title: Semi-nested real-time reverse transcription polymerase chain reaction methods for the successful quantitation of cytokeratin mRNA expression levels for the subtyping of non-small-cell lung carcinoma using paraffin-embedded and microdissected lung biopsy specimens.
abstract: In patients with inoperable advanced non-small cell lung carcinomas (NSCLCs), histological subtyping using small-mount biopsy specimens was often required to decide the indications for drug treatment. The aim of this study was to assess the utility of highly sensitive mRNA quantitation for the subtyping of advanced NSCLC using small formalin fixing and paraffin embedding (FFPE) biopsy samples. Cytokeratin (CK) 6, CK7, CK14, CK18, and thyroid transcription factor (TTF)-1 mRNA expression levels were measured using semi-nested real-time quantitative (snq) reverse-transcribed polymerase chain reaction (RT-PCR) in microdissected tumor cells collected from 52 lung biopsies. Our results using the present snqRT-PCR method showed an improvement in mRNA quantitation from small FFPE samples, and the mRNA expression level using snqRT-PCR was correlated with the immunohistochemical protein expression level. CK7, CK18, and TTF-1 mRNA were expressed at significantly higher levels (P<0.05) in adenocarcinoma (AD) than in squamous cell carcinoma (SQ), while CK6 and CK14 mRNA expression was significantly higher (P<0.05) in SQ than in AD. Each histology-specific CK, particularly CK18 in AD and CK6 in SQ, were shown to be correlated with a poor prognosis (P=0.02, 0.02, respectively). Our results demonstrated that a quantitative CK subtype mRNA analysis from lung biopsy samples can be useful for predicting the histology subtype and prognosis of advanced NSCLC.
authors.affiliation: Department of Pathology, Nihon University School of Medicine, Tokyo, Japan.
authors.forename: Yoko
authors.initials: Y
authors.lastname: Nakanishi
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Shimizu
authors.forename: Ichiro
authors.initials: I
authors.lastname: Tsujino
authors.forename: Yukari
authors.initials: Y
authors.lastname: Obana
authors.forename: Toshimi
authors.initials: T
authors.lastname: Seki
authors.forename: Fumi
authors.initials: F
authors.lastname: Fuchinoue
authors.forename: Sumie
authors.initials: S
authors.lastname: Ohni
authors.forename: Toshinori
authors.initials: T
authors.lastname: Oinuma
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Kusumi
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Yamada
authors.forename: Noriaki
authors.initials: N
authors.lastname: Takahashi
authors.forename: Shu
authors.initials: S
authors.lastname: Hashimoto
authors.forename: Norimichi
authors.initials: N
authors.lastname: Nemoto
keywords: NSCLC; cytokeratin; laser microdissection; quantitative (q) RT-PCR; subtyping