title: Detection of metastatic prostate cancer using a splice variant-specific reverse transcriptase-polymerase chain reaction assay for human glandular kallikrein.
abstract: We developed a highly sensitive splice variant-specific reverse transcriptase-PCR (RT-PCR) assay for human glandular kallikrein (hK2) mRNA and tested its ability to detect metastatic disease in men with clinically localized prostate cancer. An RT-PCR assay using primers spanning intron IV and including a significant portion of the 3' untranslated region of the hKLK2 gene, with maximum nonhomology to both hK1 and hK3, was developed. The limit of detection of the assay was five copies of hK2 cDNA and one LNCaP cell in 10(9) lymphoblasts. RT-PCR-hK2 was performed on preoperative peripheral blood specimens from 228 consecutive radical prostatectomy patients as well as 7 metastatic prostate cancer patients and 14 healthy men without prostate cancer. This new RT-PCR-hK2 assay amplifies two distinct fragments. The larger fragment (hK2-U) is approximately 680 bp in length and corresponds to the amplified product of a previously reported splice variant in the splice donor site of intron IV in the hKLK2 gene. The smaller fragment (hK2-L) is approximately 643 bp in length and corresponds to the amplified product of the native hK2 mRNA. Whereas the RT-PCR-hK2-L assay was positive in 71% of our patients with metastatic prostate cancer, 14% of healthy control men also tested positive. By univariate (P = 0.028) and multivariate (P = 0.0269) analysis, which controlled for preoperative PSA, clinical stage, and biopsy Gleason score, RT-PCR-hK2-L status added prognostic information to the prediction of lymph node-positive disease. We have developed a new RT-PCR assay which demonstrates a high sensitivity for detecting hK2 mRNA. Preoperative RT-PCR-hK2-L status helps predict pathological lymph node positivity in patients with clinically localized prostate cancer.
authors.affiliation: Matsunaga-Conte Prostate Cancer Research Center, Scott Department of Urology, Baylor College of Medicine, Houston, Texas 77030, USA. kslawin@www.urol.bcm.tmc.edu
authors.forename: K M
authors.initials: KM
authors.lastname: Slawin
authors.forename: S F
authors.initials: SF
authors.lastname: Shariat
authors.forename: C
authors.initials: C
authors.lastname: Nguyen
authors.forename: A K
authors.initials: AK
authors.lastname: Leventis
authors.forename: W
authors.initials: W
authors.lastname: Song
authors.forename: M W
authors.initials: MW
authors.lastname: Kattan
authors.forename: C Y
authors.initials: CY
authors.lastname: Young
authors.forename: D J
authors.initials: DJ
authors.lastname: Tindall
authors.forename: T M
authors.initials: TM
authors.lastname: Wheeler
mesh_terms: D000328:Adult; D000368:Aged; D017398:Alternative Splicing; D001483:Base Sequence; D016022:Case-Control Studies; D017931:DNA Primers / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007438:Introns; D007610:Kallikreins / Q000032:analysis* / Q000378:metabolism*; D008207:Lymphatic Metastasis / Q000175:diagnosis; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D015999:Multivariate Analysis; D011379:Prognosis; D011471:Prostatic Neoplasms / Q000097:blood / Q000175:diagnosis*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger; D007610:Kallikreins