title: Unambiguous detection of multiple TP53 gene mutations in AAN-associated urothelial cancer in Belgium using laser capture microdissection.
abstract: In the Balkan and Taiwan, the relationship between exposure to aristolochic acid and risk of urothelial neoplasms was inferred from the A>T genetic hallmark in TP53 gene from malignant cells. This study aimed to characterize the TP53 mutational spectrum in urothelial cancers consecutive to Aristolochic Acid Nephropathy in Belgium. Serial frozen tumor sections from female patients (n=5) exposed to aristolochic acid during weight-loss regimen were alternatively used either for p53 immunostaining or laser microdissection. Tissue areas with at least 60% p53-positive nuclei were selected for microdissecting sections according to p53-positive matching areas. All areas appeared to be carcinoma in situ. After DNA extraction, mutations in the TP53 hot spot region (exons 5-8) were identified using nested-PCR and sequencing. False-negative controls consisted in microdissecting fresh-frozen tumor tissues both from a patient with a Li-Fraumeni syndrome who carried a p53 constitutional mutation, and from KRas mutated adenocarcinomas. To rule out false-positive results potentially generated by microdissection and nested-PCR, a phenacetin-associated urothelial carcinoma and normal fresh ureteral tissues (n=4) were processed with high laser power. No unexpected results being identified, molecular analysis was pursued on malignant tissues, showing at least one mutation in all (six different mutations in two) patients, with 13/16 exonic (nonsense, 2; missense, 11) and 3/16 intronic (one splice site) mutations. They were distributed as transitions (n=7) or transversions (n=9), with an equal prevalence of A>T and G>T (3/16 each). While current results are in line with A>T prevalence previously reported in Balkan and Taiwan studies, they also demonstrate that multiple mutations in the TP53 hot spot region and a high frequency of G>T transversion appear as a complementary signature reflecting the toxicity of a cumulative dose of aristolochic acid ingested over a short period of time.
authors.affiliation: Department of Pathology, Cliniques Universitaires Saint-Luc (CUSL), Université Catholique de Louvain (UCL), Institut de Recherche Expérimentale et Clinique (IREC), Brussels, Belgium.
authors.forename: Selda
authors.initials: S
authors.lastname: Aydin
authors.affiliation: Center for Applied Molecular Technologies (CTMA), Cliniques Universitaires Saint-Luc (CUSL), Université Catholique de Louvain (UCL), Institut de Recherche Expérimentale et Clinique (IREC), Brussels, Belgium.
authors.forename: Anne-France
authors.initials: AF
authors.lastname: Dekairelle
authors.affiliation: Center for Applied Molecular Technologies (CTMA), Cliniques Universitaires Saint-Luc (CUSL), Université Catholique de Louvain (UCL), Institut de Recherche Expérimentale et Clinique (IREC), Brussels, Belgium.
authors.forename: Jérôme
authors.initials: J
authors.lastname: Ambroise
authors.affiliation: Center for Applied Molecular Technologies (CTMA), Cliniques Universitaires Saint-Luc (CUSL), Université Catholique de Louvain (UCL), Institut de Recherche Expérimentale et Clinique (IREC), Brussels, Belgium.
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Durant
authors.affiliation: Center for Applied Molecular Technologies (CTMA), Cliniques Universitaires Saint-Luc (CUSL), Université Catholique de Louvain (UCL), Institut de Recherche Expérimentale et Clinique (IREC), Brussels, Belgium.
authors.forename: Michel
authors.initials: M
authors.lastname: Heusterspreute
authors.affiliation: Department of Pathology, Cliniques Universitaires Saint-Luc (CUSL), Université Catholique de Louvain (UCL), Institut de Recherche Expérimentale et Clinique (IREC), Brussels, Belgium.
authors.forename: Yves
authors.initials: Y
authors.lastname: Guiot
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Cosyns
authors.affiliation: Center for Applied Molecular Technologies (CTMA), Cliniques Universitaires Saint-Luc (CUSL), Université Catholique de Louvain (UCL), Institut de Recherche Expérimentale et Clinique (IREC), Brussels, Belgium.
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Gala
mesh_terms: D000328:Adult; D034341:Aristolochic Acids / Q000009:adverse effects*; D001530:Belgium; D005260:Female; D006801:Humans; D061346:Laser Capture Microdissection; D008875:Middle Aged; D017354:Point Mutation; D013624:Taiwan; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*; D014571:Urologic Neoplasms / Q000139:chemically induced / Q000235:genetics* / Q000473:pathology
chemical_list: D034341:Aristolochic Acids; D016159:Tumor Suppressor Protein p53; C000228:aristolochic acid I