Neuroscience Institute Cavalieri Ottolenghi (NICO)
Neuroscience, University of Turin

Author Of 3 Presentations

Biomarkers and Bioinformatics Late Breaking Abstracts

LB1221 - Applicability of sNFL in Multiple Sclerosis as additional measure in clinical practice and implications in NEDA-3 evaluation.   (ID 2092)

Speakers
Presentation Number
LB1221
Presentation Topic
Biomarkers and Bioinformatics

Abstract

Background

Despite the increased availability of efficient therapeutic options, early and effective intervention is still mandatory for successful clinical management in Multiple Sclerosis (MS). Speaking of which, a composite measure of disease status termed "no evidence of disease activity" (NEDA) has been proposed as target for treatment in MS. However, this definition needs to be implemented with biological measures for a more personalized medicine. Serum Neurofilament light chain (sNfL) represents the most promising biomarker of disease activity and treatment efficacy in MS, reflecting axonal damage in the central nervous system. To date, sNFL correlation with clinical outcomes is well established in group analysis, yet, its implementation for individual patients is still to be addressed.

Objectives

The aim of the present real-life study is to investigate sNFL as additional measure of treatment efficacy in NEDA-3 patients.

Methods

We measured sNF-L by single molecule array (Simoa) assay (NF-light advantage kit, Quanterix) in 79 healthy participants and in 582 cross-sectionally enrolled RRMS patients, including 61 naive patients, and 521 patients treated with first- or second line therapies, demonstrating NEDA-3 status (no relapses, no disability progression, no MRI activity) during at least 12 months.

Results

1) We established clinically applicable cut-off values for each age decade testing healthy individuals, later used to interpret sNF-L levels in MS patients. 2) In MS patients, treatments notably lower sNF-L relative to untreated patients. 3) According to cut-off values, 53/521 (10%) NEDA-3 patients still demonstrate high NFL levels. These patients were distinguished in different categories: a) patients showing borderline sNFL values; b) patients with concomitant pathologies; c) patients experiencing chronic ongoing fatigability (not classifiable as disability progression); d) patients with a very active disease history; e) patients with conceivable inflammation not detectable by RMN or clinics. 4) The percentage of NEDA-3 patients with high NFL lowers with the extension of NEDA-3 status duration (down to 5% in patients with NEDA-3 status longer than 8 years).

Conclusions

The present cross-sectional study identified a subgroup of NEDA-3 patients showing high sNFL levels. High pathological sNFL levels, reflecting axonal damage, suggest the presence of chronic disease activity or subclinical transitory active inflammation not detectable by RMN or clinics in NEDA-3 patients. The establishment of simple applicable cut-off values allows sNFL applicability in everyday clinical practice. In particular, sNFL could represent an additional measure to be included in NEDA assessment for monitoring therapeutic response in individual MS patients.

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Biomarkers and Bioinformatics Late Breaking Abstracts

LB1245 - BB-CRESM: a structured institutional biobank for quality research in Multiple Sclerosis. (ID 2131)

Speakers
Presentation Number
LB1245
Presentation Topic
Biomarkers and Bioinformatics

Abstract

Background

Biobanks have recently become an important tool in clinical research. The availability of biological samples collected and stored following strict quality standards is crucial in biomedical and translational research in Multiple Sclerosis (MS), to better understand disease pathogenesis, identify biomarkers of diagnosis, prognosis and treatment response.

The Regional Reference Multiple Sclerosis Center (CRESM) at the S. Luigi Gonzaga Hospital provides comprehensive care for more than 2000 MS patients. It has been operational since 2013 in transforming the reserve of biological samples into a structured biobank.

Objectives

To describe the establishment of a structured MS biobank starting from a collection of biological samples and the process for providing scientists with biological samples and associated data.

Methods

According to guidelines by the “Biobanking and Biomolecular Resources and Research Infrastructure” (BBMRI), the steps for the BB-CRESM establishment were: Institutional commitment; Biobank management and staffing; Development of procedures to address ethical, legal, and social issues, according to the General Data Protection Regulation, in collaboration with a bioethicist.

All technical procedures were included into Standard Operating Procedures manual.

Results

BB-CRESM is a structural part of the Piedmont Regional Health Service. The General Director of San Luigi Hospital approved its Regulation, selected the BB-CRESM director and the members of the Scientific Committee.

BB-CRESM is a non-profit organization supported by the Italian Multiple Sclerosis Foundation.

Specific protocols regulate the timing and modalities of biological sample collection, ensuring privacy of subjects.

A detailed description of the biobank is in-person explained to each patient or healthy subject with the help of a leaflet; following which the informed consent is obtained. The Ethical Committee approved both the leaflet and the informed consent model.

Scientists can apply to the director of BB-CRESM specifying number, types and quantity of required samples and data. The samples can be distributed if a) they are used for a research project approved by an Ethic Committee; b) the research project is approved by the Scientific Committee; c) the scientist signs the Material Transfer Agreement; d) scientist agrees to share crude data with BB-CRESM e) scientist contributes to BB-CRESM collection and shipment expenses.

Since 2013, more than 1000 participants (healthy controls and MS patients) have enrolled in the BB-CRESM; over 20000 tubes of biological material (cerebrospinal fluid, serum, plasma, PBMCs, RNA and DNA) have been collected and stored.

Conclusions

BB-CRESM collects, stores and distributes biological samples along with associated data ensuring quality of collection and bio-banking according to BBMRI.

MS researchers can contact BB-CRESM at biobanca.cresm@sanluigi.piemonte.it.

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Reproductive Aspects and Pregnancy Poster Presentation

P1115 - A first characterization of placenta-derived extracellular vesicles in patients with multiple sclerosis. (ID 1909)

Speakers
Presentation Number
P1115
Presentation Topic
Reproductive Aspects and Pregnancy

Abstract

Background

Pregnancy is a condition of complex immunomodulation during which multiple sclerosis (MS) significantly improves. The molecular mechanisms behind this phenomenon have not been completely elucidated. A central role is played by the placenta and its released factors such as the extracellular vesicles (EV), a peculiar mechanism of communication and material exchange between cells. Studies conducted on an experimental murine model of MS have shown that EV shed into the blood during pregnancy have an inhibitory effect on the proliferation of T cells.

Objectives

To characterize the surface markers expressed by the EV secreted by the maternal (decidua) and fetal (trophoblast) side of the placenta of patients with MS and healthy donors (HC), and to evaluate the effects of the EV on the activation status of CD14+ monocytes and the activity of regulatory T lymphocytes (Treg).

Methods

Total EV shed by placental samples from 15 women with MS and 14 HC were isolated by ultracentrifugation, quantified by Nanoparticle Tracking Analysis and characterized by flow cytometry for the expression of 37 surface markers using the human MACSPlex Exosome Kit. CD14+ monocytes, Treg end CD4+ T cells were isolated from HC buffy coats. CD14+ monocytes were activated with LPS and the expression levels of pro and anti-inflammatory cytokines was evaluated by real-time PCR. CD4+ T cells were activated via CD3 and CD28 and their proliferation activity was evaluated by BrdU Cell Proliferation Assay after co-culture with EV-conditioned Treg.

Results

Most of the analyzed surface markers resulted differentially expressed between the EV released by decidua or trophoblast, but not between the EV shed by placental tissues of MS patients or HC, except for CD133, whose level was higher in the EV secreted by trophoblast of women with MS. The conditioning with EV shed by placenta samples of MS patients was able to moderately decrease the expression level of pro-inflammatory cytokines by activated CD14+ monocytes and the proliferation activity of CD4+ T cells, although the effect was not statistically significant.

Conclusions

For the first time to our knowledge, this study enabled a preliminary characterization of the EV shed by placental tissues of patients with MS. However, further studies are needed to clarify the role of placental EV in the pregnancy-related improvement of the MS disease.

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Presenter Of 3 Presentations

Biomarkers and Bioinformatics Late Breaking Abstracts

LB1219 - Normal serum NFL levels: a proposal of cut-off strategy definition for the clinical practice (ID 2090)

Speakers
Presentation Number
LB1219
Presentation Topic
Biomarkers and Bioinformatics

Abstract

Background

Serum Neurofilament light (sNFL) protein is the most promising marker of disease activity and treatment response in Multiple Sclerosis (MS). To implement sNFL in clinical practice, the definition of normal widely accepted values represents a crucial step still to be addressed. Clinically applicable cut-off values need to take into account age dependency; in addition, recent evidences suggest that physical parameters as body mass index (BMI) and blood volume (BV) might influence sNFL levels.

Objectives

The present study aims to address these crucial needs, describing sNFL levels in healthy population, their inter-individual variability in a short-term follow-up and defining reference cut-off values. The final objective is to define a strategy to allow implementation of sNFL in clinical practice.

Methods

We measured sNFL by single molecule array (Simoa) assay (NF-light advantage Kit, Quanterix) in 79 healthy individuals to define reference cut-off values. Age, BMI and BV were correlated with sNFL levels. In addition, sNFL were evaluated after a short-term follow-up time (median 67 days) to assess intra-individual variability: consecutive blood samples were tested in a subset of 27 participants (n=2-4 sample for each individual) and the coefficient of variation (CV) for NFL levels of each participant was evaluated.
sNFL were also tested in 23 naïve MS patients both at diagnostic time and immediately before treatment (median 76 days after) to evaluate the variability of sNFL in patients and the applicability of obtained cut-off values.

Results

1) Our data confirmed a strong correlation between sNFL levels and age. We found a negative correlation between sNFL levels and BV. 2) Short-term follow-up NFL assessments showed an overall intra-individual stability in sNFL levels in healthy population (median CV 15%). 3) We defined specific age decade-related cut-off values. 4) In naïve MS patients, sNFL levels were higher than control values; a high variability between diagnostic time and the beginning of treatment (median CV 39%) was shown. Cut-off values were applied in MS patients to discriminate high from normal sNFL levels: at diagnostic time, 57% of MS patients showed high sNFL levels, while at treatment start, 70% of patients demonstrated normal NFL values.

Conclusions

The present study suggests a strategy to define clinical applicable cut-offs to exploit sNFL as a personalised medicine tool in MS: specific cut-off values were calculated for each age decade. sNFL levels demonstrated an overall intra-individual stability in healthy participants in the short-term: this is relevant for a biomarker of disease activity and treatment response that, if successful, will be serially assessed during patients follow-up.

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Biomarkers and Bioinformatics Late Breaking Abstracts

LB1220 - Real-life experience with sNFL in Multiple Sclerosis patients, as monitoring and treatment decision biomarker   (ID 2091)

Speakers
Presentation Number
LB1220
Presentation Topic
Biomarkers and Bioinformatics

Abstract

Background

Neurofilament light chain (NFL) are the most promising biomarkers to investigate clinical activity and treatment efficacy in multiple sclerosis (MS), for which, to date, clinical examination and magnetic resonance imaging (MRI) are the only tools available for diagnosis and monitoring. NFL are released upon axonal damage in the cerebrospinal fluid and, in low concentration, in serum (sNFL). Whilst correlation between NFL and clinical outcomes is established, their implementation in clinical practice is still to be addressed.

Objectives

The aim of the present real-life cross-sectional study is to describe sNFL in a large cohort of MS patients as additional measure of disease activity and treatment efficacy.

Methods

We measured sNF-L by single molecule array (Simoa) assay (NF-light advantage kit, Quanterix) in 79 healthy participants and in 961 MS patients (n=1130 samples). sNFL were cross-sectionally evaluated in 830 relapsing remitting (RR), 53 primary progressive (PP) and 78 secondary progressive (SP) MS patients at different disease stages including diagnostic time, immediately before treatment, and during treatment with the main disease modifying treatments (DMTs): Interferon-beta, Glatiramer acetate, DimethylFumarate, Teriflunomide, Natalizumab, Alemtuzumab, Fingolimod, anti-CD20 . Clinical assessment was performed to evaluate correlations between sNFL, MRI and relapses.

Results

1) We established clinically applicable cut-off values for each age decade testing healthy individuals, later used to interpret sNFL levels in individual MS patients. 2) Progressive MS patients showed higher sNF-L levels and a greater prevalence of high sNFL levels (32% in PPMS and 26% in SPMS) relative to RRMS patients (16%), with respect to the previously determined cut-off values. 3) Patients experiencing MRI and/or clinical activity close to NFL dosage (+/-60 days) showed higher levels than stable patients; according to cut-off values, high NFL levels were observed in a substantial percentage of MRI active patients (72%) and clinically active patients (75%). 4) All DMTs notably lower sNF-L in RRMS patients treated for more than 12 months relative to untreated patients; though, 12% of treated patients still demonstrated high NFL levels.

Conclusions

This study provides a real-life picture of sNFL in a large cohort of MS patients. Cut-off values specific for age decade were applied to discriminate patients samples in different contexts, showing correlation with disease subtype, clinical activity and DMTs efficacy. Our study shows that clinically applicable cut-offs can enable the implementation of sNFL in everyday clinical practice in individual patients, demonstrating its potential as monitoring and treatment decision biomarker.

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Reproductive Aspects and Pregnancy Poster Presentation

P1115 - A first characterization of placenta-derived extracellular vesicles in patients with multiple sclerosis. (ID 1909)

Speakers
Presentation Number
P1115
Presentation Topic
Reproductive Aspects and Pregnancy

Abstract

Background

Pregnancy is a condition of complex immunomodulation during which multiple sclerosis (MS) significantly improves. The molecular mechanisms behind this phenomenon have not been completely elucidated. A central role is played by the placenta and its released factors such as the extracellular vesicles (EV), a peculiar mechanism of communication and material exchange between cells. Studies conducted on an experimental murine model of MS have shown that EV shed into the blood during pregnancy have an inhibitory effect on the proliferation of T cells.

Objectives

To characterize the surface markers expressed by the EV secreted by the maternal (decidua) and fetal (trophoblast) side of the placenta of patients with MS and healthy donors (HC), and to evaluate the effects of the EV on the activation status of CD14+ monocytes and the activity of regulatory T lymphocytes (Treg).

Methods

Total EV shed by placental samples from 15 women with MS and 14 HC were isolated by ultracentrifugation, quantified by Nanoparticle Tracking Analysis and characterized by flow cytometry for the expression of 37 surface markers using the human MACSPlex Exosome Kit. CD14+ monocytes, Treg end CD4+ T cells were isolated from HC buffy coats. CD14+ monocytes were activated with LPS and the expression levels of pro and anti-inflammatory cytokines was evaluated by real-time PCR. CD4+ T cells were activated via CD3 and CD28 and their proliferation activity was evaluated by BrdU Cell Proliferation Assay after co-culture with EV-conditioned Treg.

Results

Most of the analyzed surface markers resulted differentially expressed between the EV released by decidua or trophoblast, but not between the EV shed by placental tissues of MS patients or HC, except for CD133, whose level was higher in the EV secreted by trophoblast of women with MS. The conditioning with EV shed by placenta samples of MS patients was able to moderately decrease the expression level of pro-inflammatory cytokines by activated CD14+ monocytes and the proliferation activity of CD4+ T cells, although the effect was not statistically significant.

Conclusions

For the first time to our knowledge, this study enabled a preliminary characterization of the EV shed by placental tissues of patients with MS. However, further studies are needed to clarify the role of placental EV in the pregnancy-related improvement of the MS disease.

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