A Novel Gene Signature for Molecular Diagnosis of Human Prostate Cancer by RT-qPCR

Abstract

BackgroundProstate cancer (CaP) is one of the most relevant causes of cancer death in Western Countries. Although detection of CaP at early curable stage is highly desirable, actual screening methods present limitations and new molecular approaches are needed. Gene expression analysis increases our knowledge about the biology of CaP and may render novel molecular tools, but the identification of accurate biomarkers for reliable molecular diagnosis is a real challenge. We describe here the diagnostic power of a novel 8-genes signature: ornithine decarboxylase (ODC), ornithine decarboxylase antizyme (OAZ), adenosylmethionine decarboxylase (AdoMetDC), spermidine/spermine N(1)-acetyltransferase (SSAT), histone H3 (H3), growth arrest specific gene (GAS1), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and Clusterin (CLU) in tumour detection/classification of human CaP.Methodology/Principal FindingsThe 8-gene signature was detected by retrotranscription real-time quantitative PCR (RT-qPCR) in frozen prostate surgical specimens obtained from 41 patients diagnosed with CaP and recommended to undergo radical prostatectomy (RP). No therapy was given to patients at any time before RP. The bio-bank used for the study consisted of 66 specimens: 44 were benign-CaP paired from the same patient. Thirty-five were classified as benign and 31 as CaP after final pathological examination. Only molecular data were used for classification of specimens. The Nearest Neighbour (NN) classifier was used in order to discriminate CaP from benign tissue. Validation of final results was obtained with 10-fold crossvalidation procedure. CaP versus benign specimens were discriminated with (80±5)% accuracy, (81±6)% sensitivity and (78±7)% specificity. The method also correctly classified 71% of patients with Gleason score<7 versus ≥7, an important predictor of final outcome.Conclusions/SignificanceThe method showed high sensitivity in a collection of specimens in which a significant portion of the total (13/31, equal to 42%) was considered CaP on the basis of having less than 15% of cancer cells. This result supports the notion of the “cancer field effect”, in which transformed cells extend beyond morphologically evident tumour. The molecular diagnosis method here described is objective and less subjected to human error. Although further confirmations are needed, this method posses the potential to enhance conventional diagnosis.

Regulation of miR106b cluster through the RB pathway

“…[40][41][42][43] The mRNA signals were normalized to GAPDH mRNA internal control and the primers used are presented in the Table S1. Mir 25, miR93 and mir106b were analyzed by QRT-PCR by using specific RT-PCR primers from Applied Biosystem, Inc.…”

Therapeutic CDK4/6 inhibition in breast cancer: key mechanisms of response and failure

“…A measure of 150 ng of cDNA was used to quantify and compare the transcript levels among the above-mentioned cell lines. ABI Step-One apparatus and Power SYBR Green Master Mix with primers were directed against human p21, p27, and GAPDH mRNA (Rizzi et al, 2008;Wakimoto et al, 2008;Vivar et al, 2009). The samples were analyzed with a standard 2-h run with an annealing temperature of 55 1C.…”

The clusterin paradigm in prostate and breast carcinogenesis

“…In different kinds of cancers, CLU has been reported to be up-or down-regulated (Redondo et al 2000, Xie et al 2002, Zhang et al 2003, Chen et al 2004, Pucci et al 2004. We have found that CLU mRNA and protein products are down-regulated in both low-grade and high-grade prostate cancer (Bettuzzi et al 2000, Scaltriti et al 2004a, Rizzi et al 2008. The study was conducted in surgical prostate specimens from patients affected by prostatic adenocarcinoma graded from 1 to 5 (Scaltriti et al 2004a).…”