Participants
This retrospective study included a consecutive sample of 563 participants from the Centre for Neurodegenerative Disorders, Department of Clinical and Experimental Sciences, University of Brescia, Italy.
Each participant underwent a neurological evaluation, routine laboratory examination and a standardized neuropsychological and behavioural assessment, as previously reported [20].
In all FTD cases, the diagnosis was supported by brain structural imaging, while CSF concentrations of tau, p-Tau181 and Aβ1-42 or PET amyloid were measured in a subset of cases (n = 223), to rule out Alzheimer’s disease, as previously reported [21]. Furthermore, in familial cases (based on the presence of at least one dementia case among first-degree relatives) and early onset sporadic cases, genetic screening for GRN, C9orf72 and MAPT P301L mutations was performed; given the low frequency of MAPT mutations in Italy [22], we considered only the P301L mutation and we sequenced the entire MAPT gene only in selected cases.
Clinical evaluation
At baseline, patients underwent a standardized neuropsychological battery which included the Mini-Mental State Examination (MMSE), the short story recall test, the Rey complex figure (copy and recall), phonemic and semantic fluencies, the token test, the clock-drawing test and the trail making test (part A and part B) [23].
The level of functional independence was assessed with the basic activities of daily living (BADL) and the instrumental activities of daily living (IADL) questionnaires, whereas neuropsychiatric and behavioural disturbances were evaluated with the Frontal Behaviour Inventory (FBI) [24].
Disease severity was assessed using the global CDR plus NACC FTLD [8, 9]. Prodromal FTD was defined to reflect mild cognitive or behavioural impairment with relatively preserved functional independence, corresponding to a global CDR plus NACC = 0.5; patients with a mild dementia syndrome were classified with a global CDR plus NACC = 1; patients with a moderate or severe dementia syndrome were classified with a global CDR plus NACC = 2 or 3, respectively [8]. To confirm the diagnosis of prodromal FTD, all patients had a follow-up evaluation that confirmed eventual conversion to dementia, or were carriers of a pathogenic FTD mutation.
Disease progression was defined as a transition to a higher global CDR plus NACC score at 1-year follow-up, whereas no progression was defined when an equal or reduced global CDR plus NACC score was recorded at follow-up. One-year follow-up data was available for 258 participants.
NfL measurements
A subgroup of patients (n = 192) underwent blood collection for serum NfL measurement; 63 healthy controls (HC) (age 67.0, IQR 61.0–74.0 years) were recruited among spouses or caregivers as reference.
Serum was collected by venipuncture, processed and stored in aliquots at – 80 °C according to standardized procedures. Serum NfL was measured by single-molecule array (Simoa) technology on an HD-X Analyzer using the commercial NF-light® assay according to the manufacturer’s instructions (Quanterix, Billerica, MA). Detailed analytical procedures and assay validation have been previously described [15]. The lower limits of quantitation for serum NfL were 0.174 pg/mL. The measurements were performed out in one round of experiments using the same batch of reagents, and the operators were blinded to all clinical information. Quality control samples had intra-assay and inter-assay coefficients of variation of less than 8 and 20%, respectively.
Statistical analysis
Continuous and categorical variables are reported as median (interquartile range) and n (%) respectively. Baseline demographic and clinical variables were compared across groups using one-way Kruskal-Wallis or Fisher’s tests, as appropriate. Differences in NfL levels between groups were compared by using the rank-based nonparametric analysis of covariance (ANCOVA) method, with age as a covariate [25]. We reported marginal mean differences with 95% confidence intervals (95% CI) for relevant comparisons.
To assess the contributions of patient characteristics (sex, age, education, presence of genetic mutation, disease duration, clinical phenotype, FBI, MMSE) and NfL levels to disease progression, we developed multilevel univariable and multivariable logistic regression models considering the time- and severity-dependent nature of independent variables throughout different global CDR plus NAAC FTLD groups. To avoid overfitting in the model, variables were chosen based on previous findings [19, 26,27,28,29,30]. Moreover, multicollinearity was checked and only variables with a variance inflation factors (VIF) < 3 were included. Linearity of the continuous variables with respect to the logit of the dependent variable was assessed via the Box-Tidwell procedure [31]. Based on this assessment, all continuous independent variables were found to be linearly related to the logit of the dependent variable. For each factor, odds ratios (ORs) and 95% confidence intervals (CIs) are reported.
For Fig. 1, we calculated standardized differences compared with HC (for NfL measures) or published Italian normative data for neuropsychological tests [32, 33].
A two-sided p value < 0.05 was considered significant and corrected for multiple comparisons using the Benjamini-Hochberg false discovery rate (FDR) [34]. Data analyses were carried out using IBM SPSS 25.0 and GraphPad Prism 8.0 software.