title: Quantitative analysis of hTERT mRNA levels in cells microdissected from cytological specimens.
abstract: Clinicians frequently require cytopathological assessment of tumor samples for preoperative diagnosis, but in some specimens, diagnosis remains inconclusive after cytological examination. To date, several molecular markers, including human telomerase reverse transcriptase (hTERT), have been assessed for the ability to detect malignancy. However, analyses using whole cytological specimens are generally affected by contamination of untargeted cells. The present study investigated the feasibility of more sensitive examination by quantitative mRNA analysis of target cells microdissected from cytological specimens. Laser capture microdissection (LCM) was used to obtain target cells from cytological specimens. hTERT mRNA levels were then measured in target cells by quantitative real-time RT-PCR (qRT-PCR). The effect of RNA fragmentation on qRT-PCR was also assessed. Total RNA from cytological specimens was sometimes fragmented to a large degree. To avoid the effect of RNA fragmentation, gene specific priming and PCR primers generating short PCR products were used and no difference in delta Ct values between fragmented and non-fragmented RNA were found. hTERT mRNA levels were measured in cells microdissected from 33 cytological specimens. The levels of hTERT mRNA were significantly higher in malignant cases compared to those in non-malignant cases (P = 0.0003). The sensitivity was 96.2%, even when the specificities were 100%. High levels of hTERT mRNA were also found in three cases that were not diagnosed as malignant by cytological examination. Quantitative assessment of hTERT mRNA levels in cells microdissected from cytological specimens is a potential diagnostic tool to potentiate cytological examination in diagnosing malignancy.
authors.affiliation: Department of Surgery and Oncology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
authors.forename: Hayato
authors.initials: H
authors.lastname: Fujita
authors.forename: Kenoki
authors.initials: K
authors.lastname: Ohuchida
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Mizumoto
authors.forename: Takuya
authors.initials: T
authors.lastname: Egami
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Miyasaka
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Yamaguchi
authors.forename: Jun
authors.initials: J
authors.lastname: Yu
authors.forename: Lin
authors.initials: L
authors.lastname: Cui
authors.forename: Manabu
authors.initials: M
authors.lastname: Onimaru
authors.forename: Shunichi
authors.initials: S
authors.lastname: Takahata
authors.forename: Masazumi
authors.initials: M
authors.lastname: Tsuneyoshi
authors.forename: Masao
authors.initials: M
authors.lastname: Tanaka
mesh_terms: D045744:Cell Line, Tumor; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D012333:RNA, Messenger / Q000378:metabolism*; D012334:RNA, Neoplasm / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019098:Telomerase / Q000235:genetics* / Q000378:metabolism
chemical_list: D012333:RNA, Messenger; D012334:RNA, Neoplasm; C509186:TERT protein, human; D019098:Telomerase
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