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.

TP53 mutations in p53-negative dysplastic urothelial cells from Belgian AAN patients: New evidence for aristolochic acid-induced molecular pathogenesis and carcinogenesis

“…Clinical and pathological characteristics of 5 AAN patients referred to Cliniques Universitaires Saint-Luc (Brussels, Belgium) have been detailed elsewhere [7]. Assessment of the time period to ingest a cumulative toxic dose of AA [13] was compared with data from the BEN [8,14] and Taiwanese [10,12,15] AAN series.…”

“…The current complementary study was approved by the Research Ethics Committee of the Université Catholique de Louvain as a continuation of the previous work [7]. Identification of TP53 mutations was carried out on the same tissue samples as in latter study [7].…”

“…Identification of TP53 mutations was carried out on the same tissue samples as in latter study [7]. Tissues from five pelvi-ureterectomies and a single cystectomy were selected on the basis of the availability of frozen material.…”

“…AAN was first reported in the early 1990's in Belgian patients having undertaken a weight-loss regimen contaminated with AA [5,6]. In a recent work on Belgian AAN, fresh frozen tissues and laser microdissection were used to characterize the mutational spectrum in the TP53 hotspot region after DNA extraction from malignant urothelial cells overexpressing p53 protein by immunohistochemistry (IHC) [7]. Beside the presence of A > T transversion considered as the hallmark of AA-exposure in humans [8][9][10], the previous study revealed two striking observations: the poly-or multiclonal TP53 alterations and an unusual high prevalence of G > T transversions in the Belgian series compared to the International Agency for Research on Cancer (IARC) TP53 database [11] and other clinical series of AA-associated UC [8][9][10]12].…”

In vitro genotoxicity tests point to an unexpected and harmful effect of a Magnolia and Aristolochia association

“…Several studies confirmed that aristolochic acids are genotoxic, in both bacterial and mammalian cells, through the generation of aristolactams that yield highly persistent and non-or poorly-repaired DNA adducts (Fang et al 2011;Sidorenko et al 2012;Aydin et al 2014). Among the Belgian patients, 70 evolved into end-stage renal disease, requiring treatment by dialysis and/or renal transplantation.…”