|803-2||IMPROVED DETECTION OF Helicobacter pylori DNA IN FORMALIN FIXED PARAFFIN-EMBEDDED (FFPE) TISSUE OF PATIENTS WITH HEPATOCELLULAR CARCINOMA USING LASER CAPTURE MICRODISSECTION TECHNIQUE|
|Autores:||Elizabeth Maria Afonso Rabelo Gonçalves (UNICAMP - State University of Campinas) ; Ilária C. Sgardioli (UNICAMP - State University of Campinas) ; Iscia Lopes Cendes (UNICAMP - State University of Campinas) ; Ana Lúcia Rosseto Pereira (UNICAMP - State University of Campinas) ; Cecília Amélia Fazzio Escanhoela (UNICAMP - State University of Campinas) ; Jazon Romilson de Souza Almeida (UNICAMP - State University of Campinas) ; José Murilo Robilotta Zeitune (UNICAMP - State University of Campinas) |
INTRODUCTION: genomic DNA extraction from FFPE tissues is challenging since nucleic acids are commonly fragmented and crosslinked with proteins. Furthermore, methods of DNA extraction from FFPE tissue are generally laborious and time consuming. Laser capture microdissection (LCM) is a recently developed technique for isolation of pure populations of cells from tissue sections by microscopic visualization. Considering that Helicobacter (H.) pylori DNA has been detected in hepatocellular carcinoma (HCC) tissues, the aim of this study was to use LCM technique to improve the detection of this bacterium in FFPE liver from Brazilian patients with HCC. METHODS: for this purpose, six samples of FFPE liver were stained with carbol fuchsin and microdissected. After microdissection, DNA extraction was performed using a simple digestion buffer and the crude lysate was directly employed as template for polymerase chain reaction (PCR). The samples were amplified using H. pylori specific 16S rRNA primers and PCR products were further sequenced. DISCUSSION: results showed that all six microdissected samples were positive for 16S rRNA gene that showed 98% similarity to H. pylori by sequence analysis. These results demonstrated that LCM technique can be extensively applied for identification of H. pylori in FFPE liver tissue of patients with hepatocellular carcinoma. CONCLUSIONS: considering that bacteria were mainly found in peritumoral tissue, this technique was highly effective for obtaining a targeted bacterial population within a selected area in the HCC tissue. Beyond that, LCM technique simplified the H. pylori detection since extracted DNA was used directly as a template for PCR amplification. Further studies will be performed in order to isolate H. pylori from gastric biopsies and other tissues using LCM technique.
Palavras-chave: FFPE tissue, Helicobacter pylori, Hepatocellular carcinoma, Laser capture microdissection