Multiomics Blood-Based Biomarkers Predict Alzheimer's Predementia with High Specificity in a Multicentric Cohort Study.

BACKGROUND: The primary criteria for diagnosing mild cognitive impairment (MCI) due to Alzheimer's Disease (AD) or probable mild AD dementia rely partly on cognitive assessments and the presence of amyloid plaques. Although these criteria exhibit high sensitivity in predicting AD among cognitively impaired patients, their specificity remains limited. Notably, up to 25% of non-demented patients with amyloid plaques may be misdiagnosed with MCI due to AD, when in fact they suffer from a different brain disorder. The introduction of anti-amyloid antibodies complicates this scenario. Physicians must prioritize which amyloid-positive MCI patients receive these treatments, as not all are suitable candidates. Specifically, those with non-AD amyloid pathologies are not primary targets for amyloid-modifying therapies. Consequently, there is an escalating medical necessity for highly specific blood biomarkers that can accurately detect pre-dementia AD, thus optimizing amyloid antibody prescription. OBJECTIVES: The objective of this study was to evaluate a predictive model based on peripheral biomarkers to identify MCI and mild dementia patients who will develop AD dementia symptoms in cognitively impaired population with high specificity. DESIGN: Peripheral biomarkers were identified in a gene transfer-based animal model of AD and then validated during a retrospective multi-center clinical study. SETTING: Participants from 7 retrospective cohorts (US, EU and Australia). PARTICIPANTS: This study followed 345 cognitively impaired individuals over up to 13 years, including 193 with MCI and 152 with mild dementia, starting from their initial visits. The final diagnoses, established during their last assessments, classified 249 participants as AD patients and 96 as having non-AD brain disorders, based on the specific diagnostic criteria for each disorder subtype. Amyloid status, assessed at baseline, was available for 82.9% of the participants, with 61.9% testing positive for amyloid. Both amyloid-positive and negative individuals were represented in each clinical group. Some of the AD patients had co-morbidities such as metabolic disorders, chronic diseases, or cardiovascular pathologies. MEASUREMENTS: We developed targeted mass spectrometry assays for 81 blood-based biomarkers, encompassing 45 proteins and 36 metabolites previously identified in AAV-AD rats. METHODS: We analyzed blood samples from study participants for the 81 biomarkers. The B-HEALED test, a machine learning-based diagnostic tool, was developed to differentiate AD patients, including 123 with Prodromal AD and 126 with mild AD dementia, from 96 individuals with non-AD brain disorders. The model was trained using 70% of the data, selecting relevant biomarkers, calibrating the algorithm, and establishing cutoff values. The remaining 30% served as an external test dataset for blind validation of the predictive accuracy. RESULTS: Integrating a combination of 19 blood biomarkers and participant age, the B-HEALED model successfully distinguished participants that will develop AD dementia symptoms (82 with Prodromal AD and 83 with AD dementia) from non-AD subjects (71 individuals) with a specificity of 93.0% and sensitivity of 65.4% (AUROC=81.9%, p<0.001) during internal validation. When the amyloid status (derived from CSF or PET scans) and the B-HEALED model were applied in association, with individuals being categorized as AD if they tested positive in both tests, we achieved 100% specificity and 52.8% sensitivity. This performance was consistent in blind external validation, underscoring the model's reliability on independent datasets. CONCLUSIONS: The B-HEALED test, utilizing multiomics blood-based biomarkers, demonstrates high predictive specificity in identifying AD patients within the cognitively impaired population, minimizing false positives. When used alongside amyloid screening, it effectively identifies a nearly pure prodromal AD cohort. These results bear significant implications for refining clinical trial inclusion criteria, facilitating drug development and validation, and accurately identifying patients who will benefit the most from disease-modifying AD treatments.

Pharmacological and structural integrity of muscarinic M2 acetylcholine receptors produced in Sf9 insect cells.

Muscarinic acetylcholine receptors (human m2 subtype), expressed in Sf9 cells, using the baculovirus system, were purified and found to display the expected ligand binding properties, whether membrane-bound or affinity-purified. The purified recombinant receptors were specifically photolabelled with p-N,N-[3H]dimethylamino and p-N,N-[3H]dibutylamino benzene diazonium derivatives. Electrophoretic patterns for covalent radioactive incorporation of the probes were essentially similar to those for [3H]propylbenzilylcholine mustard-labelled receptor sites but were dependent on the infection time of Sf9 cells. Pharmacological properties of the recombinant receptors being unaltered did not reflect structural integrity of the protein as substantial proteolytic fragmentation was detected at a prolonged infection time, i.e., at the highest level of expression. Selection of overexpression conditions, as illustrated here for muscarinic receptors, thus requires not only pharmacological controls, but also analysis of the covalently labelled protein under strongly dissociating conditions.

Covalent labeling of muscarinic acetylcholine receptors by tritiated aryldiazonium photoprobes.

p-dimethylamino (A) and p-dibutylamino (B) benzenediazonium salts, previously characterized as efficient labels of membrane-bound and solubilized muscarinic receptor sites, are endowed with overall interesting photochemical and alkylating properties that allow their use as structural probes of the muscarinic ligand binding domain to be considered. Under reversible binding conditions, these antagonists display no binding selectivity towards the 5 muscarinic acetylcholine receptor (mAChR) subtypes. They were used here, in a tritiated form, as photoaffinity labels of purified muscarinic receptors from porcine striatum, and their irreversible binding was assessed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. When irradiated under energy transfer conditions, [3H]A and [3H]B were both found to covalently label purified muscarinic receptor sites in a light-dependent and atropine-protectable manner. The electrophoretic migration properties of the alkylated sites were similar to those of [3H]propylbenzilylcholine mustard (PrBCM)-labeled mAChRs. Specific radioactive incorporation showed a clear dependency on probe concentration. Labeling efficiency was rather high, with up to 30% and even 60% of the receptor population being photolabeled by [3H]A and [3H]B, respectively. These two photoactivatable ligands have proven to be powerful tools for the structural analysis of other cholinergic targets (acetylcholinesterase and the nicotinic acetylcholine receptor) by allowing the characterization of a number of different residues belonging to their acetylcholine-binding domain. Altogether, these results reinforce the interest of our site-directed labeling approach because [3H]A- and [3H]B-alkylated mAChRs may now be considered as suitable materials to investigate the muscarinic receptor-binding pocket through peptide mapping, sequence analyses, and identification of radiolabeled amino acid residues.

[Latent pathology of the thyroid: an epidemiological and statistical study of thyroids sampled during 507 consecutive autopsies]. / Patologia latente della tiroide: studio epidemiologico e statistico di tiroidi prelevate in corso di 507 autopsie consecutive.

In order to highlight the occult pathology of the thyroid in the aged, the authors examined all the glands withdrawn from 507 consecutive autopsies on subjects of 67.10 years of median age. In 10 tables there are weight and measurements of the thyroids, macro and microscopic details and pathologic appearances. Adenomas were found in 17.1% of cases and histologically besides the known cases referred also to the Hürtle cells type, 4 clear cells adenomas and 5 adenolipomas were found. In 27.42% the subjects were affected or were dead for a malignant extrathyroidal neoplasm, and in 26% of these there was a metastasis in the thyroid. Never a primitive thyroid carcinoma, macroscopically and clinically evident, but in 53 thyroid glands, 54 occult carcinomas (OC) were found, particularly 37 papillary and 17 medullary. IN 57% of 165 histologically treated thyroids, to evidence C-cells, hyperplasia of these cells was found associated with various pathologic conditions, more in aged subjects. In 147 glands were found 170 nodules of various number of cells, at times positive for calcitonin. These solid cellular nodes (SCN) were evaluated as nodular C-cells hyperplasia. Besides isolated cases of acute thyroiditis (also the mycotic type), of tubercular thyroiditis and Hashimoto's, in 12% of the glands a lymphocytic chronic thyroiditis was found, frequently with Hürtle cells. Others observations were: basophilic thickening of colloid also with calcium oxalate crystals, lipoid degeneration of follicular cells and fat interfollicular and interlobular infiltration, thyroid amyloidosis, inner and media elastic calcification of thyroid arteries, presence of cysts with squamous cells coating, parathyroid glands and cartilage intrathyroidal plaques.

Cystic mesothelioma of the peritoneum. A report of three cases.

Three cases of peritoneal cystic mesothelioma are reported. All patients were women who had undergone previous abdominal surgery for unrelated conditions. Tumors consisted of solitary and multiple cystic masses involving the abdominal and pelvic peritoneum. The cysts focally infiltrated the muscularis externa of the small intestine in case 1, the outer muscular layer of the uterus in case 2, and the omental tissue in case 3. These findings give morphologic support to the borderline clinical behavior of this tumor that often recurs and support the hypothesis that previous surgery may play a role in its pathogenesis.

[Occult carcinoma of the thyroid gland: an epidemiological study of autopsy material]. / Carcinoma occulto della tiroide, studio epidemiologico su materiale autoptico.

The occurrence of occult thyroid carcinoma at autopsy was examined in 507 consecutive autopsies performed over one-year in subjects without clinical evidence of thyroid cancer, from different regions of Italy, including areas of endemic goiter. We found 54 (10.65%) occult thyroid carcinomas. In 37 cases the histologic pattern was of the papillary type, with diameter ranging between 176 and 6000 microns, 12 of these cases showed a typical papillary pattern, 6 had a marked fibrosis, 2 had a cystic pattern, one showed a lymphoid stroma, and 17 had a follicular pattern. The remaining 17 cases were medullary carcinomas, with a diameter ranging from 50 to 1600 microns. The percentage of occult thyroid carcinomas reported in the present study may constitute real value of the occurrence of this tumor in the Italian population.

[Critical reflections on differentiated carcinoma of the thyroid: difficulties, doubts and differential-diagnosis problems]. / Riflessioni critiche sul carcinoma differenziato della tiroide: difficoltà, dubbi e problemi diagnostico-differenziali.

This work reviews the problems associated with the diagnosis of well differentiated carcinomas of the thyroid (follicular and papillary), which anatomically and clinically can show characteristics not readily distinguishable from those found in thyroid hyperplasia and adenomas. Some features of atypical adenomas and Hurthle cell tumors are detailed, in particular the borderline malignancy of the latter. We have examined the histological parameters useful in diagnosis of follicular carcinoma (cellular polymorphism and size variability with increased and atypical mitoses, invasion of the capsule and vessels, metastasis to lymph nodes and distant organs) and of papillary carcinoma (true papillae, large and crowded nuclei displaying a "ground glass" appearance with grooves and cytoplasmic inclusions and psammoma bodies). The limitations of these parameters are discussed with emphasis on frozen section examination when the lack of time limits examination to few and small tissue fragments. Invasion of the capsule and vessels is not easily detected and the "ground glass" nucleus and presence of grooves are not evident. The biological behavior of well differentiated carcinomas is discussed and, although unpredictable and variable in the single case, is generally that of a slow growing tumor. It is partially influenced by the age of the patient, size and stage of the tumor, invasion of the capsule and vessels and metastatic spread. We have observed that the biological behavior, apparently different in the follicular and papillary forms, appears identical for both tumors when a large number of cases are analyzed. Mention is made of the various surgical choices (total thyroidectomy, subtotal thyroidectomy, lobectomy, different surgical procedures which take into account the various risk factors). No statistically significant differences in recurrences and metastatic spread are obtained by electing more or less aggressive surgery with or without extensive dissection of cervical lymph nodes.

Immunohistochemical study of solid cell nests of the thyroid gland found from an autopsy study.

An immunohistochemical study was performed to identify the histogenesis of solid cell nests (SCN) found in 30 of 202 thyroids obtained at autopsy. Immunoperoxidase staining was used to detect the presence of calcitonin, thyroglobulin, thyroxin, low and high molecular weight keratins, and carcinoembryonic antigen (CEA). Results showed that cells forming solid nests had immunoreactivity for calcitonin, low molecular weight keratin, and CEA, but not for thyroglobulin, thyroxin and high molecular weight keratin. Thus, SCN do not result from tangentially cut thyroid follicles (absence of staining for thyroglobulin and thyroxin), nor from a squamous metaplastic process (absence of staining for high molecular weight keratin), but instead they are formed by C-cells because they showed calcitonin immunoreactivity, and neurosecretory granules.