Improved Detection of Helicobacter pylori DNA in Formalinfixed Paraffin-embedded (FFPE) Tissue of Patients with Hepatocellular Carcinoma Using Laser Capture Microdissection (LCM)

摘要

DNA has been detected in hepatic tissues from patients with various hepatobiliary diseases, mainly cirrhosis and hepatocellular carcinoma (HCC). Although the role of Helicobacter spp. in pathogenesis of these diseases remains unclear, the available data suggest that Helicobacter infection may play a role in hepatic carcinogenesis [1]. Considering that HCC is one of the most common malignancies with more than 500.000 new tumors diagnosed annually [2], further studies related to H. pylori and development of HCC have fundamental importance on the understanding of its pathogenesis. Formalin-fixed paraffin-embedded (FFPE) tissue represents an extraordinary source for molecular studies as genomic DNA can be extracted from this sample. However, DNA extraction from FFPE tissues is challenging because nucleic acids are commonly fragmented and cross-linked 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. Because of its high precision and accuracy, LCM has been employed in cancer-related studies. In this work, we used LCM technique to improve the detection of H. pylori in FFPE liver from patients with HCC. With this aim, six H. pylori-positive samples detected by polymerase chain reaction (PCR) with H. pylori-specific 16S rRNA primers were selected. The sequence of the sense primer (JW21) was 5'-GCGACCTGCTGGAACATTAC-3'(position 691-710) and the antisense primer (JW22) was 5'-CGTTAGCTCCATTACTGGAGA-3' (position 829-809) [3]. Tissue samples were cut on 0.17 mm PEN membrane-covered slides (Carl Zeiss, MicroImaging GmbH, G?ttingen, Germany) and then routine staining with carbol fuchsin was performed [4]. Thereafter, stained bacteria were microdissected using a PALM MicroBeam system (Carl Zeiss, MicroImaging GmbH, G?ottingen, Germany) and then ejected into the Eppendorf tube cap by a single laser shot (Fig. 1C,D). After microdissection, a digestion buffer was added into Eppendorf for DNA extraction. The crude lysate was directly employed as template for PCR [4]. The samples were further amplified using H. pylori 16S rRNA primers as previously described [3], and amplicons were identified by sequence analysis. Microorganisms resembling H. pylori were observed in hepatic sinus from HCC samples (Fig. 1A,B). The number of cocci was greater than of bacilli as previously described [5]. PCR results showed that all six microdissected samples were positive for 16S rRNA gene and showed 98% similarity to 16S rRNA gene of H. pylori by sequence analysis (GeneBank accession number CP003419.1). Nevertheless, we cannot exclude the possibility of cross-reaction of these primers with other Helicobacter spp. These results demonstrated that LCM can be extensively applied for identification of H. pylori in FFPE liver tissue of HCC patients. 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 simplified the H. pylori detection because extracted DNA was used directly as a template for PCR amplification. Further studies will be performed to isolate H. pylori from other tissues using LCM technique.