From Cerebrospinal Fluid Neurochemistry to Clinical Diagnosis of Alzheimer’s Disease in the Era of Anti-Amyloid Treatments. Report of Four Patients

Analysis of classical cerebrospinal fluid biomarkers, especially when incorporated in a classification/diagnostic system such as the AT(N), may offer a significant diagnostic tool allowing correct identification of Alzheimer’s disease during life. We describe four patients with more or less atypical or mixed clinical presentation, in which the classical cerebrospinal fluid biomarkers amyloid peptide with 42 and 40 amino acids (Aβ42 and Aβ40, respectively), phospho-tau (τP-181) and total tau (τΤ) were measured. Despite the unusual clinical presentation, the biomarker profile was compatible with Alzheimer’s disease in all four patients. The measurement of classical biomarkers in the cerebrospinal fluid may be a useful tool in identifying the biochemical fingerprints of Alzheimer’s disease, especially currently, due to the recent approval of the first disease-modifying treatment, allowing not only typical but also atypical cases to be enrolled in trials of such treatments.


Introduction
Alzheimer's disease (AD), the most common cause of dementia, is a neurodegenerative disorder characterized by neuronal and synaptic loss and eventually brain atrophy, due to extracellular polymerization and the accumulation of amyloid peptide with 40 and especially 42 amino acids (Aβ 40 and Aβ 42, respectively) in the form of amyloid plaques and intracellular polymerization of hyper-phosphorylated tau protein in the form of paired helical filaments, viewed microscopically as neurofibrillary tangles [1]. This pathophysiological/pathobiochemical process of AD starts many years before, and likely, one to three decades prior to symptom onset [2,3]. Following this long asymptomatic or "preclinical" phase of the disease [4], the symptomatic phase starts [5] initially with mild cognitive impairment (MCI) [6] and finally dementia [7]. At the symptomatic phase, the typical presentation of AD is usually of the "hippocampal amnestic-type", characterized by a deficit in episodic memory with difficulty in both free and cued recall [8]. However, in approximately 10-15% of AD patients, atypical (non-amnestic) presentations have been described [5] and this percentage may rise to 22-64% in early-onset (pre-senile) cases [9]. Such atypical presentations include primary progressive aphasia (PPA) [10], frontal dementia

Patients
The four patients presented here were examined at the 2nd Department of Neurology. They had cognitive impairment with an atypical presentation, creating clinical diagnostic uncertainty, with CSF biomarkers resolving the problem by revealing the CSF "neurochemical fingerprint" of AD (otherwise, there were no specific selection criteria).
Initially, history, neurological and complete physical examination were recorded routinely. Secondary causes including thyroid disease, B12 deficiency, neurosyphilis, brain tumor, or subdural hematoma (but not normal pressure hydrocephalus) were excluded. Written informed consent was obtained for all cases. The study had the approval of the Scientific Board and Ethics Committee of "Attikon" Hospital (project identification codes of approval: A13, 7 April 2021 and 157, 16 March 2021 respectively) and was conducted according to the ethical guidelines of the 1964 Declaration of Helsinki.

Neuropsychological Approach
Following history and clinical examination, a battery of neuropsychological tests was performed. Global tests for the assessment of cognition and activities of daily living included the Addenbrooke's Cognitive Examination-Revised version (ACE-R), the Mini Mental State Examination (MMSE), and the Instrumental Activities of Daily Living (IADL), all of which have been validated in Greece [37][38][39]. Brief bed-side tests for memory (free and cued recall), frontal function, visuospatial skills, and possible depression included the 5-words memory test [40], the Frontal Assessment Battery (FAB) [41], the CLOX (1 and 2) [42], and the short version of the Geriatric Depression Scale (GDS) [43], respectively. Finally, as a tool for the concomitant assessment of cognitive and functional status, the Clinical Dementia Rating (CDR, both sum of boxes and overall score) was used [44].

Neuroimaging
A routine 1.5 or 3T brain magnetic resonance imaging (MRI) scan was the preferred method of neuroimaging, including 3D T1W sequences, suitable for assessing cortical and central atrophy, including medial temporal atrophy, according to a visual scale [45]. The Evans index and callosal angle were also calculated as appropriate [46]. Alternatively, a brain computerized (CT) scan was obtained in cases with MRI contraindication (orthopedic prostheses).

Lumbar Puncture and CSF Biomarker Measurements
A lumbar puncture was performed using a standard, 21-22G, Quincke-type needle, at the L4-L5 interspace, at 9-12 a.m. according to widely accepted recommendations on standardized operative procedures for CSF biomarkers [47]. In brief, CSF was collected in six polypropylene tubes. The first and second tubes (1 mL each) were used for routine CSF cytology and biochemistry, respectively. The third tube (2 mL) was used for oligoclonal bands and IgG index determinations. The following two tubes (5 mL each) were used for biomarker determinations. The last tube (~2 mL) was used for syphilis serology or other tests according to clinical indications. All CSF samples had <500 red blood cells/µL.
The two tubes intended for CSF biomarker analysis were immediately centrifuged (2000× g 15 min), aliquoted in polypropylene tubes (1 mL each), and finally stored at −80 • C. Aliquots were thawed only once, just before analysis, which was performed within three months of storage.
The CSF AD profile ("fingerprint") was defined as decreased Aβ 42 or decreased Aβ 42 /Aβ 40 and increased τ P-181 , and thus, compatible with the A + T + (N) + or A + T + (N) − profiles of the AT(N) classification system [18], according to Figure 1.
The CSF AD profile ("fingerprint") was defined as decreased Aβ42 or decreased Aβ42/Aβ40 and increased τP-181, and thus, compatible with the A + T + (N) + or A + T + (N) − profiles of the AT(N) classification system [18], according to Figure 1.

Results
The demographic, clinical, neuropsychological, and CSF neurochemical data of the four patients are summarized in Table 1.

Patient 1
A seventy-six-year-old female was examined due to four years of "memory problems". She increasingly had to keep memos and frequently repeated the same questions. According to the results of the neuropsychological testing, she had incipient dementia, with a profile more compatible with a frontal or frontal-subcortical syndrome (decreased attention and concentration and executive function) rather than the typical hippocampal amnestic syndrome (Table 1). Neuroimaging showed frontal-frontoparietal atrophy and asymmetric hippocampal atrophy (Figure 2a). Biomarker assessment showed decreased Aβ 42 and Aβ 42 /Aβ 40 ratio and increased both τ P-181 and τ T , compatible with AD. Biomedicines 2021, 9, x FOR PEER REVIEW 6 of 14

Patient 2
This seventy-six-year-old female suffered gradually progressive difficulty in speech for three years. Upon examination, she had a perfect understanding of language, but during spontaneous speech she made many pauses in an effort to "recall" the appropriate word. Upon naming testing, anomic (word-finding) difficulty was obvious, with object knowledge and single-word comprehension completely spared. Phonological errors were frequent and sentence repetition was severely affected. The motor and grammatical aspects of speech were normal. No difficulty in other cognitive domains was reported and decreased scores in neuropsychological testing were attributed mainly to the language (aphasic) disorder. She had no other significant difficulty in activities of daily living except in communication due to the aphasic disorder, which was compatible with Primary Progressive Aphasia (PPA) of the logopenic-type [49]. Atrophy was predominant in the left perisylvian and parietal areas (Figure 2b). Biomarker analysis revealed normal Aβ 42 with reduced Aβ 42 /Aβ 40 ratio, together with increased τ P-181 and τ T , compatible with AD.

Patient 3
An eighty-one-year-old male developed a gradually progressive cognitive decline during the last four years. He had apathy, social withdrawal difficulty in performing complex tasks, mental "slowness", and reduced attention. The previous year, progressive gait difficulty was noticed, with slow and short steps, sometimes a "magnetic" gait, and occasional falls with one fracture. The previous month, urinary urgency and sometimes incontinence was added into the clinical picture. Neuropsychological testing revealed moderate-stage dementia showing a mixed profile, including significant frontal, amnestic, and visuoconstructive components. Neuroimaging revealed an increased Evans index, acute callosal angle, tight convexity and periventricular caps, suggestive of normal pressure hydrocephalus [46], but cerebral small vessel disease was also evident (Figure 2c). Consistently with the suspicion of normal pressure hydrocephalus, a spinal taping test (removal of 40 mL of CSF) resulted in a significant improvement of gait and cognition. However, CSF biomarkers analysis revealed decreased Aβ 42 and increased τ P-181 and τ T , compatible with the additional presence of AD.

Patient 4
This eighty-three-year-old female developed gradually progressive gait difficulty with slow and short steps, postural instability, and frequent falls during the last three years and was unresponsive to L-dopa treatment. In addition, apathy, mental "slowness" and reduced attention were reported. In the previous year, urinary incontinence was noted. Upon clinical examination, she was practically bed-ridden, with asymmetric parkinsonism, including limb bradykinesia and rigidity more evident in the left limbs, while pyramidal signs were additionally present, more evident in the left limbs. Frequent myoclonic jerks were observed in the upper limbs, especially the left. Cortical sensory loss and sensory neglect were present in the right limbs. Primitive reflexes (especially grasping) were also present. Neuropsychological testing revealed moderate-stage dementia showing a mixed profile, including significant frontal, amnestic and visuoconstructive components, while significant upper limb apraxia was present. The patient met clinical criteria for corticobasal syndrome [50]. Despite some degree of asymmetrical atrophy, neuroimaging revealed an increased Evans index, acute callosal angle, and periventricular caps, suggestive of normal pressure hydrocephalus [46], while some degree of cerebral small vessel disease was also evident (Figure 2d). The spinal taping test (removal of 40 mL of CSF) resulted in a significant improvement of cognition, but there was no change in gait. Analysis of CSF biomarkers showed reduced Aβ 42 /Aβ 40 ratio, together with increased τ P-181 and τ T , compatible with the presence of AD.

Discussion
In the present study, we present four cognitively impaired patients with clinical presentations creating diagnostic uncertainty. The first patient was at the transition from MCI to mild dementia and, while she complained of memory problems, the total delayed recall (including memory cues) was normal, which is considered not compatible with the hippocampal amnestic disorder (typically expected in AD), but more compatible with a frontal-subcortical-type of memory decline. Despite a senile onset of disease and a presumably higher probability for AD, this is estimated to be no more than~70% in such cases with early-stage disease and non-typical presentation [21,22], with other pathologies entering in the differential diagnosis. In the second patient, the clinical profile was compatible with PPA of the logopenic-type, which is due to AD in approximately 50-80% of patients [10,51]. However, it should be not considered synonymous with AD [49], since, in~25%, it is caused by one of the frontotemporal pathologies [51].
Thus, in both patients 1 and 2, there was still a significant chance (at the level of 25-30%) that a non-AD pathology may be the cause of the cognitive decline. Since both patients had MMSE > 20, making them eligible for aducanumab treatment, it is necessary to increase the diagnostic certainty from 70-75% to as high as possible, in order to initiate such a specific, expensive, and with potentially serious complications, treatment. In both patients, the CSF biomarker results, according to the AT(N) classification system [18], were compatible with the presence of AD.
In patients 3 and 4, the case was quite different since they were mixed cases of dementia. Patient 3 had typical clinical and imaging characteristics of normal pressure hydrocephalus and the positive taping test was consistent with this notion. Normal-pressure hydrocephalus may occur alone, but in three-quarters of cases, AD and/or cerebrovascular disease (usual of the small vessel-type) may be additionally present [52]. In the additional presence of AD, a shunting operation may offer some degree of gait improvement, which may positively affect the quality of life [53]; however, cognitive improvement may be modest [53] and the overall improvement is traditionally thought to be moderate at best and short-lived [54]. Thus, the possible co-occurrence of AD should be known prior to the selection of optimal treatment (or treatment combinations). In patient 3, the whole picture was compatible with NPH and concomitant small vessel disease, both of which may contribute to the clinical picture. However, CSF biomarkers revealed a third significant component in this patient's dementia, that of AD. Patient 4 was the most intriguing. She had a mixed movement and cognitive disorder, with a clinical picture typical of corticobasal syndrome, while neuroimaging revealed a normal pressure hydrocephalus-like picture and some degree of small vessel disease. A taping test resulted in the improvement of cognition only, but not of gait, probably because the corticobasal component of the motor disability was already severe enough to oppose any improvement. The corticobasal syndrome is not a disease, but a clinical picture that can be due to many neurodegenerative diseases, the most common being corticobasal degeneration which belongs to the 4-repeat tauopathies [50]. However, it can be caused by AD, Lewy body pathology, progressive supranuclear palsy, and even Creutzfeldt-Jakob disease [12], with AD accounting for a significant percentage of cases with corticobasal syndrome [55]. CSF biomarker analysis in patient 4 revealed that AD was indeed the underlying cause. Normal-pressure hydrocephalus was probably present as well (hence the cognitive improvement following the taping test), however, it was superimposed on AD.
Classical CSF biomarkers are useful in identifying the AD biochemical fingerprint in typical and atypical AD cases [27,28]. Their diagnostic performance has been validated in autopsy-proven cases [56]. They have been proven useful in cases with primary progressive aphasia [51], corticobasal syndrome [57], and cases of AD mixed with Lewy body pathology [58] or cerebrovascular disease [14,59]. They can identify the concomitant presence of AD in cases with normal pressure hydrocephalus [60,61], and possibly predict a worse neurosurgical prognosis [62], although recent data suggest that they may predict the opposite [16].
When incorporated in the AT(N) classification system, CSF biomarkers may be used effectively not only in research but also in clinical practice [36,63]. It should be noted that in patients 2 and 4, CSF levels of Aβ 42 were normal. However, the Aβ 42 /Aβ 40 ratio was abnormally reduced in both, allowing the diagnosis of AD. Despite some concerns about the interchangeability between Aβ 42 and the Aβ 42 /Aβ 40 ratio in the AT(N) system [64], the ratio shows better diagnostic accuracy compared to Aβ 42 alone [32,65], correlates better with amyloid imaging by positron emission tomography [32], and its better diagnostic performance has been confirmed in pathologically proven cases [32].
There are some limitations in classical CSF biomarker determination. Preanalytical factors, including CSF sampling and storage, may affect test results and internationally accepted guidelines have been formulated for this reason [47]. International quality control programs and projects have been organized, in order to identify and control for confounding factors, improve the methodologies used, optimize analytical performance, and harmonize the levels of biomarkers [66][67][68]. However, there is still a significant intraand inter-laboratory variability [67,69] and each laboratory should have its own cut-off values [28]. Discordant biomarker results have been observed in different reference laboratories, especially for Aβ 42 [70]. Diagnostically gray zones also exist and, when added to the possible measurement error, they may lead to a variability of ±25% [70]. Normal levels of all three CSF classical biomarkers may be observed in normal aging, but also in psychiatric disorders which may present with cognitive complaints, sometimes entering in the differential diagnosis of frontotemporal dementia. Furthermore, the classical CSF biomarkers cannot identify additional neurodegenerative pathologies, which are not rare in older patients with AD [71]. Finally, determination of CSF biomarkers requires a lumbar puncture which is a cause of concern and anxiety in many patients and caregivers, and it cannot be easily repeated for frequent follow-up.
Other molecules are under intense investigation in an effort to optimize the differential diagnostic value of the classic biomarkers and identify possible additional neurodegenerative pathologies. They include markers of neuroinflammation such as the triggering receptor expressed on myeloid cells 2 (TREM2), progranulin, and chitinase-3-like protein-1 (YKL-40), markers of synaptic dysfunction such as neurogranin, and markers of neuronal injury such as neurofilament light (NfL) and visinin-like protein 1 (VILIP-1), while miR-NAs could also be helpful [72][73][74][75][76][77]. Oligomeric forms of Aβ 42 [78], α-synuclein [79], and TAR DNA-Binding Protein 43 (TDP43) [80] are emerging biomarkers, but work must still be carried out to achieve adequate diagnostic performance. Especially for α-synuclein, which has been traditionally considered as a marker of synuclein pathology, results are conflicting [79], partially due to the effect of preanalytical and analytical factors, including differences in a-synuclein species detected by different methods [81]. Recent evidence suggests that α-, and also βand γ-synuclein, may be effective markers of AD rather than synucleinopathy [82]. Both αand β-synuclein may be early markers of AD, even in non-demented elder subjects [83,84], while the ratio of total tau/α-synuclein may serve as a marker of tau phosphorylation, even allowing patients with the A − T + (N + ) profile to re-enter the AD diagnostic group [85]. Blood-based classical [86,87] and exosomal [88] biomarkers may prove helpful, especially for frequent monitoring of the biochemical effects of anti-amyloid antibodies. The AT(N) system is flexible and may expand to an ATX(N) form, incorporating such new or evolving biomarkers of AD-related or additional non-AD pathologies [89].

Conclusions
Biomarkers are not stand-alone tools and should always be interpreted along with clinical, neuropsychological, and imaging data. Keeping this in mind, analysis of classical CSF biomarkers, especially when incorporated in a classification/diagnostic system such as the AT(N), may offer a significant diagnostic tool [90,91], with both added [92] and prognostic [36] value, allowing the correct identification of AD during life, especially in cases with atypical or mixed presentations [93]. This is always important for correct therapeutic decisions, and it is of paramount importance currently, due to the recent approval of aducanumab as a disease-modifying treatment. Whether atypical cases are going to have the same benefit (from classical or newer treatments) as the typical ones, remains to be elucidated. Funding: E.K. has received research funding from ELPEN Pharmaceutical Co. Inc. and NUTRICIA.

Institutional Review Board Statement:
The study had the approval of the Bioethics Committee and the Scientific Board of "Attikon" Hospital (approval numbers 157/16-03-2021 and A13/07-04-2021, respectively). It was performed according to the 1964 Declaration of Helsinki.
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study and/or next of kin caregivers (depending on the severity of cognitive impairment).

Data Availability Statement:
The data presented in this study are available upon request from the corresponding author. The data are not publicly available due to privacy restrictions.