Clear-cell renal cell carcinoma (ccRCC) presents challenges to clinical management because of late-stage detection, treatment resistance, and frequent disease recurrence. Metabolically, ccRCC has a well-described Warburg effect utilization of glucose, but how this affects complex carbohydrate synthesis and alterations to protein and cell surface glycosylation is poorly defined. Using an imaging mass spectrometry approach, N-glycosylation patterns and compositional differences were assessed between tumor and nontumor regions of formalin-fixed clinical ccRCC specimens and tissue microarrays. Regions of normal kidney tissue samples were also evaluated for N-linked glycan-based distinctions between cortex, medullar, glomeruli, and proximal tubule features. Most notable was the proximal tubule localized detection of abundant multiantennary N-glycans with bisecting N-acetylglucosamine and multziple fucose residues. These glycans are absent in ccRCC tissues, while multiple tumor-specific N-glycans were detected with tri-and tetra-antennary structures and varying levels of fucosylation and sialylation. A polycystic kidney disease tissue was also characterized for N-glycan composition, with specific nonfucosylated glycans detected in the cyst fluid regions. Complementary to the imaging mass spectrometry analyses was an assessment of transcriptomic gene array data focused on the fucosyltransferase gene family and other glycosyltransferase genes. The transcript levels of the FUT3 and FUT6 genes responsible for the enzymes that add fucose to N-glycan antennae were significantly decreased in all ccRCC tissues relative to matching nontumor tissues. These striking differences in glycosylation associated with ccRCC could lead to new mechanistic insight into the glycobiology underpinning kidney malignancies and suggest the potential for new therapeutic interventions and diagnostic markers. K E Y W O R D Sclear-cell renal cell carcinoma, fucosylation, MALDI imaging, N-glycosylation, polycystic kidney disease
• la cara c t é ri s ation d'une pollution (réhab i l i t ation de site, ...)• la recherche à l'état de traces d'un ou plusieurs polluants.Sous différentes configurations, le couplage CPG/SM permet donc de répondre à ces attentes. Différents modes d'ionisation et d'acquisition Modes d'ionisationLe laboratoire est équipé de systèmes CPG/SM quadripolaires pouvant fonctionner :• en impact électronique (bombardement d'électrons), • en ionisation chimique en mode positif ou négatif (ionisation d'un gaz réactant). Modes d'acquisitionDeux modes d'acquisition sont généralement employés :• le mode spectre complet où l'appareil balaye une gamme de masse, • le mode SIM (selected ion monitoring) où l'ap p a re i l acquiert le courant ionique (TIC) d'un ou quelques ions. Exemples d'applicationsLa possibilité de configurer le couplage CPG/SM selon différents modes d'ionisation et d'acquisition permet d'adapter l'appareillage selon les objectifs visés :• impact électronique et spectre complet pour des scre enings qualitatifs• SIM, impact électronique ou ionisation chimique pour des analyses quantitatives. Screening qualitatif : spectre complet/ impact électroniqueLors de campagnes de réhabilitation de sites, il est impéra-tif d'identifier les polluants organiques dans les sols ou les eaux souterraines.Un screening en spectrométrie de masse permet de mieux connaître le type de pollution rencontrée et d'en déterminer les composés traceurs. Mode opératoire :• extraction (liquide/liquide ; A.S.E ; Soxhlet).• Analyse en impact électronique EI 70 eV. Rejet de chlorophénols et chlorobenzènes : impact électronique et SIMDans le cadre d'une étude de rejets industriels (sidérurgie) nous avons quantifié ces composés sur différents supports (résines, condensats). Mode opératoire :Après acétylation les chlorophénols et chlorobenzènes sont quantifiés en mode SIM (Figs. 2 et 3). Organo-halogénés volatils : Purge/trap-MS impact électronique et SIMD i rectement issue des méthodes EPA (env i ronmental protection age n cy) cette confi g u ration permet d'at t e i n d re des niveaux de sensibilité trés intéressants (ppb).
Changes in cell surface protein glycosylation are common alterations that occur with tumor progression and reflect the use of many cancer biomarkers like PSA and CA19-9. However, identification of specific glycans associated with tumor regions is still poorly defined. Our group has developed a two-dimensional glycan tissue imaging mass spectrometry approach that can be used with any clinical formalin-fixed, paraffin-embedded tumor tissue used in pathology. Based on the analysis of over one thousand prostate cancer FFPE tissue blocks and tissue microarray samples across the spectrum of disease, it was found that the presence of multi-fucosylated branched N-glycans is associated with advanced tumors with neuroendocrine and metastatic features. These glycans are not present in indolent and lower-grade adenocarcinomas. The hypothesis currently being tested is to determine whether detection of higher numbers of these fucosylated structures predicts a worse prognosis for progressive metastatic disease. Race could be a factor for increased presentation at the time of diagnosis with more advanced disease. The goal of the present study was to assess whether more advanced tumors containing multi-fucosylated N-glycans are detected more frequently in a racial cohort of 307 samples. A series of 14 prostate tumor tissue microarrays (TMA) representing African-American (n=105), Hispanic (n=101) and Caucasian (n=101) subjects was evaluated with this method. The 307 individual tumor samples represented the spectrum of tumor stage, Gleason grade, and status of disease recurrence. For select cases, the original source tumor block tissues were analyzed to confirm the TMA core results. Each TMA slide was processed for antigen retrieval and peptide N-glycosidase F digestions to release N-glycans. Samples were analyzed by MALDI-FTICR mass spectrometry, and data were visualized and analyzed by SCiLS Lab software. Tumor tissues having increased levels of fucosylation were detected in each cohort, but with an overall increase in numbers detected in the African-American cohort. Multitiered analyses are ongoing to identify glycan signatures associated with age, grade, stage, race, and recurrence. In summary, the presence and detection of a distinct panel of multi-fucosylated tissue N-glycans associated with the most lethal forms of prostate cancer can be detected in low-grade tumor samples at the time of diagnosis. This could be developed into a tissue-based prognostic biomarker panel to be applied at the time of initial diagnosis, and impact earlier treatment decisions. Citation Format: Richard R. Drake, Fred David, Cameron Miller, Melanie Jefferson, Laura Spruill, Michael Liss, Brandi Weaver, Peggi M. Angel, Robin Leach, Chanita Hughes-Halbert. Distinguishing lethal from indolent prostate cancer using N-glycan imaging mass spectrometry in a racial tissue microarray cohort [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr C032.
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