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BACKGROUND: Observations in people with cerebral cavernous malformations, and in preclinical models of this disorder, suggest that the β-blocker propranolol might reduce the risk of intracerebral haemorrhage. We aimed to evaluate the safety and efficacy of prolonged treatment with propranolol to reduce the incidence of symptomatic intracerebral haemorrhage or focal neurological deficit in people with familial cerebral cavernous malformations.METHODS: We conducted a randomised, open-label, blinded-endpoint, phase 2 pilot trial (Treat_CCM) at six national reference centres for rare diseases in Italy. People aged 18 years or older with symptomatic familial cerebral cavernous malformation were eligible for enrolment. Participants were randomly assigned (2:1) to receive either oral propranolol (20-320 mg daily) plus standard care (intervention group), or standard care alone (control group), for 24 months. Participants, caregivers, and investigators were aware of treatment group assignment. Participants had clinical assessments and 3 T brain MRI at baseline and at 12 and 24 months. The primary outcome was new occurrence of symptomatic intracerebral haemorrhage or focal neurological deficit attributable to cerebral cavernous malformation over 24 months. Outcome assessors were masked to treatment group assignment. The primary analysis was done in the intention-to-treat population. Because of the pilot study design, we chose a one-sided 80% CI, which could either exclude a clinically meaningful effect or show a signal of efficacy. This trial is registered with EudraCT, 2017-003595-30, and ClinicalTrials.gov, NCT03589014, and is closed to recruitment.FINDINGS: Between April 11, 2018, and Dec 5, 2019, 95 people were assessed for eligibility and 83 were enrolled, of whom 57 were assigned to the propranolol plus standard care group and 26 to the standard care alone group. The mean age of participants was 46 years (SD 15); 48 (58%) were female and 35 (42%) were male. The incidence of symptomatic intracerebral haemorrhage or focal neurological deficit was 1·7 (95% CI 1·4-2·0) cases per 100 person-years (two [4%] of 57 participants) in the propranolol plus standard care group and 3·9 (3·1-4·7) per 100 person-years (two [8%] of 26) in the standard care alone group (univariable hazard ratio [HR] 0·43, 80% CI 0·18-0·98). The univariable HR showed a signal of efficacy, according to predefined criteria. The incidence of hospitalisation did not differ between groups (8·2 cases [95% CI 7·5-8·9] per 100 person-years in the propranolol plus standard care group vs 8·2 [95% CI 7·1-9·3] per 100 person-years in the standard care alone group). One participant in the standard care alone group died of sepsis. Three participants in the propranolol plus standard care group discontinued propranolol due to side-effects (two reported hypotension and one reported weakness).INTERPRETATION: Propranolol was safe and well tolerated in this population. Propranolol might be beneficial for reducing the incidence of clinical events in people with symptomatic familial cerebral cavernous malformations, although this trial was not designed to be adequately powered to investigate efficacy. A definitive phase 3 trial of propranolol in people with symptomatic familial cerebral cavernous malformations is justified.FUNDING: Italian Medicines Agency, Associazione Italiana per la Ricerca sul Cancro, Swedish Science Council, Knut and Alice Wallenberg Foundation, CARIPLO Foundation, Italian Ministry of Health.

The secreted products of the metastasis suppressor geneKiSS1-derived peptide levels in liquid biopsies from 60 NSCLC patients were assayed by ELISA. Preclinical experiments were carried out using quantitative real time polymerase chain reaction (qRT-PCR), ELISA, annexin V-binding and caspase activation assays.We compared KiSS1 release in 3 different matrices (serum, plasma and urine) and the highest levels were detectable in serum (range, 0-4.5 ng/mL). We observed increased levels of seric KiSS1 in NSCLC patients as compared to healthy donors. KiSS1 serum concentrations, after surgical procedure and/or adjuvant therapy. We observed differences among disease stages in urine samples. In preclinical models,Our results showing a peculiar modulation of KiSS1 levels in liquid biopsies of NSCLC patients and a regulation of cisplatin-induced apoptosis by KiSS1-derived peptides support an involvement of KiSS1 in cell response to treatment and highlight its promising features as a potential biomarker in NSCLC.

Mesenchymal stromal cells (MSCs) prepared as advanced therapies medicinal products (ATMPs) have been widely used for the treatment of different diseases. The latest developments concern the possibility to use MSCs as carrier of molecules, including chemotherapeutic drugs. Taking advantage of their intrinsic homing feature, MSCs may improve drugs localization in the disease area. However, for cell therapy applications, a significant number of MSCs loaded with the drug is required. We here investigate the possibility to produce a large amount of Good Manufacturing Practice (GMP)-compliant MSCs loaded with the chemotherapeutic drug Paclitaxel (MSCs-PTX), using a closed bioreactor system. Cells were obtained starting from 13 adipose tissue lipoaspirates. All samples were characterized in terms of number/viability, morphology, growth kinetics, and immunophenotype. The ability of MSCs to internalize PTX as well as the antiproliferative activity of the MSCs-PTX in vitro was also assessed. The results demonstrate that our approach allows a large scale expansion of cells within a week, the MSCs-PTX, despite a different morphology from MSCs, displayed the typical features of MSCs in terms of viability, adhesion capacity, and phenotype. In addition, MSCs showed the ability to internalize PTX and finally to kill cancer cells, inhibiting the proliferation of tumor lines in vitro. In summary our results demonstrate for the first time that it is possible to obtain, in a short time, large amounts of MSCs loaded with PTX to be used in clinical trials for the treatment of patients with oncological diseases.

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&ndash, 7 days of differentiation with GM-CSF and IL-4 followed by 2&ndash, 3 days of activation/maturation. In attempts to shorten the vaccine&rsquo, 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 PGE2 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.

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 PGE2 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.

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.

Purpose: Deep venous thrombosis/pulmonary embolism (DVT/PE) is a frequent complication in the course of cancer, particularly in brain tumors. We investigated genetic and plasma factors possibly associated with risk of DVT/PE in patients with high-grade glioma. Experimental Design: In a case-control study, we studied polymorphisms of the genes coding for factor II (G20210A), factor V (G1691A), methylenetetrahydrofolate-reductase (C677T), tissue-type plasminogen activator (tPA; insertion/deletion), plasminogen activator inhibitor-1 (PAI-1; 4G/5G), and vascular endothelial growth factor (VEGF; C936T). We also measured plasma levels of d-dimer, lipoprotein (lp) (a), homocysteine, VEGF, tPA, and PAI-1, comparing healthy control patients with patients with glioma or with patients with neurological nonneoplastic disease (multiple sclerosis). Results: Genotype frequencies of polymorphisms analyzed were similar in patients with glioma and in healthy matched population. d-dimer, lp (a), homocysteine, VEGF, tPA, and PAI-1 plasma levels were significantly higher in patients with glioma than in healthy controls, whereas patients having neurological nonneoplastic disease had plasma values of these molecules not significantly different from healthy controls. VEGF, tPA, and PAI-1 were also found at high-plasma levels in patients carrying genotypes that, in healthy controls, were associated with “low-producing” phenotypes. Conclusions: Genetic risk factors alone did not explain the high incidence of DVT/PE observed in patients with glioma. Higher plasma levels of molecules influencing the coagulation pathways indicate that the tumor itself might confer an increased risk of DVT/PE; thus, d-dimer, homocysteine, lp (a), VEGF, tPA, and PAI-1 look like good candidates to be evaluated as DVT/PE prognostic factors.

Immune-based treatments represent a promising new class of therapy designed to boost the immune system to specifically eradicate malignant cells. Immunotherapy may generate specific anti-tumor immune responses, and dendritic cells (DC), professional antigen-presenting cells, are widely used in experimental cancer immunotherapy. Several reports describe methods for the generation of mature, antigen-pulsed DC for clinical use. Improved quality and standardization are desirable to obtain GMP-compliant protocols. In this study we describe the generation of DC from 31 Glioblastoma (GB) patients starting from their monocytes isolated by immunomagnetic CD14 selection using the CliniMACS® device. Upon differentiation of CD14+ with IL-4 and GM-CSF, DC were induced to maturation with TNF-α, PGE(2), IL-1β, and IL-6. Whole tumor lysate was obtained, for the first time, in a closed system using the semi-automated dissociator GentleMACS®. The yield of proteins improved by 130% compared to the manual dissociation method. Interestingly the Mean Fluorescence Intensity for CD83 increased significantly in DC pulsed with "new method" lysate compared to DC pulsed with "classical method" lysate. Our results indicate that immunomagnetic isolation of CD14(+) monocytes using the CliniMACS® device and their pulsing with whole tumor lysate proteins is a suitable method for clinical-scale generation of high quality, functional DC under GMP-grade conditions.