in glioblastoma patients. The feasibility of generating Fast-DC pulsed with whole tumor lysate was assessed using a series of small-scale cultures performed in parallel with clinical grade large scale standard method preparations. Our results demonstrate that this Fast protocol is effective only in the presence of PGE in the maturation cocktail to guarantee that Fast-DC cells exhibit a mature phenotype and fulfill all requirements for in vivo use in immunotherapy approaches. Fast-DC generated following this protocol were equally potent to standard DC in inducing Ag-specific T cell proliferation in vitro. Generation of Fast-DC not only reduces labor, cost, and time required for in vitro clinical grade DC development, but can also minimizes inter-preparations variability and the risk of contamination.
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http://dx.doi.org/10.3390/pharmaceutics12030215DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7150800PMC
March 2020

ABCC3 Expressed by CD56 CD16 NK Cells Predicts Response in Glioblastoma Patients Treated with Combined Chemotherapy and Dendritic Cell Immunotherapy.

Int J Mol Sci 2019 11 23;20(23). Epub 2019 Nov 23.

Unit of Molecular Neuro-Oncology, Fondazione IRCCS Istituto Neurologico Carlo Besta, 20133 Milan, Italy.

Recently, we found that temozolomide (TMZ) can upregulate the expression of the multidrug-resistance protein ABCC3 in NK cells from both glioma-bearing mice and glioblastoma patients treated with dendritic cell immunotherapy combined with TMZ, allowing NK cells to escape apoptosis and favoring their role as antitumor effector cells. Here, we demonstrate that CD56 NK cells expressing CD16 are predominant in patients surviving more than 12 months after surgery without disease progression. CD56 CD16 NK cells co-expressed high levels of ABCC3 and IFN-. Notably, not only basal but also TMZ-induced ABCC3 expression was related to a strong, long-term NK cell response and a better prognosis of patients. The identification of the single nucleotide polymorphism (SNP) rs35467079 with the deletion of a cytosine (-897DelC) in the promoter region of the ABCC3 gene resulted associated with a better patient outcome. ABCC3 expression in patients carrying DelC compared to patients with reference haplotype was higher and modulated by TMZ. The transcription factor NRF2, involved in ABCC3 induction, was phosphorylated in CD56 CD16 NK cells expressing ABCC3 under TMZ treatment. Thus, ABCC3 protein and the SNP -897DelC can play a predictive role in patients affected by GBM, and possibly other cancers, treated with dendritic cell immunotherapy combined with chemotherapy.
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http://dx.doi.org/10.3390/ijms20235886DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6928625PMC
November 2019

Advanced MRI Assessment during Dendritic Cell Immunotherapy Added to Standard Treatment Against Glioblastoma.

J Clin Med 2019 Nov 17;8(11). Epub 2019 Nov 17.

Neuro-oncology Unit, Fondazione IRCCS Istituto Neurologico Carlo Besta, 20133 Milan, Italy.

Evaluating changes induced by immunotherapies (IT) on conventional magnetic resonance imaging (MRI) is difficult because those treatments may produce inflammatory responses. To explore the potential contribution of advanced MRI to distinguish pseudoprogression (PsP) and true tumor progression (TTP), and to identify patients obtaining therapeutic benefit from IT, we examined aMRI findings in newly diagnosed glioblastoma treated with dendritic cell IT added to standard treatment. We analyzed longitudinal MRIs obtained in 22 patients enrolled in the EUDRACT N° 2008-005035-15 trial. According to RANO criteria, we observed 18 TTP and 8 PsP. Comparing MRI performed at the time of TTP/PsP with the previous exam performed two months before, a difference in cerebral blood volume ΔrCBV ≥ 0.47 distinguished TTP from PsP with a sensitivity of 67% and specificity of 75% (p = 0.004). A decrease in minimal apparent diffusion coefficient rADC (1.15 vs. 1.01, = 0.003) was observed after four vaccinations only in patients with a persistent increase of natural killer cells (response effectors during IT) in peripheral blood. Basal rADC > 1 was independent predictor of longer progression free (16.1 vs. 9 months, = 0.0001) and overall survival (32.8 vs. 17.5 months, = 0.0005). In conclusion, rADC predicted response to immunotherapy and survival; Apparent Diffusion Coefficient (ADC) and Cerebral Blood Volume (CBV) modifications over time help differentiating PsP from TTP at onset.
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http://dx.doi.org/10.3390/jcm8112007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6912338PMC
November 2019

B7-H3-redirected chimeric antigen receptor T cells target glioblastoma and neurospheres.

EBioMedicine 2019 Sep 26;47:33-43. Epub 2019 Aug 26.

Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC, USA; Department of Microbiology and Immunology, University of North Carolina, Chapel Hill, NC, USA. Electronic address:

Background: The dismal survival of glioblastoma (GBM) patients urgently calls for the development of new treatments. Chimeric antigen receptor T (CAR-T) cells are an attractive strategy, but preclinical and clinical studies in GBM have shown that heterogeneous expression of the antigens targeted so far causes tumor escape, highlighting the need for the identification of new targets. We explored if B7-H3 is a valuable target for CAR-T cells in GBM.

Methods: We compared mRNA expression of antigens in GBM using TCGA data, and validated B7-H3 expression by immunohistochemistry. We then tested the antitumor activity of B7-H3-redirected CAR-T cells against GBM cell lines and patient-derived GBM neurospheres in vitro and in xenograft murine models.

Findings: B7-H3 mRNA and protein are overexpressed in GBM relative to normal brain in all GBM subtypes. Of the 46 specimens analyzed by immunohistochemistry, 76% showed high B7-H3 expression, 22% had detectable, but low B7-H3 expression and 2% were negative, as was normal brain. All 20 patient-derived neurospheres showed ubiquitous B7-H3 expression. B7-H3-redirected CAR-T cells effectively targeted GBM cell lines and neurospheres in vitro and in vivo. No significant differences were found between CD28 and 4-1BB co-stimulation, although CD28-co-stimulated CAR-T cells released more inflammatory cytokines.

Interpretation: We demonstrated that B7-H3 is highly expressed in GBM specimens and neurospheres that contain putative cancer stem cells, and that B7-H3-redirected CAR-T cells can effectively control tumor growth. Therefore, B7-H3 represents a promising target in GBM. FUND: Alex's Lemonade Stand Foundation; Il Fondo di Gio Onlus; National Cancer Institute; Burroughs Wellcome Fund.
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http://dx.doi.org/10.1016/j.ebiom.2019.08.030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6796553PMC
September 2019

NG2/CSPG4 in glioblastoma: about flexibility.

Neuro Oncol 2019 06;21(6):697-698

Unit of Molecular Neuro-Oncology, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan, Italy.

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http://dx.doi.org/10.1093/neuonc/noz055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6556905PMC
June 2019

Altered function of the glutamate-aspartate transporter GLAST, a potential therapeutic target in glioblastoma.

Int J Cancer 2019 05 5;144(10):2539-2554. Epub 2019 Jan 5.

Unit of Molecular Neuro-Oncology, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan, Italy.

In glioma patients, high levels of glutamate can cause brain edema and seizures. GLAST, a glutamate-aspartate transporter expressed by astrocytes with a role in glutamate uptake, is highly expressed on the plasma membrane of glioblastoma (GBM) cells, and its expression significantly correlates with shortened patient survival. Here, it was demonstrated that inhibition of GLAST expression limited the progression and invasion of GBM xenografts. Magnetic resonance spectroscopy was used to measure glutamate in GLAST-expressing gliomas showing that these tumors exhibit increased glutamate concentration compared to GLAST-depleted glioma. Despite their GLAST expression, GBM stem-like cells (GSCs) released rather than taking up glutamate due to their lack of Na+/K+-ATPase. Overexpression of Na+/K+-ATPase in these cells restored glutamate uptake and induced apoptosis. The therapeutic relevance of targeting GLAST in gliomas was assessed using the inhibitor UCPH-101. In glioma-bearing mice, a single intratumoral injection of UCPH-101 significantly increased survival by decreasing GLAST expression and inducing apoptosis. Thus, GLAST has a novel role in GBM that appears to have crucial relevance in glutamate trafficking and may thus be a new therapeutic target.
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http://dx.doi.org/10.1002/ijc.31985DOI Listing
May 2019

Survival gain in glioblastoma patients treated with dendritic cell immunotherapy is associated with increased NK but not CD8 T cell activation in the presence of adjuvant temozolomide.

Oncoimmunology 2018;7(4):e1412901. Epub 2018 Jan 29.

Unit of Molecular Neuro-Oncology, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan, Italy.

In a two-stage phase II study, 24 patients with first diagnosis of glioblastoma (GBM) were treated with dendritic cell (DC) immunotherapy associated to standard radiochemotherapy with temozolomide (TMZ) followed by adjuvant TMZ. Three intradermal injections of mature DC loaded with autologous GBM lysate were administered before adjuvant TMZ, while 4 injections were performed during adjuvant TMZ. According to a two-stage Simon design, to proceed to the second stage progression-free survival (PFS) 12 months after surgery was expected in at least 8 cases enrolled in the first stage. Evidence of immune response and interaction with chemotherapy were investigated. After a median follow up of 17.4 months, 9 patients reached PFS12. In these patients (responders, 37.5%), DC vaccination induced a significant, persistent activation of NK cells, whose increased response was significantly associated with prolonged survival. CD8 T cells underwent rapid expansion and priming but, after the first administration of adjuvant TMZ, failed to generate a memory status. Resistance to TMZ was associated with robust expression of the multidrug resistance protein ABCC3 in NK but not CD8 T cells. The negative effect of TMZ on the formation of T cell-associated antitumor memory deserves consideration in future clinical trials including immunotherapy.
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http://dx.doi.org/10.1080/2162402X.2017.1412901DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5889286PMC
January 2018

Constitutive and TNFα-inducible expression of chondroitin sulfate proteoglycan 4 in glioblastoma and neurospheres: Implications for CAR-T cell therapy.

Sci Transl Med 2018 02;10(430)

Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599, USA.

The heterogeneous expression of tumor-associated antigens limits the efficacy of chimeric antigen receptor (CAR)-redirected T cells (CAR-Ts) for the treatment of glioblastoma (GBM). We have found that chondroitin sulfate proteoglycan 4 (CSPG4) is highly expressed in 67% of the GBM specimens with limited heterogeneity. CSPG4 is also expressed on primary GBM-derived cells, grown in vitro as neurospheres (GBM-NS), which recapitulate the histopathology and molecular characteristics of primary GBM. CSPG4.CAR-Ts efficiently controlled the growth of GBM-NS in vitro and in vivo upon intracranial tumor inoculation. Moreover, CSPG4.CAR-Ts were also effective against GBM-NS with moderate to low expression of CSPG4. This effect was mediated by the in vivo up-regulation of CSPG4 on tumor cells, induced by tumor necrosis factor-α (TNFα) released by the microglia surrounding the tumor. Overall, the constitutive and TNFα-inducible expression of CSPG4 in GBM may greatly reduce the risk of tumor cell escape observed when targeted antigens are heterogeneously expressed on tumor cells.
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http://dx.doi.org/10.1126/scitranslmed.aao2731DOI Listing
February 2018

Genetic Evolution of Glioblastoma Stem-Like Cells From Primary to Recurrent Tumor.

Stem Cells 2017 11 29;35(11):2218-2228. Epub 2017 Sep 29.

Laboratory of Cancer Stem Cell Research, Candiolo Cancer Institute, FPO-IRCCS, Candiolo, Italy.

Glioblastoma (GBM) is a lethal tumor that displays remarkable genetic heterogeneity. It is also known that GBM contains a cell hierarchy driven by GBM stem-like cells (GSCs), responsible for tumor generation, therapeutic resistance, and relapse. An important and still open issue is whether phylogenetically related GSCs can be found in matched primary and recurrent GBMs, and reflect tumor genetic evolution under therapeutic pressure. To address this, we analyzed the mutational profile of GSCs isolated from either human primary GBMs (primary GSCs) or their matched tumors recurring after surgery and chemoradiotherapy (recurrent GSCs). We found that recurrent GSCs can accumulate temozolomide-related mutations over primary GSCs, following both linear and branched patterns. In the latter case, primary and recurrent GSCs share a common set of lesions, but also harbor distinctive mutations indicating that primary and recurrent GSCs derive from a putative common ancestor GSC by divergent genetic evolution. Interestingly, TP53 mutations distinctive of recurrent GSCs were detectable at low frequency in the corresponding primary tumors and likely marked pre-existent subclones that evolved under therapeutic pressure and expanded in the relapsing tumor. Consistently, recurrent GSCs displayed in vitro greater therapeutic resistance than primary GSCs. Overall, these data indicate that (a) phylogenetically related GSCs are found in matched primary and recurrent GBMs and (b) recurrent GSCs likely pre-exist in the untreated primary tumor and are both mutagenized and positively selected by chemoradiotherapy. Stem Cells 2017;35:2218-2228.
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http://dx.doi.org/10.1002/stem.2703DOI Listing
November 2017

Hypermutations in gliomas: a potential immunotherapy target.

Discov Med 2017 02;23(125):113-120

Unit of Molecular Neuro-Oncology, Fondazione IRCCS Istituto Neurologico Besta, 20133 Milano, Italy.

Checkpoint inhibitors, like ipilimumab, nivolumab, and pembrolizumab, have provided a breakthrough in cancer immunotherapy, such as in the treatment of melanoma and colorectal and lung cancer. The close relationship between the number of mutations (mutational load) and the response to checkpoint immunotherapy has been convincingly demonstrated in these cancers. Hypermutations in tumors are caused by environmental factors, like UV radiations or cigarette smoking, or by germinal mutations affecting genes of the Mismatch Repair (MMR) machinery, as in the Lynch syndrome. In the context of a high mutational load, a number of neoantigens become visible to the immune system, creating the basis for effective T cell responses. In low- and high-grade gliomas, the most frequent brain tumors, germinal MMR defects are rare; however, hypermutations associated with mutations or decreased expression of MMR genes are rather frequent, occurring in 20-60% of the tumors at recurrence after alkylating chemotherapy with temozolomide. Ongoing clinical trials and genomic investigations will clarify if temozolomide-induced hypermutations, which usually occur in the presence of methylation of the methylguanine methyltransferase gene (MGMT), will be effectively targeted by immunotherapy with checkpoint inhibitors or dendritic cell immunotherapy, thus improving the survival expectations for patients affected by these tumors.
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February 2017

ER-mitochondria contacts control surface glycan expression and sensitivity to killer lymphocytes in glioma stem-like cells.

EMBO J 2017 06 10;36(11):1493-1512. Epub 2017 Mar 10.

Department of Cell Physiology and Metabolism, University of Geneva, Geneva, Switzerland

Glioblastoma is a highly heterogeneous aggressive primary brain tumor, with the glioma stem-like cells (GSC) being more sensitive to cytotoxic lymphocyte-mediated killing than glioma differentiated cells (GDC). However, the mechanism behind this higher sensitivity is unclear. Here, we found that the mitochondrial morphology of GSCs modulates the ER-mitochondria contacts that regulate the surface expression of sialylated glycans and their recognition by cytotoxic T lymphocytes and natural killer cells. GSCs displayed diminished ER-mitochondria contacts compared to GDCs. Forced ER-mitochondria contacts in GSCs increased their cell surface expression of sialylated glycans and reduced their susceptibility to cytotoxic lymphocytes. Therefore, mitochondrial morphology and dynamism dictate the ER-mitochondria contacts in order to regulate the surface expression of certain glycans and thus play a role in GSC recognition and elimination by immune effector cells. Targeting the mitochondrial morphology, dynamism, and contacts with the ER could be an innovative strategy to deplete the cancer stem cell compartment to successfully treat glioblastoma.
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http://dx.doi.org/10.15252/embj.201695429DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5452011PMC
June 2017

Resetting cancer stem cell regulatory nodes upon MYC inhibition.

EMBO Rep 2016 12 16;17(12):1872-1889. Epub 2016 Nov 16.

IBPM - CNR, Rome, Italy

MYC deregulation is common in human cancer and has a role in sustaining the aggressive cancer stem cell populations. MYC mediates a broad transcriptional response controlling normal biological programmes, but its activity is not clearly understood. We address MYC function in cancer stem cells through the inducible expression of Omomyc-a MYC-derived polypeptide interfering with MYC activity-taking as model the most lethal brain tumour, glioblastoma. Omomyc bridles the key cancer stemlike cell features and affects the tumour microenvironment, inhibiting angiogenesis. This occurs because Omomyc interferes with proper MYC localization and itself associates with the genome, with a preference for sites occupied by MYC This is accompanied by selective repression of master transcription factors for glioblastoma stemlike cell identity such as OLIG2, POU3F2, SOX2, upregulation of effectors of tumour suppression and differentiation such as ID4, MIAT, PTEN, and modulation of the expression of microRNAs that target molecules implicated in glioblastoma growth and invasion such as EGFR and ZEB1. Data support a novel view of MYC as a network stabilizer that strengthens the regulatory nodes of gene expression networks controlling cell phenotype and highlight Omomyc as model molecule for targeting cancer stem cells.
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http://dx.doi.org/10.15252/embr.201541489DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5283599PMC
December 2016

Decitabine Treatment of Glioma-Initiating Cells Enhances Immune Recognition and Killing.

PLoS One 2016 31;11(8):e0162105. Epub 2016 Aug 31.

Centre of Oncology, Geneva University Hospitals and University of Geneva, Geneva, Switzerland.

Malignant gliomas are aggressive brain tumours with very poor prognosis. The majority of glioma cells are differentiated (glioma-differentiated cells: GDCs), whereas the smaller population (glioma-initiating cells, GICs) is undifferentiated and resistant to conventional therapies. Therefore, to better target this pool of heterogeneous cells, a combination of diverse therapeutic approaches is envisaged. Here we investigated whether the immunosensitising properties of the hypomethylating agent decitabine can be extended to GICs. Using the murine GL261 cell line, we demonstrate that decitabine augments the expression of the death receptor FAS both on GDCs and GICs. Interestingly, it had a higher impact on GICs and correlated with an enhanced sensitivity to FASL-mediated cell death. Moreover, the expression of other critical molecules involved in cognate recognition by cytotoxic T lymphocytes, MHCI and ICAM-1, was upregulated by decitabine treatment. Consequently, T-cell mediated killing of both GDCs and GICs was enhanced, as was T cell proliferation after reactivation. Overall, although GICs are described to resist classical therapies, our study shows that hypomethylating agents have the potential to enhance glioma cell recognition and subsequent destruction by immune cells, regardless of their differentiation status. These results support the development of combinatorial treatment modalities including epigenetic modulation together with immunotherapy in order to treat heterogenous malignancies such as glioblastoma.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0162105PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5007044PMC
August 2017

The multidrug-resistance transporter Abcc3 protects NK cells from chemotherapy in a murine model of malignant glioma.

Oncoimmunology 2016 May 29;5(5):e1108513. Epub 2015 Oct 29.

Unit of Molecular Neuro-Oncology , Milan, Italy.

Abcc3, a member of the ATP-binding cassette transporter superfamily, plays a role in multidrug resistance. Here, we found that Abcc3 is highly expressed in blood-derived NK cells but not in CD8(+) T cells. In GL261 glioma-bearing mice treated with the alkylating agent temozolomide (TMZ) for 5 d, an early increased frequency of NK cells was observed. We also found that Abcc3 is strongly upregulated and functionally active in NK cells from mice treated with TMZ compared to controls. We demonstrate that Abcc3 is critical for NK cell survival during TMZ administration; more importantly, Akt, involved in lymphocyte survival, is phosphorylated only in NK cells expressing Abcc3. The resistance of NK cells to chemotherapy was accompanied by increased migration and homing in the brain at early time points. Cytotoxicity, evaluated by IFNγ production and specific lytic activity against GL261 cells, increased peripherally in the later phases, after conclusion of TMZ treatment. Intra-tumor increase of the NK effector subset as well as in IFNγ, granzymes and perforin-1 expression, were found early and persisted over time, correlating with a profound modulation on glioma microenvironment induced by TMZ. Our findings reveal an important involvement of Abcc3 in NK cell resistance to chemotherapy and have important clinical implications for patients treated with chemo-immunotherapy.
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http://dx.doi.org/10.1080/2162402X.2015.1108513DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4910710PMC
May 2016

MET inhibition overcomes radiation resistance of glioblastoma stem-like cells.

EMBO Mol Med 2016 05 2;8(5):550-68. Epub 2016 May 2.

Laboratory of Cancer Stem Cell Research, Candiolo Cancer Institute, FPO-IRCCS, Candiolo, Italy Department of Oncology, University of Torino, Candiolo, Italy

Glioblastoma (GBM) contains stem-like cells (GSCs) known to be resistant to ionizing radiation and thus responsible for therapeutic failure and rapidly lethal tumor recurrence. It is known that GSC radioresistance relies on efficient activation of the DNA damage response, but the mechanisms linking this response with the stem status are still unclear. Here, we show that the MET receptor kinase, a functional marker of GSCs, is specifically expressed in a subset of radioresistant GSCs and overexpressed in human GBM recurring after radiotherapy. We elucidate that MET promotes GSC radioresistance through a novel mechanism, relying on AKT activity and leading to (i) sustained activation of Aurora kinase A, ATM kinase, and the downstream effectors of DNA repair, and (ii) phosphorylation and cytoplasmic retention of p21, which is associated with anti-apoptotic functions. We show that MET pharmacological inhibition causes DNA damage accumulation in irradiated GSCs and their depletion in vitro and in GBMs generated by GSC xenotransplantation. Preclinical evidence is thus provided that MET inhibitors can radiosensitize tumors and convert GSC-positive selection, induced by radiotherapy, into GSC eradication.
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http://dx.doi.org/10.15252/emmm.201505890DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5130292PMC
May 2016

Immunotherapy with dendritic cells loaded with glioblastoma stem cells: from preclinical to clinical studies.

Cancer Immunol Immunother 2016 Jan 16;65(1):101-9. Epub 2015 Sep 16.

Unit of Molecular Neuro-Oncology, Fondazione I.R.C.C.S. Istituto Neurologico C. Besta, Via Celoria 11, 20133, Milan, Italy.

Different approaches have been explored to raise effective antitumor responses against glioblastoma (GBM), the deadliest of primary brain tumors. In many clinical studies, cancer vaccines have been based on dendritic cells (DCs) loaded with peptides, representing one or more specific tumor antigens or whole lysates as a source of multiple antigens. Randomized clinical trials using DCs are ongoing, and results of efficacy are not yet available. Such strategies are feasible and safe; however, immune-suppressive microenvironment, absence of appropriate specific epitopes to target, and cancer immunoediting can limit their efficacy. The aim of this review is to describe how the definition of novel and more specific targets may increase considerably the possibility of successful DC immunotherapy. By proposing to target glioblastoma stem-like cells (GSCs), the immune response will be pointed to eradicating factors and pathways highly relevant to GBM biology. Preclinical observations on efficacy, and preliminary results of immunotherapy trials, encourage exploring the clinical efficacy of DC immunotherapy in GBM patients using high-purity, GSC-loaded DC vaccines.
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http://dx.doi.org/10.1007/s00262-015-1754-9DOI Listing
January 2016

Novel mechanisms and approaches in immunotherapy for brain tumors.

Discov Med 2015 Jul-Aug;20(108):7-15

Unit of Molecular Neuro-Oncology, Fondazione IRCCS Istituto Neurologico Besta, via Celoria 11, 20133 Milano, Italy.

Converging data indicate that the immune system is able to recognize cancer epitopes as non-self and mount an immune reaction that may erase, or temporarily block, tumor growth. The immune pressure supports the amplification of immune resistant tumor clones, creating an immune suppressive environment that leads to the formation of a clinically relevant tumor. These general observations also apply to brain tumors and specifically to gliomas. Cancer immunotherapy strategies are aimed at reverting such immune suppression. Two approaches are already used in the clinics. The first one, peptide immunotherapy, has been oriented to the most aggressive glioma, glioblastoma (GBM) where, in the context of EGFR (epidermal growth factor receptor) amplification, a large deletion arises and creates a novel, cancer-specific antigen, EGFRvIII. The second one is dendritic cell immunotherapy. Dendritic cells are potent antigen presenting cells that can be pulsed with autologous tumor lysate or peptide pp65 from cytomegalovirus (CMV) that is present in GBM but not in normal brain. Antigen presentation by dendritic cells is bolstered by preconditioning their injection site with the tetanus/diphtheria toxoid. The third approach is adoptive cell therapy (ACT) in which tumor-specific T cells can be amplified ex vivo and subsequently re-injected to the patient to lyse cells expressing tumor antigens, increasing survival durably in a fraction of melanoma patients. ACT may also be based on T cell transduction of tumor specific receptors or chimeric antigen receptors (CARs). CARs are powerful tools for immunotherapy but off-target toxicity may be an issue as they do not request MHC presentation for activation. Upcoming clinical trial results will clarify the most effective direction for cancer immunotherapy in gliomas and other cancers with poor prognosis.
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May 2016

Safe and Reproducible Preparation of Functional Dendritic Cells for Immunotherapy in Glioblastoma Patients.

Stem Cells Transl Med 2015 Oct 13;4(10):1164-72. Epub 2015 Aug 13.

Cell Therapy Production Unit, Laboratory of Cellular Neurobiology, Cerebrovascular Unit, and Unit of Molecular Neuro-Oncology, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan, Italy.

Unlabelled: Cell therapy based on dendritic cells (DCs) pulsed with tumor lysate is a promising approach in addition to conventional therapy for the treatment of patients with glioblastoma (GB). The success of this approach strongly depends on the ability to generate high-quality, functionally mature DCs (mDCs), with a high level of standardization and in compliance with Good Manufacturing Practices. In the cell factory of the Carlo Besta Foundation, two phase I clinical trials on immunotherapy with tumor lysate-loaded DCs as treatment for GB are ongoing. From 2010 to 2014, 54 patients were enrolled in the studies and 54 batches of DCs were prepared. We retrospectively analyzed the results of the quality control tests carried out on each produced batch, evaluating yield of mDCs and their quality in terms of microbiological safety and immunological efficacy. The number of mDCs obtained allowed the treatment of all the enrolled patients. All 54 batches were sterile, conformed to acceptable endotoxin levels, and were free of Mycoplasma species and adventitious viruses. During culture, cells maintained a high percentage of viability (87%-98%), and all batches showed high viability after thawing (mean±SD: 94.6%±2.9%). Phenotype evaluation of mDCs showed an evident upregulation of markers typical of DC maturation; mixed lymphocyte reaction tests for the functional evaluation of DCs demonstrated that all batches were able to induce lymphocyte responses. These results demonstrated that our protocol for DC preparation is highly reproducible and permits generation of large numbers of safe and functional DCs for in vivo use in immunotherapy approaches.

Significance: Cell therapy based on antigen-pulsed dendritic cells (DCs) is a promising approach for the treatment of glioblastoma patients. The success of this approach strongly depends on the ability to generate high-quality, functional DCs with a high level of standardization, ensuring reproducibility, efficacy, and safety of the final product. This article summarizes the results of the quality controls on 54 batches, to demonstrate the feasibility of producing a therapeutic cell-based vaccine via a well-controlled Good Manufacturing Practices (GMP)-compliant production process. The findings may be of scientific interest to those working in the field of preparation of GMP-compliant products for cell-therapy applications.
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http://dx.doi.org/10.5966/sctm.2015-0091DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4572908PMC
October 2015

Effective immuno-targeting of the IDH1 mutation R132H in a murine model of intracranial glioma.

Acta Neuropathol Commun 2015 Jan 21;3. Epub 2015 Jan 21.

The R132H mutation of cytosolic isocitrate dehydrogenase (IDH1) is present in the majority of low grade gliomas.Immunotherapy in these tumors has an interesting, still unexploited, therapeutic potential, as they are less immunosuppressive than glioblastomas. Using site-directed mutagenesis we introduced the R132H mutation into the murine glioma cell line GL261,creating mIDH1-GL261. Presence of the mutation was confirmed by immunoblotting and production of the oncometabolite 2-hydroxyglutarate (2HG), demonstrated by mass spectrometry (LC-MS/MS) performed on cell supernatant. In vitro mIDH1-GL261 had different morphology but similar growth rate than parental GL261 (p-GL261). After intracranial injection, MRI suggested that the initial growth rate was slower in mIDH1-GL261 than p-GL261 gliomas but overall survival was similar. mIDH1-GL261 gliomas showed evidence of R132H expression and of intratumoral 2HG production (evaluated by MRS and LC-MS/MS). Immunizations were performed nine days after intracranial implantation of mIDH1- or p-GL261 cells by three subcutaneous injections of five different peptides encompassing the IDH1 mutation site, all emulsified with Montanide ISA-51, in association with GM-CSF. Control mice were injected with four ovalbumin peptides or vehicle. Mice with mIDH1-GL261 but not p-GL261 gliomas treated with mIDH1 peptides survived longer than controls; 25% of them were cured. Immunized mice showed higher amounts of peripheral CD8+ T cells, higher production of IFN-γ, and evidence of anti-mIDH1 antibodies.Immunizations led to intratumoral up-regulation of IFN-γ, granzyme-b and perforin-1 and down-regulation of TGF-β2 and IL-10. These results support the translational potential of immunotherapeutic targeting of gliomas carrying IDH1 mutations.
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http://dx.doi.org/10.1186/s40478-014-0180-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4359524PMC
January 2015

EGFR amplified and overexpressing glioblastomas and association with better response to adjuvant metronomic temozolomide.

J Natl Cancer Inst 2015 May 3;107(5). Epub 2015 Mar 3.

Pathology (MC, BL, MFB, DM, VP, FF, PLP) and Pharmacology Units (CB, MP), Department of Molecular and Translational Medicine, University of Brescia and National Institute of Neuroscience, Italy; Medical Oncology (SG), Neurosurgery (LB), Radiation Oncology (MB), and Neuroradiology Departments (RL), Spedali Civili of Brescia, University of Brescia, Italy; Neural Stem Cell Biology Unit, Division of Regenerative Medicine, Stem Cells & Gene Therapy, San Raffaele Scientific Institute, Milan (SM, RG); Pathology Unit, Department of Surgical and Morphological Sciences, University of Insubria, Italy (DF); Neurological Institute Besta, Milan, Italy (SP, GF); Herbert Irving Comprehensive Cancer Center, Department of Pathology & Cell Biology and Department of Medicine, Division of Digestive and Liver Diseases, Columbia University, New York, NY (PD); IRCCS San Camillo Hospital, Venice, Italy (MP).

Background: Lack of robust predictive biomarkers, other than MGMT promoter methylation, makes temozolomide responsiveness in newly diagnosed glioblastoma (GBM) patients difficult to predict. However, we identified patients with long-term survival (≥35 months) within a group of newly diagnosed GBM patients treated with standard or metronomic adjuvant temozolomide schedules. We thus investigated possible molecular profiles associated with longer survival following temozolomide treatment.

Methods: We investigated the association of molecular features with progression-free (PFS) and overall survival (OS). Human-derived GBM cancer stem cells (CSCs) were used to investigate in vitro molecular mechanisms associated with temozolomide responsiveness. Surgically removed recurrences allowed investigation of molecular changes occurring during therapy in vivo. Statistical analyses included one- and two-way analysis of variance, Student's t test, Cox proportional hazards, and the Kaplan-Meier method. All statistical tests were two-sided.

Results: No association was found between survival and gene classifiers associated with different molecular GBM subtypes in the standard-treated group, while in metronomic-treated patients robust association was found between EGFR amplification/overexpression and PFS and OS (OS, EGFR-high vs low: hazard ratiodeath = 0.22, 95% confidence interval = 0.09 to 0.55, P = .001). The result for OS remained statistically significant after Bonferroni correction (P interaction < .0005). Long-term survival following metronomic temozolomide was independent from MGMT and EGFRvIII status and was more pronounced in EGFR-overexpressing GBM patients with PTEN loss. In vitro findings confirmed a selective dose- and time-dependent decrease in survival of temozolomide-treated EGFR+ human-derived glioblastoma CSCs, which occurred through inhibition of NF-κB transcriptional activity. In addition, reduction in EGFR-amplified cells, along with a statistically significant decrease in NF-κB/p65 expression, were observed in specimens from recurrent metronomic-treated EGFR-overexpressing GBM patients.

Conclusions: EGFR-amplified/overexpressing glioblastomas strongly benefit from metronomic temozolomide-based therapies.
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http://dx.doi.org/10.1093/jnci/djv041DOI Listing
May 2015

Perspectives for immunotherapy in glioblastoma treatment.

Curr Opin Oncol 2014 Nov;26(6):608-14

Unit of Molecular Neuro-Oncology, Fondazione I.R.C.C.S. Istituto Neurologico C. Besta, Milano, Italy.

Purpose Of Review: Avoiding immune destruction is one emerging hallmark of cancer, including glioblastoma. The number of immunotherapy approaches to fight glioblastoma is growing. Here, we review the recent progress in four main areas: dendritic cell immunotherapy, peptide vaccination, chimeric antigen receptors and immune checkpoints.

Recent Findings: We and others are using dendritic cells to present glioblastoma antigens (whole tumor lysate) to the immune system; our initial data indicate that clinical benefit is associated to increased presence of natural killer cells in the periphery. A pilot study loading dendritic cells with glioblastoma stem-like cells will start soon. Peptide vaccination targeting the epidermal growth factor receptor variant III (EGFRvIII) epitope, present in 25% of glioblastomas, is ongoing. Intriguing results have been obtained by vaccination with three other peptides in pediatric gliomas. Another clinical trial is targeting EGFRvIII by adoptive cell transfer of chimeric antigen receptor. This exciting technology could be suited for a number of other potential epitopes discovered through next-generation sequencing. Finally, antibodies against the immune checkpoints cytotoxic T lymphocyte antigen-4 and programmed cell death-1, which demonstrated efficacy in advanced melanomas, will be used in novel trials for recurrent glioblastoma.

Summary: In all these studies attention to novel side-effects and to MRI as immunological follow-up to distinguish progression or pseudoprogression will be of critical relevance.
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http://dx.doi.org/10.1097/CCO.0000000000000135DOI Listing
November 2014

Sox2 is required to maintain cancer stem cells in a mouse model of high-grade oligodendroglioma.

Cancer Res 2014 Mar 5;74(6):1833-44. Epub 2014 Mar 5.

Authors' Affiliations: Department of Biotechnology and Biosciences, University of Milano-Bicocca; Department of Molecular Neuro-Oncology, Fondazione I.R.C.C.S. Istituto Neurologico C.Besta; Department of Experimental Oncology, European Institute of Oncology at IFOM-IEO Campus; Tissue Processing Unit, The FIRC Institute of Molecular Oncology Foundation-IFOM, IFOM-IEO Campus, Milano; and IRCCS Azienda Ospedaliera Universitaria San Martino, IST Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy.

The stem cell-determining transcription factor Sox2 is required for the maintenance of normal neural stem cells. In this study, we investigated the requirement for Sox2 in neural cancer stem-like cells using a conditional genetic deletion mutant in a mouse model of platelet-derived growth factor-induced malignant oligodendroglioma. Transplanting wild-type oligodendroglioma cells into the brain generated lethal tumors, but mice transplanted with Sox2-deleted cells remained free of tumors. Loss of the tumor-initiating ability of Sox2-deleted cells was reversed by lentiviral-mediated expression of Sox2. In cell culture, Sox2-deleted tumor cells were highly sensitive to differentiation stimuli, displaying impaired proliferation, increased cell death, and aberrant differentiation. Gene expression analysis revealed an early transcriptional response to Sox2 loss. The observed requirement of oligodendroglioma stem cells for Sox2 suggested its relevance as a target for therapy. In support of this possibility, an immunotherapeutic approach based on immunization of mice with SOX2 peptides delayed tumor development and prolonged survival. Taken together, our results showed that Sox2 is essential for tumor initiation by mouse oligodendroglioma cells, and they illustrated a Sox2-directed strategy of immunotherapy to eradicate tumor-initiating cells.
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http://dx.doi.org/10.1158/0008-5472.CAN-13-1942DOI Listing
March 2014

Frequency of NFKBIA deletions is low in glioblastomas and skewed in glioblastoma neurospheres.

Mol Cancer 2013 Dec 11;12:160. Epub 2013 Dec 11.

Molecular Neuro-Oncology Unit, IRCCS Foundation "C,Besta" Neurological Institute, via Celoria 11, 20133 Milan, Italy.

The NF-kB family of transcription factors is up-regulated in inflammation and different cancers. Recent data described heterozygous deletions of the NF-kB Inhibitor alpha gene (NFKBIA) in about 20% of glioblastomas (GBM): deletions were mutually exclusive with epidermal growth factor receptor (EGFR) amplification, a frequent event in GBM. We assessed the status of NFKBIA and EGFR in 69 primary GBMs and in corresponding neurospheres (NS). NFKBIA deletion was investigated by the copy number variation assay (CNV); EGFR amplification by CNV ratio with HGF; expression of EGFR and EGFRvIII by quantitative PCR or ReverseTranscriptase PCR. Heterozygous deletions of NFKBIA were present in 3 of 69 primary GBMs and, surprisingly, in 30 of 69 NS. EGFR amplification was detected in 36 GBMs: in corresponding NS, amplification was lost in 13 cases and reduced in 23 (10 vs 47 folds in NS vs primary tumors; p < 0.001). The CNV assay was validated investigating HPRT1 on chromosome X in females and males. Results of array-CGH performed on 3 primary GBMs and 1 NS line were compatible with the CNV assay. NS cells with NFKBIA deletion had increased nuclear activity of p65 (RelA) and increased expression of the NF-kB target IL-6. In absence of EGF in the medium, EGFR amplification was more conserved and NFKBIA deletion less frequent point to a low frequency of NFKBIA deletions in GBM and suggest that EGF in the culture medium of NS may affect frequency not only of EGFR amplifications but also of NFKBIA deletions.
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http://dx.doi.org/10.1186/1476-4598-12-160DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4029392PMC
December 2013

Prognostic value of CD109+ circulating endothelial cells in recurrent glioblastomas treated with bevacizumab and irinotecan.

PLoS One 2013 12;8(9):e74345. Epub 2013 Sep 12.

Department of Neuro-Oncology Unit, Fondazione IRCCS Istituto Neurologico C, Besta, Milan, Italy.

Background: Recent data suggest that circulating endothelial and progenitor cells (CECs and CEPs, respectively) may have predictive potential in cancer patients treated with bevacizumab, the antibody recognizing vascular endothelial growth factor (VEGF). Here we report on CECs and CEPs investigated in 68 patients affected by recurrent glioblastoma (rGBM) treated with bevacizumab and irinotecan and two Independent Datasets of rGBM patients respectively treated with bevacizumab alone (n=32, independent dataset A: IDA) and classical antiblastic chemotherapy (n=14, independent dataset B: IDB).

Methods: rGBM patients with KPS ≥50 were treated until progression, as defined by MRI with RANO criteria. CECs expressing CD109, a marker of tumor endothelial cells, as well as other CEC and CEP subtypes, were investigated by six-color flow cytometry.

Results: A baseline count of CD109+ CEC higher than 41.1/ml (1(st) quartile) was associated with increased progression free survival (PFS; 20 versus 9 weeks, P=0.008) and overall survival (OS; 32 versus 23 weeks, P=0.03). Longer PFS (25 versus 8 weeks, P=0.02) and OS (27 versus 17 weeks, P=0.03) were also confirmed in IDA with CD109+ CECs higher than 41.1/ml but not in IDB. Patients treated with bevacizumab with or without irinotecan that were free from MRI progression after two months of treatment had significant decrease of CD109+ CECs: median PFS was 19 weeks; median OS 29 weeks. The presence of two non-contiguous lesions (distant disease) at baseline was an independent predictor of shorter PFS and OS (P<0.001).

Conclusions: Data encourage further studies on the predictive potential of CD109+ CECs in GBM patients treated with bevacizumab.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0074345PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3772091PMC
May 2014

DNA damage in mammalian neural stem cells leads to astrocytic differentiation mediated by BMP2 signaling through JAK-STAT.

Stem Cell Reports 2013 25;1(2):123-38. Epub 2013 Jul 25.

IFOM Foundation-The FIRC Institute of Molecular Oncology Foundation, Via Adamello 16, 20139 Milan, Italy.

The consequences of DNA damage generation in mammalian somatic stem cells, including neural stem cells (NSCs), are poorly understood despite their potential relevance for tissue homeostasis. Here, we show that, following ionizing radiation-induced DNA damage, NSCs enter irreversible proliferative arrest with features of cellular senescence. This is characterized by increased cytokine secretion, loss of stem cell markers, and astrocytic differentiation. We demonstrate that BMP2 is necessary to induce expression of the astrocyte marker GFAP in irradiated NSCs via a noncanonical signaling pathway engaging JAK-STAT. This is promoted by ATM and antagonized by p53. Using a SOX2-Cre reporter mouse model for cell-lineage tracing, we demonstrate irradiation-induced NSC differentiation in vivo. Furthermore, glioblastoma assays reveal that irradiation therapy affects the tumorigenic potential of cancer stem cells by ablating self-renewal and inducing astroglial differentiation.
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http://dx.doi.org/10.1016/j.stemcr.2013.06.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3757751PMC
May 2015

The integrated landscape of driver genomic alterations in glioblastoma.

Nat Genet 2013 Oct 5;45(10):1141-9. Epub 2013 Aug 5.

1] Institute for Cancer Genetics, Columbia University Medical Center, New York, New York, USA. [2].

Glioblastoma is one of the most challenging forms of cancer to treat. Here we describe a computational platform that integrates the analysis of copy number variations and somatic mutations and unravels the landscape of in-frame gene fusions in glioblastoma. We found mutations with loss of heterozygosity in LZTR1, encoding an adaptor of CUL3-containing E3 ligase complexes. Mutations and deletions disrupt LZTR1 function, which restrains the self renewal and growth of glioma spheres that retain stem cell features. Loss-of-function mutations in CTNND2 target a neural-specific gene and are associated with the transformation of glioma cells along the very aggressive mesenchymal phenotype. We also report recurrent translocations that fuse the coding sequence of EGFR to several partners, with EGFR-SEPT14 being the most frequent functional gene fusion in human glioblastoma. EGFR-SEPT14 fusions activate STAT3 signaling and confer mitogen independence and sensitivity to EGFR inhibition. These results provide insights into the pathogenesis of glioblastoma and highlight new targets for therapeutic intervention.
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http://dx.doi.org/10.1038/ng.2734DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3799953PMC
October 2013