title: Laser microdissection and primary cell cultures improve pharmacogenetic analysis in pancreatic adenocarcinoma.
abstract: A key focus of research on pancreatic ductal adenocarcinoma (PDAC) is identifying new techniques to tailor gemcitabine and 5-fluorouracil treatments. Availability of tumor tissue is critical for the accurate assessment of gene expression, and laser microdissection (LMD) and primary cell cultures may be useful tools to separate tumor cells from the stromal reaction. The aim of this study was (1) to address the genetic profile relevant to drug activity and (2) to evaluate differences between microdissected and non-microdissected tumors, normal tissues, and primary cell cultures. Quantitative PCR of seven key genes was performed on mRNA from 113 microdissected and 28 non-microdissected tumors, a pool of normal tissues and four established primary cell lines. Protein expression was evaluated by western blot and immunocytochemistry and cytotoxicity by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. LMD allowed the analysis of 110 samples and revealed significant differences in mRNA levels between microdissected tumors and normal tissues, as well as between non-microdissected and microdissected tumors from the same patients. In contrast, primary cell lines showed similar expression profiles with respect to their respective microdissected tumors. In particular, expression levels of human equilibrative nucleoside transporter-1 and thymydilate synthase were significantly related to gemcitabine and 5-fluorouracil cytotoxicity. We conclude that LMD is a reliable technique for mRNA extraction, and allows detection of significant differences in the expression of specific target genes when compared to non-microdissected specimens and normal tissues. Moreover, expression levels in microdissected tumors are similar to those observed in primary tumor cell cultures, both at mRNA and protein level, and are related to drug chemosensitivity. The use of these ex vivo techniques for molecular analysis of tumors therefore appears to be of some value in implementing the clinical management of PDAC.
authors.affiliation: Department of Oncology, Transplants and Advanced Technologies in Medicine, Division of Surgical Pathology, Molecular and Ultrastructural Pathology, University of Pisa, Pisa, Italy. n.funel@ao-pisa.toscana.it
authors.forename: Niccola
authors.initials: N
authors.lastname: Funel
authors.forename: Elisa
authors.initials: E
authors.lastname: Giovannetti
authors.forename: Marco
authors.initials: M
authors.lastname: Del Chiaro
authors.forename: Valentina
authors.initials: V
authors.lastname: Mey
authors.forename: Luca E
authors.initials: LE
authors.lastname: Pollina
authors.forename: Sara
authors.initials: S
authors.lastname: Nannizzi
authors.forename: Ugo
authors.initials: U
authors.lastname: Boggi
authors.forename: Simona
authors.initials: S
authors.lastname: Ricciardi
authors.forename: Mario
authors.initials: M
authors.lastname: Del Tacca
authors.forename: Generoso
authors.initials: G
authors.lastname: Bevilacqua
authors.forename: Franco
authors.initials: F
authors.lastname: Mosca
authors.forename: Romano
authors.initials: R
authors.lastname: Danesi
authors.forename: Daniela
authors.initials: D
authors.lastname: Campani
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000970:Antineoplastic Agents / Q000494:pharmacology*; D014408:Biomarkers, Tumor / Q000378:metabolism; D021441:Carcinoma, Pancreatic Ductal / Q000378:metabolism* / Q000473:pathology; D002478:Cells, Cultured; D003841:Deoxycytidine / Q000031:analogs & derivatives / Q000494:pharmacology; D005260:Female; D005472:Fluorouracil / Q000494:pharmacology; D006801:Humans; D007834:Lasers; D008297:Male; D042282:Microdissection / Q000379:methods*; D008875:Middle Aged; D020411:Oligonucleotide Array Sequence Analysis; D010190:Pancreatic Neoplasms / Q000378:metabolism* / Q000473:pathology; D012333:RNA, Messenger / Q000096:biosynthesis / Q000302:isolation & purification; D013778:Tetrazolium Salts / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology; D000093542:Gemcitabine
chemical_list: D000970:Antineoplastic Agents; D014408:Biomarkers, Tumor; D012333:RNA, Messenger; D013778:Tetrazolium Salts; D013844:Thiazoles; D003841:Deoxycytidine; C022616:thiazolyl blue; D005472:Fluorouracil; D000093542:Gemcitabine