Demographics.
Demographic and clinical data of participants are shown in Table 1 (cohort 1) and Table 2 (cohort 2). In cohort 1 (Table 1, n=116 subjects), 50 out of 116 participants were positive for amyloid–PET (PET+) (43.1% prevalence) and 66 out of 116 were negative (PET-; 56% prevalence). From the PET+ group, 21 out of 50 were clinically diagnosed with MCI due to AD (MCI-PET+; 42.0% prevalence), and 25 out of 50 were diagnosed with AD dementia (AD-PET+; 50.0% prevalence). Four controls showed a PET+ status (Control-PET+), representing the 7.7% of all controls in cohort 1. About the PET- subjects, 18 out of 66 were clinically diagnosed with FTD (FTD-PET-; 27.3% prevalence) and 48 out of 66 were found asymptomatic (Control-PET-; 72.7% prevalence).
In the independent validation cohort (cohort 2; Table 2, n=142 subjects), 43 out of 142 participants were PET+ (30.3% prevalence) and 99 out of 142 were PET-; 73.9% prevalence). All PET+ subjects received the diagnosis of MCI due to AD except for 4 participants, which stayed cognitively healthy. This group represented the 5.4% of all controls in cohort 2. From the PET- group, 29 out of 99 were diagnosed with MCI probably not due to AD (MCI-PET-; 29.3% prevalence) and 70 out of 99 were found asymptomatic (70.7% prevalence).
No significant sex differences were found between groups. Mean age was statistically similar in all groups except for the FTD group from cohort 1, which was higher (Table 1). All patients showed lower MMSE scores than controls, specially AD and FTD groups. As expected, ApoE ε4 genotype was significantly higher in MCI-PET+ group and AD compared with asymptomatic subjects and FTD patients, according to previous publications [33-34].
In cohort 2, mean age was statistically similar in all groups. MCI patients showed lower MMSE scores than controls. Prevalence of ApoE ε4 genotype was significantly higher in MCI-PET+ group and AD compared with asymptomatic subjects.
MCI-PET- group also showed a significantly higher prevalence of ApoE ε4 genotype compared with the healthy control group.
Salivary Lf levels across diagnostic groups.
In cohort 1, salivary Lf levels were significantly lower in MCI-PET+ (3.8 ± 2.0 µg/ml) and AD (3.6 ± 1.5 µg/ml) groups compared with cognitively normal elderly subjects (7.7 ± 2.4 µg/ml; p < 0.0001), and FTD patients (mean 9.7 ± 2.9 µg/ml; p < 0.0001; Fig. 1A). Lf levels in FTD group were slightly increased compared with controls (p < 0.05) but the difference was much more relevant when comparing with MCI-PET+ and AD-PET+ groups. No significant differences in salivary Lf levels were found between MCI-PET+ and AD patients (Table 1, Fig. 1A). Regarding the control-PET+ group, levels of Lf were inconsistent, showing values of 16.0, 12.6, 6.0 and 4.6 µg/ml.
In cohort 2, saliva Lf levels were significantly lower in MCI-PET+ (3.5 ± 2.5 µg/ml) group compared with healthy controls (9.2 ± 2.9 µg/ml; p < 0.0001), and MCI-PET- (mean 7.4 ± 4.6 µg/ml; p < 0.0001; Table 2, Fig. 1B). No differences in salivary Lf levels were found between control and MCI-PET- groups. Regarding the control-PET+ group, levels of Lf tended to be down-regulated, and the 4 subjects showed values of 6.5, 4.4, 3.8 and 3.7 µg/ml.
Cohort 1 and cohort 2 combination (n=250, excluding the 8 control-PET+ subjects), resulted in 165 out of 250 PET- and 85 out of 250 PET+ subjects. Salivary Lf levels were significantly higher in the PET- group (8.5 ± 3.2 µg/ml) compared with the PET+ group (3.6 ± 2.1 µg/ml; p < 0.0001; Fig. 1C). By excluding the control group, analysis also reported significant differences in salivary Lf levels between PET-, covering FTD and some MCI patients (8.3 ± 4.2 µg/ml), and PET+ patients (p < 0.0001; Fig. 1D).
Correlations between salivary Lf and demographic, clinical, and ApoE genotype.
We then investigated correlations between salivary Lf concentrations and clinical and demographic data, including age, MMSE score, disease duration and CDR, in separated groups within cohorts and combining all of them. We also assessed the effects of ApoE ε4 genotype in Lf levels. We found that higher salivary Lf levels were associated with higher MMSE scores (r = 0.69; p < 0.01) and lower CDR scores (r = -0.59; p < 0.01) in the FTD group. No correlations were found between salivary Lf levels and age or disease duration in the combined or in the separated groups.
According to ApoE genotype, no differences were found between Lf levels in saliva in ApoE ε4 carriers compared with ApoE ε2 and ε3 carriers within the diagnostic groups, suggesting that the ApoE ε4 genotype is not related to salivary Lf levels.
Diagnostic value for Lf.
Lf results were analysed using logistic regression models in cohort 1 and the validation cohort 2, excluding control PET- from both cohorts. Using the Youden’s index, we found that the optimal cut-off point to differentiate salivary Lf from MCI/AD-PET+ patients and control-PET- group was 5.63 µg/ml in cohort 1. At this cut-off point, specificity and sensitivity were estimated in 91.67% and 86.96%, respectively. The obtained area under the curve (AUC) was 0.952 (95% CI, 0.911 - 0.992) (Table 3, Fig. 2A). Using the same cut-off point, the obtained sensitivity and specificity differentiating MCI/AD-PET+ from FTD-PET- patients were 86.96% and 88.89%, respectively. The AUC was 0.97 (95% CI, 0.924 - 1.00) (Table 3, Fig. 2B). Combining Lf levels from all subjects in cohort 1 grouped as PET- and PET+ and keeping the cut-off point of 5.63 µg/ml, we obtained 86.96% sensitivity and 90.91% specificity (Table 3, Fig. 2C).
In the validation cohort 2 analyses, the cut-off estimated from cohort 1 was replicated. The AUC to differentiate MCI-PET+ patients from control-PET- was 0.93 (95% CI, 0.876 - 0.989), with 92.86% specificity and 87.05% sensitivity (Table 3, figure 2D). Both, the accuracy and specificity to differentiate MCI-PET+ from MCI-PET- groups were lower (Table 3, Fig. 2E), due to the higher variability in the salivary Lf levels observed in the MCI-PET- group. That could be explained because MCI patients with negative amyloid PET comprise a heterogeneous group with different and/or mixed non-amyloid aetiologies. Combining Lf levels from all subjects in cohort 2 grouped as PET- and MCI-PET+ and keeping the cut-off point of 5.63 µg/ml from cohort 1, we obtained 82.05% sensitivity and 82.83% specificity with an AUC of 0.88 (95% CI, 0.817 - 0.945) (Table 3, Fig. 2F). Overall, these results revealed that salivary Lf is able to differentiate between subjects with positive and negative cerebral amyloidosis with high accuracy.