Punctual and kinetic MRD analysis from the Fondazione Italiana Linfomi MCL0208 phase 3 trial in mantle cell lymphoma.

Minimal residual disease (MRD) analysis is a known predictive tool in mantle cell lymphoma (MCL). We describe MRD results from the Fondazione Italiana Linfomi phase 3 MCL0208 prospective clinical trial assessing lenalidomide (LEN) maintenance vs observation after autologous stem cell transplantation (ASCT) in the first prospective comprehensive analysis of different techniques, molecular markers, and tissues (peripheral blood [PB] and bone marrow [BM]), taken at well-defined time points. Among the 300 patients enrolled, a molecular marker was identified in 250 (83%), allowing us to analyze 234 patients and 4351 analytical findings from 10 time points. ASCT induced high rates of molecular remission (91% in PB and 83% in BM, by quantitative real-time polymerase chain reaction [RQ-PCR]). Nevertheless, the number of patients with persistent clinical and molecular remission decreased over time in both arms (up to 30% after 36 months). MRD predicted early progression and long-term outcome, particularly from 6 months after ASCT (6-month time to progression [TTP] hazard ratio [HR], 3.83; P < .001). In single-timepoint analysis, BM outperformed PB, and RQ-PCR was more reliable, while nested PCR appeared applicable to a larger number of patients (234 vs 176). To improve MRD performance, we developed a time-varying kinetic model based on regularly updated MRD results and the MIPI (Mantle Cell Lymphoma International Prognostic Index), showing an area under the ROC (Receiver Operating Characteristic) curve (AUROC) of up to 0.87 using BM. Most notably, PB reached an AUROC of up to 0.81; with kinetic analysis, it was comparable to BM in performance. MRD is a powerful predictor over the entire natural history of MCL and is suitable for models with a continuous adaptation of patient risk. The study can be found in EudraCT N. 2009-012807-25 (https://eudract.ema.europa.eu/).

IELSG38: phase II trial of front-line chlorambucil plus subcutaneous rituximab induction and maintenance in mucosa-associated lymphoid tissue lymphoma.

The IELSG38 trial was conducted to investigate the effects of subcutaneous (SC) rituximab on the complete remission (CR) rate and the benefits of SC maintenance in patients with extranodal marginal zone lymphoma (MZL) who received frontline treatment with chlorambucil plus rituximab. Study treatment comprised an induction phase with chlorambucil 6 mg/m2/day orally on weeks 1-6, 9-10, 13-14, 17-18, and 21-22, and rituximab 375 mg/m2 intravenously on day 1 of weeks 1-4, and 1400 mg SC on weeks 9, 13, 17, and 21. Then, a maintenance phase followed with rituximab administered at 1400 mg SC every two months for two years. Of the 112 patients enrolled, 109 were evaluated for efficacy. The CR rates increased from 52% at the end of the induction phase to 70% upon completion of the maintenance phase. With a median follow-up of 5.8 years, the 5-year event-free, progression-free, and overall survival rates were 87% (95% CI, 78-92), 84% (95% CI, 75-89), and 93% (95% CI, 86-96), respectively. The most common grade ≥3 toxicities were neutropenia (33%) and lymphocytopenia (16%). Six patients experienced treatment-related serious adverse events, including fever of unknown origin, sepsis, pneumonia, respiratory failure, severe cerebellar ataxia, and fatal acute myeloid leukemia. The trial showed that subcutaneous rituximab did not improve the complete remission rate at the conclusion of the induction phase, which was the main endpoint. Nevertheless, SC maintenance might have facilitated long-term disease control, potentially contributing to enhanced event-free and progression-free survival.

In Vitro Biocompatibility and Endothelial Permeability of Branched Polyglycidols Generated by Ring-Opening Polymerization of Glycidol with B(C6F5)3 under Dry and Wet Conditions.

Polyglycidol or polyglycerol (PG), a polyether widely used in biomedical applications, has not been extensively studied in its branched cyclic form (bcPG), despite extensive research on hyperbranched PG (HPG). This study explores the biomedical promise of bcPG, particularly its ability to cross the blood-brain barrier (BBB). We evaluate in vitro biocompatibility, endothelial permeability, and formation of branched linear PG (blPG) as topological impurities in the presence of water. Small angle X-ray scattering in solution revealed a fractal dimension of approximately two for bcPG and the mixture bc+blPG, suggesting random branching. Comparisons of cytotoxicity and endothelial permeability between bcPG, bc+blPG, and HPG in a BBB model using hCMEC/D3 cells showed different biocompatibility profiles and higher endothelial permeability for HPG. bcPG showed a tendency to accumulate around cell nuclei, in contrast to the behavior of HPG. This study contributes to the understanding of the influence of polymer topology on biological behavior.

Impact of immunochemotherapy with R-bendamustine or R-CHOP for treatment naïve advanced-stage follicular lymphoma: A subset analysis of the FOLL12 trial by Fondazione Italiana Linfomi.

We conducted a post hoc analysis of the FOLL12 trial to determine the impact of different initial immunochemotherapy (ICT) regimens on patient outcomes. Patients were selected from the FOLL12 trial, which included adults with stage II-IV follicular lymphoma (FL) grade 1-3a and high tumor burden. Patients were randomized 1:1 to receive either standard ICT followed by rituximab maintenance (RM) or the same ICT followed by a response-adapted approach. ICT consisted of rituximab-bendamustine (RB) or rituximab, cyclophosphamide, doxorubicin, and prednisone (R-CHOP), per physician's decision. A total of 786 patients were included in this analysis, 341 of whom received RB and 445 R-CHOP. RB was more frequently prescribed to older subjects, females, patients without bulky disease, and those with grade 1-2 FL. After a median of 56 months of follow-up, R-CHOP and RB had similar progression-free survival (PFS) (Hazard Ratio for RB 1.11, 95% CI 0.87-1.42, p = 0.392). Standard RM was associated with improved PFS compared to response-adapted management both after R-CHOP and RB. Grade 3-4 hematologic adverse events were more frequent with R-CHOP during induction treatment and more frequent with RB during RM. Grade 3-4 infections were more frequent with RB. RB was also associated with a higher incidence of transformed FL. R-CHOP and RB showed similar activity and efficacy, but with different safety profiles and long-term events, suggesting that the treating physician should carefully select the most appropriate chemotherapy regimen for each patient based on patient's individual characteristics, choices, and risk profile.

SPRi analysis of molecular interactions of mApoE-functionalized liposomes as drug delivery systems for brain diseases.

The application of liposomes (LPs) to central nervous system disorders could represents a turning point in the therapy and quality of life of patients. Indeed, LPs have demonstrated their ability to cross the blood-brain barrier (BBB) and, as a consequence, to enhance the therapeutics delivery into the brain. Some approaches for BBB crossing involve the modification of LP surfaces with biologically active ligands. Among them, the Apolipoprotein E-modified peptide (mApoE) has been used for several LP-based nanovectors under investigation. In this study, we propose Surface Plasmon Resonance imaging (SPRi) for the characterization of multifunctionalized LPs for Glioblastoma treatment. LPs were functionalized with mApoE and with a metallo-protease sensitive lipopeptide to deliver and guarantee the localized release of an encapsulated drug in diseased areas. The SPRi analysis was optimized in order to evaluate the binding affinity between LPs and mApoE receptors, finding that mApoE-LPs generated SPRi signals referred to interactions between mApoE and receptors mainly present in the brain. Moreover, a significant binding between LPs and VCAM-1 (endothelial receptor) was observed, whereas LPs did not interact significantly with peripheral receptors expressed on monocytes and lymphocytes. SPRi results confirmed not only the presence of mApoE on LP surfaces, but also its binding affinity, thanks to the specific interaction with selected receptors. In conclusion, the high sensitivity and the multiplexing capability associated with the low volumes of sample required and the minimal sample preparation, make SPRi an excellent technique for the characterization of multifunctionalized nanoparticles-based formulations.

Early progression as a predictor of survival in marginal zone lymphomas: an analysis from the FIL-NF10 study.

Marginal zone lymphomas (MZLs) are indolent nonfollicular B-cell lymphomas (INFLs) and have heterogeneous clinical behavior. Recently, time to progression of disease at 24 months (POD24) was identified to stratify overall survival (OS) in follicular non-Hodgkin lymphoma and in INFL. Here, we examined the ability of POD24 to predict subsequent OS in a large, international cohort of MZL as part of the NF10 prospective international registry headed by Fondazione Italiana Linfomi (FIL). POD24 was only calculated for MZL patients requiring immediate therapy and was defined as experiencing lymphoma progression within 24 months from diagnosis. Among the 1325 patients enrolled in the NF10 study, we identified 321 patients with MZL for whom immediate therapy was planned right after lymphoma diagnosis. Overall, POD24 was confirmed in 59 patients (18%). Three-year OS for patients with POD24 was 53% with a hazard ratio of 19.5 (95% confidence interval, 8.4-45) compared with patients without POD24 (3-year OS, 95%). Association of POD24 with OS was confirmed for the subgroup of splenic and extranodal MZLs. Assessment of POD24 stratifies subsequent outcome in MZL and identifies a high-risk population. This trial was registered at www.clinicaltrials.gov as #NCT02904577.

Dietary Nanoparticles Interact with Gluten Peptides and Alter the Intestinal Homeostasis Increasing the Risk of Celiac Disease.

The introduction of metallic nanoparticles (mNPs) into the diet is a matter of concern for human health. In particular, their effect on the gastrointestinal tract may potentially lead to the increased passage of gluten peptides and the activation of the immune response. In consequence, dietary mNPs could play a role in the increasing worldwide celiac disease (CeD) incidence. We evaluated the potential synergistic effects that peptic-tryptic-digested gliadin (PT) and the most-used food mNPs may induce on the intestinal mucosa. PT interaction with mNPs and their consequent aggregation was detected by transmission electron microscopy (TEM) analyses and UV-Vis spectra. In vitro experiments on Caco-2 cells proved the synergistic cytotoxic effect of PT and mNPs, as well as alterations in the monolayer integrity and tight junction proteins. Exposure of duodenal biopsies to gliadin plus mNPs triggered cytokine production, but only in CeD biopsies. These results suggest that mNPs used in the food sector may alter intestinal homeostasis, thus representing an additional environmental risk factor for the development of CeD.

Reduced Levels of ABCA1 Transporter Are Responsible for the Cholesterol Efflux Impairment in ß-Amyloid-Induced Reactive Astrocytes: Potential Rescue from Biomimetic HDLs.

The cerebral synthesis of cholesterol is mainly handled by astrocytes, which are also responsible for apoproteins' synthesis and lipoproteins' assembly required for the cholesterol transport in the brain parenchyma. In Alzheimer disease (AD), these processes are impaired, likely because of the astrogliosis, a process characterized by morphological and functional changes in astrocytes. Several ATP-binding cassette transporters expressed by brain cells are involved in the formation of nascent discoidal lipoproteins, but the effect of beta-amyloid (Aß) assemblies on this process is not fully understood. In this study, we investigated how of Aß1-42-induced astrogliosis affects the metabolism of cholesterol in vitro. We detected an impairment in the cholesterol efflux of reactive astrocytes attributable to reduced levels of ABCA1 transporters that could explain the decreased lipoproteins' levels detected in AD patients. To approach this issue, we designed biomimetic HDLs and evaluated their performance as cholesterol acceptors. The results demonstrated the ability of apoA-I nanodiscs to cross the blood-brain barrier in vitro and to promote the cholesterol efflux from astrocytes, making them suitable as a potential supportive treatment for AD to compensate the depletion of cerebral HDLs.

The Clustering of mApoE Anti-Amyloidogenic Peptide on Nanoparticle Surface Does Not Alter Its Performance in Controlling Beta-Amyloid Aggregation.

The deposition of amyloid-ß (Aß) plaques in the brain is a significant pathological signature of Alzheimer's disease, correlating with synaptic dysfunction and neurodegeneration. Several compounds, peptides, or drugs have been designed to redirect or stop Aß aggregation. Among them, the trideca-peptide CWG-LRKLRKRLLR (mApoE), which is derived from the receptor binding sequence of apolipoprotein E, is effectively able to inhibit Aß aggregation and to promote fibril disaggregation. Taking advantage of Atomic Force Microscopy (AFM) imaging and fluorescence techniques, we investigate if the clustering of mApoE on gold nanoparticles (AuNP) surface may affect its performance in controlling Aß aggregation/disaggregation processes. The results showed that the ability of free mApoE to destroy preformed Aß fibrils or to hinder the Aß aggregation process is preserved after its clustering on AuNP. This allows the possibility to design multifunctional drug delivery systems with clustering of anti-amyloidogenic molecules on any NP surface without affecting their performance in controlling Aß aggregation processes.

The synergistic effect of chlorotoxin-mApoE in boosting drug-loaded liposomes across the BBB.

We designed liposomes dually functionalized with ApoE-derived peptide (mApoE) and chlorotoxin (ClTx) to improve their blood-brain barrier (BBB) crossing. Our results demonstrated the synergistic activity of ClTx-mApoE in boosting doxorubicin-loaded liposomes across the BBB, keeping the anti-tumour activity of the drug loaded: mApoE acts promoting cellular uptake, while ClTx promotes exocytosis of liposomes.

A New Approach for Glyco-Functionalization of Collagen-Based Biomaterials.

The cell microenvironment plays a pivotal role in mediating cell adhesion, survival, and proliferation in physiological and pathological states. The relevance of extracellular matrix (ECM) proteins in cell fate control is an important issue to take into consideration for both tissue engineering and cell biology studies. The glycosylation of ECM proteins remains, however, largely unexplored. In order to investigate the physio-pathological effects of differential ECM glycosylation, the design of affordable chemoselective methods for ECM components glycosylation is desirable. We will describe a new chemoselective glycosylation approach exploitable in aqueous media and on non-protected substrates, allowing rapid access to glyco-functionalized biomaterials.

Efficacy of bendamustine and rituximab in splenic marginal zone lymphoma: results from the phase II BRISMA/IELSG36 study.

Splenectomy in addition to immunotherapy with rituximab can provide quick and sometimes durable disease control in patients with splenic marginal zone lymphoma (SMZL). However, systemic chemotherapy is ultimately required in many cases. The BRISMA (Bendamustine-rituximab as first-line treatment of splenic marginal zone lymphoma)/IELSG (International Extranodal Lymphoma Study Group)36 trial is an open-label, single arm phase II study designed by the IELSG in cooperation with the Fondazione Italiana Linfomi and the lymphoma Study Association according to Simon's two-stage method. The primary endpoint was complete response rate. Fifty-six patients with SMZL diagnosis confirmed on central revision were treated with bendamustine (90 mg/m2  days 1, 2) and rituximab (375 mg/m2  day 1) every 28 days for six cycles (B-R). The overall response and CR rates were 91% and 73%, respectively. Duration of response, progression-free survival and overall survival at 3 years were 93% (95% confidence interval [CI] 81-98), 90% (95% CI 77-96) and 96% (95% CI 84-98), respectively. Toxicity was mostly haematological. Neutropenia grade ≥3 was recorded in 43% of patients; infections and febrile neutropenia in 5·4% and 3·6%. Overall, 14 patients (25%) experienced serious adverse events. Five patients (9%) went off-study because of toxicity and one patient died from infection. In conclusion, B-R resulted in a very effective first-line regimen for SMZL. Based on the results achieved in the BRISMA trial, B-R should be considered when a chemotherapy combination with rituximab is deemed necessary for symptomatic SMZL patients.

Efficacy of bendamustine and rituximab as first salvage treatment in chronic lymphocytic leukemia and indirect comparison with ibrutinib: a GIMEMA, ERIC and UK CLL FORUM study.

We performed an observational study on the efficacy of ben-damustine and rituximab (BR) as first salvage regimen in chronic lymphocytic leukemia (CLL). In an intention-to-treat analysis including 237 patients, the median progression-free survival (PFS) was 25 months. The presence of del(17p), unmutated IGHV and advanced stage were associated with a shorter PFS at multivariate analysis. The median time-to-next treatment was 31.3 months. Front-line treatment with a chemoimmunotherapy regimen was the only predictive factor for a shorter time to next treatment at multivariate analysis. The median overall survival (OS) was 74.5 months. Advanced disease stage (i.e. Rai stage III-IV or Binet stage C) and resistant disease were the only parameters significantly associated with a shorter OS. Grade 3-5 infections were recorded in 6.3% of patients. A matched-adjusted indirect comparison with ibrutinib given second-line within Named Patient Programs in the United Kingdom and in Italy was carried out with OS as objective end point. When restricting the analysis to patients with intact 17p who had received chemoimmunotherapy in first line, there was no difference in OS between patients treated with ibrutinib (63% alive at 36 months) and patients treated with BR (74.4% alive at 36 months). BR is an efficacious first salvage regimen in CLL in a real-life population, including the elderly and unfit patients. BR and ibrutinib may be equally effective in terms of OS when used as first salvage treatment in patients without 17p deletion.

Antibody-functionalized polymer nanoparticle leading to memory recovery in Alzheimer's disease-like transgenic mouse model.

Alzheimer's disease (AD) is a neurodegenerative disorder related, in part, to the accumulation of amyloid-ß peptide (Aß) and especially the Aß peptide 1-42 (Aß1-42). The aim of this study was to design nanocarriers able to: (i) interact with the Aß1-42 in the blood and promote its elimination through the "sink effect" and (ii) correct the memory defect observed in AD-like transgenic mice. To do so, biodegradable, PEGylated nanoparticles were surface-functionalized with an antibody directed against Aß1-42. Treatment of AD-like transgenic mice with anti-Aß1-42-functionalized nanoparticles led to: (i) complete correction of the memory defect; (ii) significant reduction of the Aß soluble peptide and its oligomer level in the brain and (iii) significant increase of the Aß levels in plasma. This study represents the first example of Aß1-42 monoclonal antibody-decorated nanoparticle-based therapy against AD leading to complete correction of the memory defect in an experimental model of AD.

Fluorimetric detection of the earliest events in amyloid ß oligomerization and its inhibition by pharmacologically active liposomes.

BACKGROUND: Amyloid ß (Aß) peptide aggregation is the main molecular mechanism underlying the development of Alzheimer's disease, the most widespread form of senile dementia worldwide. Increasing evidence suggests that the key factor leading to impaired neuronal function is accumulation of water-soluble Aß oligomers rather than formation of the senile plaques created by the deposition of large fibrillary aggregates of Aß. However, several questions remain about the preliminary steps and the progression of Aß oligomerization. METHODS: We show that the initial stages of the aggregation of fluorescently labeled Aß can be determined with a high degree of precision and at physiological (i.e., nanomolar) concentrations by using either steady-state fluorimetry or time-correlated single-photon counting. RESULTS: We study the dependence of the oligomerization extent and rate on the Aß concentration. We determine the chemical binding affinity of fluorescently labeled Aß for liposomes that have been recently shown to be pharmacologically active in vivo, reducing the Aß burden within the brain. We also probe their capacity to hinder the Aß oligomerization process in vitro. CONCLUSIONS: We introduced a fluorescence assay allowing investigation of the earliest steps of Aß oligomerization, the peptide involved in Alzheimer's disease. The assay proved to be sensitive even at Aß concentrations as low as those physiologically observed in the cerebrospinal fluid. GENERAL SIGNIFICANCE: This work represents an extensive and quantitative study on the initial events of Aß oligomerization at physiological concentration. It may enhance our comprehension of the molecular mechanisms leading to Alzheimer's disease, thus paving the way to novel therapeutic strategies.

Retro-inverso peptide inhibitor nanoparticles as potent inhibitors of aggregation of the Alzheimer's Aß peptide.

Aggregation of amyloid-ß peptide (Aß) is a key event in the pathogenesis of Alzheimer's disease (AD). We investigated the effects of nanoliposomes decorated with the retro-inverso peptide RI-OR2-TAT (Ac-rGffvlkGrrrrqrrkkrGy-NH2) on the aggregation and toxicity of Aß. Remarkably low concentrations of these peptide inhibitor nanoparticles (PINPs) were required to inhibit the formation of Aß oligomers and fibrils in vitro, with 50% inhibition occurring at a molar ratio of ~1:2000 of liposome-bound RI-OR2-TAT to Aß. PINPs also bound to Aß with high affinity (Kd=13.2-50 nM), rescued SHSY-5Y cells from the toxic effect of pre-aggregated Aß, crossed an in vitro blood-brain barrier model (hCMEC/D3 cell monolayer), entered the brains of C57 BL/6 mice, and protected against memory loss in APPSWE transgenic mice in a novel object recognition test. As the most potent aggregation inhibitor that we have tested so far, we propose to develop PINPs as a potential disease-modifying treatment for AD.

The hunt for brain Aß oligomers by peripherally circulating multi-functional nanoparticles: Potential therapeutic approach for Alzheimer disease.

We previously showed the ability of liposomes bi-functionalized with phosphatidic acid and an ApoE-derived peptide (mApoE-PA-LIP) to reduce brain Aß in transgenic Alzheimer mice. Herein we investigated the efficacy of mApoE-PA-LIP to withdraw Aß peptide in different aggregation forms from the brain, using a transwell cellular model of the blood-brain barrier and APP/PS1 mice. The spontaneous efflux of Aß oligomers (Aßo), but not of Aß fibrils, from the 'brain' side of the transwell was strongly enhanced (5-fold) in presence of mApoE-PA-LIP in the 'blood' compartment. This effect is due to a withdrawal of Aßo exerted by peripheral mApoE-PA-LIP by sink effect, because, when present in the brain side, they did not act as Aßo carrier and limit the oligomer efflux. In vivo peripheral administration of mApoE-PA-LIP significantly increased the plasma Aß level, suggesting that Aß-binding particles exploiting the sink effect can be used as a therapeutic strategy for Alzheimer disease. From the Clinical Editor: Alzheimer disease (AD) at present is an incurable disease, which is thought to be caused by an accumulation of amyloid-ß (Aß) peptides in the brain. Many strategies in combating this disease have been focused on either the prevention or dissolving these peptides. In this article, the authors showed the ability of liposomes bi-functionalized with phosphatidic acid and with an ApoE- derived peptide to withdraw amyloid peptides from the brain. The data would help the future design of more novel treatment for Alzheimer disease.

Multifunctional liposomes reduce brain ß-amyloid burden and ameliorate memory impairment in Alzheimer's disease mouse models.

Alzheimer's disease is characterized by the accumulation and deposition of plaques of ß-amyloid (Aß) peptide in the brain. Given its pivotal role, new therapies targeting Aß are in demand. We rationally designed liposomes targeting the brain and promoting the disaggregation of Aß assemblies and evaluated their efficiency in reducing the Aß burden in Alzheimer's disease mouse models. Liposomes were bifunctionalized with a peptide derived from the apolipoprotein-E receptor-binding domain for blood-brain barrier targeting and with phosphatidic acid for Aß binding. Bifunctionalized liposomes display the unique ability to hinder the formation of, and disaggregate, Aß assemblies in vitro (EM experiments). Administration of bifunctionalized liposomes to APP/presenilin 1 transgenic mice (aged 10 months) for 3 weeks (three injections per week) decreased total brain-insoluble Aß1-42 (-33%), assessed by ELISA, and the number and total area of plaques (-34%) detected histologically. Also, brain Aß oligomers were reduced (-70.5%), as assessed by SDS-PAGE. Plaque reduction was confirmed in APP23 transgenic mice (aged 15 months) either histologically or by PET imaging with [(11)C]Pittsburgh compound B (PIB). The reduction of brain Aß was associated with its increase in liver (+18%) and spleen (+20%). Notably, the novel-object recognition test showed that the treatment ameliorated mouse impaired memory. Finally, liposomes reached the brain in an intact form, as determined by confocal microscopy experiments with fluorescently labeled liposomes. These data suggest that bifunctionalized liposomes destabilize brain Aß aggregates and promote peptide removal across the blood-brain barrier and its peripheral clearance. This all-in-one multitask therapeutic device can be considered as a candidate for the treatment of Alzheimer's disease.