Multi-Class Cancer Subtyping in Salivary Gland Carcinomas with MALDI Imaging and Deep Learning.

Salivary gland carcinomas (SGC) are a heterogeneous group of tumors. The prognosis varies strongly according to its type, and even the distinction between benign and malign tumor is challenging. Adenoid cystic carcinoma (AdCy) is one subgroup of SGCs that is prone to late metastasis. This makes accurate tumor subtyping an important task. Matrix-assisted laser desorption/ionization (MALDI) imaging is a label-free technique capable of providing spatially resolved information about the abundance of biomolecules according to their mass-to-charge ratio. We analyzed tissue micro arrays (TMAs) of 25 patients (including six different SGC subtypes and a healthy control group of six patients) with high mass resolution MALDI imaging using a 12-Tesla magnetic resonance mass spectrometer. The high mass resolution allowed us to accurately detect single masses, with strong contributions to each class prediction. To address the added complexity created by the high mass resolution and multiple classes, we propose a deep-learning model. We showed that our deep-learning model provides a per-class classification accuracy of greater than 80% with little preprocessing. Based on this classification, we employed methods of explainable artificial intelligence (AI) to gain further insights into the spectrometric features of AdCys.

High-resolution MRI of the human palatine tonsil and its schematic anatomic 3D reconstruction.

The palatine tonsils form an important part of the human immune system. Together with the other lymphoid tonsils of Waldeyer's tonsillar ring, they act as the first line of defense against ingested or inhaled pathogens. Although histologically stained sections of the palatine tonsil are widely available, they represent the tissue only in two dimensions and do not provide reference to three-dimensional space. Such a representation of a tonsillar specimen based on imaging data as a 3D anatomical reconstruction is lacking both in scientific publications and especially in textbooks. As a first step in this direction, the objective of the present work was to image a resected tonsil specimen with high spatial resolution in a 9.4 T small-bore pre-clinical MRI and to combine these data with data from the completely sectioned and H&E stained same palatine tonsil. Based on the information from both image modalities, a 3D anatomical sketch was drawn by a scientific graphic artist. In perspective, such studies could help to overcome the difficulty of capturing the spatial extent and arrangement of anatomical structures from 2D images and to establish a link between three-dimensional anatomical preparations and two-dimensional sections or illustrations, as they have been found so far in common textbooks and anatomical atlases.

Identification of Proteomic Markers in Head and Neck Cancer Using MALDI-MS Imaging, LC-MS/MS, and Immunohistochemistry.

PURPOSE: The heterogeneity of squamous cell carcinoma tissue greatly complicates diagnosis and individualized therapy. Therefore, characterizing the heterogeneity of tissue spatially and identifying appropriate biomarkers is crucial. MALDI-MS imaging (MSI) is capable of analyzing spatially resolved tissue biopsies on a molecular level. EXPERIMENTAL DESIGN: MALDI-MSI is used on snap frozen and formalin-fixed and paraffin-embedded (FFPE) tissue samples from patients with head and neck cancer (HNC) to analyze m/z values localized in tumor and nontumor regions. Peptide identification is performed using LC-MS/MS and immunohistochemistry (IHC). RESULTS: In both FFPE and frozen tissue specimens, eight characteristic masses of the tumor's epithelial region are found. Using LC-MS/MS, the peaks are identified as vimentin, keratin type II, nucleolin, heat shock protein 90, prelamin-A/C, junction plakoglobin, and PGAM1. Lastly, vimentin, nucleolin, and PGAM1 are verified with IHC. CONCLUSIONS AND CLINICAL RELEVANCE: The combination of MALDI-MSI, LC-MS/MS, and subsequent IHC furnishes a tool suitable for characterizing the molecular heterogeneity of tissue. It is also suited for use in identifying new representative biomarkers to enable a more individualized therapy.

Preservation of urine free catecholamines and their free O-methylated metabolites with citric acid as an alternative to hydrochloric acid for LC-MS/MS-based analyses.

BACKGROUND: Measurements of urinary fractionated metadrenalines provide a useful screening test to diagnose phaeochromocytoma. Stability of these compounds and their parent catecholamines during and after urine collection is crucial to ensure accuracy of the measurements. Stabilisation with hydrochloric acid (HCl) can promote deconjugation of sulphate-conjugated metadrenalines, indicating a need for alternative preservatives. METHODS: Urine samples with an intrinsically acidic or alkaline pH (5.5-6.9 or 7.1-8.7, respectively) were used to assess stability of free catecholamines and their free O-methylated metabolites over 7 days of room temperature storage. Stabilisation with HCl was compared with ethylenediaminetetraacetic acid/metabisulphite and monobasic citric acid. Catecholamines and metabolites were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: Free catecholamines and their O-methylated metabolites were stable in acidic urine samples over 7 days of room temperature storage, independent of the presence or absence of any stabilisation method. In contrast, free catecholamines, but not the free O-methylated metabolites, showed rapid degradation within 24 h and continuing degradation over 7 days in urine samples with an alkaline pH. Adjustment of alkaline urine samples to a pH of 3-5 with HCl or 4.8-5.4 with citric acid completely blocked degradation of catecholamines. Ethylenediaminetetraacetic acid/metabisulphite, although reducing the extent of degradation of catecholamines in alkaline urine, was largely ineffectual as a stabiliser. CONCLUSIONS: Citric acid is equally effective as HCl for stabilisation of urinary free catecholamines and minimises hazards associated with use of strong inorganic acids while avoiding deconjugation of sulphate-conjugated metabolites during simultaneous LC-MS/MS measurements of free catecholamines and their free O-methylated metabolites.

Levodopa therapy in Parkinson's disease: influence on liquid chromatographic tandem mass spectrometric-based measurements of plasma and urinary normetanephrine, metanephrine and methoxytyramine.

BACKGROUND: Medication-related interferences with measurements of catecholamines and their metabolites represent important causes of false-positive results during diagnosis of phaeochromocytomas and paragangliomas (PPGLs). Such interferences are less troublesome with measurements by liquid chromatography with tandem mass-spectrometry (LC-MS/MS) than by other methods, but can still present problems for some drugs. Levodopa, the precursor for dopamine used in the treatment of Parkinson's disease, represents one potentially interfering medication. METHODS: Plasma and urine samples, obtained from 20 Parkinsonian patients receiving levodopa, were analysed for concentrations of catecholamines and their O-methylated metabolites by LC-MS/MS. Results were compared with those from a group of 120 age-matched subjects and 18 patients with PPGLs. RESULTS: Plasma and urinary free and deconjugated (free + conjugated) methoxytyramine, as well as urinary dopamine, showed 22- to 148-fold higher (P < 0.0001) concentrations in patients receiving levodopa than in the reference group. In contrast, plasma normetanephrine, urinary noradrenaline and urinary free and deconjugated normetanephrine concentrations were unaffected. Plasma free metanephrine, urinary adrenaline and urinary free and deconjugated metanephrine all showed higher (P < 0.05) concentrations in Parkinsonian patients than the reference group, but this was only a problem for adrenaline. Similar to normetanephrine, plasma and urinary metanephrine remained below the 97.5 percentiles of the reference group in almost all Parkinsonian patients. CONCLUSIONS: These data establish that although levodopa treatment confounds identification of PPGLs that produce dopamine, the therapy is not a problem for use of LC-MS/MS measurements of plasma and urinary normetanephrine and metanephrine to diagnose more commonly encountered PPGLs that produce noradrenaline or adrenaline.

Simultaneous liquid chromatography tandem mass spectrometric determination of urinary free metanephrines and catecholamines, with comparisons of free and deconjugated metabolites.

OBJECTIVE: We introduce a novel liquid chromatographic tandem-mass spectrometric method for simultaneous measurements of urinary catecholamines and their free O-methylated metabolites, which we compare to the deconjugated metabolites. METHODS: Method performance was validated for recovery, linearity, precision and accuracy, analyte stability, ion suppression and carry over. Results from 53 patients with and 138 volunteers without pheochromocytoma were compared. RESULTS: Analyte recoveries ranged from 60 to 96% and intra- and inter-assay coefficients of variation from 2.7 to 13.2%. The method showed excellent linearity over 3 orders of magnitude with analytical sensitivity sufficient to measure to 1.2 nmol/L. Free O-methylated metabolites were excreted at less than 20% the rates of the deconjugated metabolites, but were easily measureable. Increases in urinary normetanephrine in pheochromocytoma patients relative to volunteers were higher for free than deconjugated metabolites and higher for both than for norepinephrine (10 vs 5.5 vs 3.7 fold increases). In contrast, relative increases in urinary free versus deconjugated metanephrine (2.7 and 3.2) and methoxytyramine (2.1 and 1.9) did not differ, but for methoxytyramine were larger than for dopamine (1.2). CONCLUSION: Measurements of urinary catecholamines and their free O-methylated metabolites by our method provide potential advantages over urinary deconjugated metanephrines for diagnosis of pheochromocytoma.