Analytic and clinical validation of a prostate cancer-enhanced messenger RNA detection assay in whole blood as a prognostic biomarker for survival

Eur Urol. 2014 Jun;65(6):1191-7. doi: 10.1016/j.eururo.2013.07.006. Epub 2013 Jul 26.


Background: Biomarkers based on detecting prostate cancer (PCa)-specific transcripts in blood are associated with inferior outcomes, but their validation in a clinical context is lacking.

Objective: To determine whether detecting enhanced transcripts for PCa in whole blood using an analytically valid assay has prognostic significance relative to circulating tumor cell (CTC) enumeration.

Design, setting, and participants: The detection of KLK3, KLK2, HOXB13, GRHL2, and FOXA1 in whole blood by reverse transcription polymerase chain reaction (RT-PCR) was studied in 97 men with metastatic castration-resistant PCa (mCRPC) as a prognostic factor for overall survival.

Intervention: The 2.5 ml of blood was collected in PAXgene tubes for total RNA extraction and 7.5 ml for CTC enumeration from patients with progressive mCRPC.

Outcome measurements and statistical analysis: PCa-enriched genes were detected using a sensitive RT-PCR assay in whole blood from patients with mCRPC. Analytical validity of the assay was established in a clinical laboratory environment. The frequency of detecting transcripts was compared to CTC enumeration using CellSearch in an independent data set and survival associations were explored by concordance probability estimate (CPE).

Results and limitations: Two or more genes were detected by PCR in 53% of patients (51 of 97; 95% confidence interval [CI], 43-63%), and unfavorable CTC counts (five of more cells) were seen in 46% (45 of 97; 95% CI, 36-56%). Importantly, transcripts were detectable in 11 of 52 patients with favorable CTC counts (21%; 95% CI, 8-35%). Transcript detection predicted overall survival in a proportional hazards model. Significantly, the predictive accuracy of RT-PCR detection in combination with CTC enumeration had a CPE of 0.752 (standard error: 0.038), although this was limited by the number of patients evaluated.

Conclusions: This validated RT-PCR assay detecting prostate-specific RNA in whole blood is prognostic for survival and may assess patient risk in tandem with CellSearch CTC enumeration. Its clinical utility is being prospectively explored.

Keywords: Biomarker; Circulating tumor cells; Prostate cancer; Prostate-specific markers.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Validation Study

MeSH terms

  • Aged
  • Aged, 80 and over
  • Biomarkers, Tumor / blood*
  • DNA-Binding Proteins / genetics
  • Hepatocyte Nuclear Factor 3-alpha / genetics
  • Homeodomain Proteins / genetics
  • Humans
  • Kallikreins / genetics
  • Male
  • Middle Aged
  • Neoplasm Metastasis
  • Neoplastic Cells, Circulating*
  • Prognosis
  • Prostate-Specific Antigen / genetics
  • Prostatic Neoplasms, Castration-Resistant / blood
  • Prostatic Neoplasms, Castration-Resistant / genetics*
  • Prostatic Neoplasms, Castration-Resistant / pathology*
  • RNA, Messenger / blood*
  • Reverse Transcriptase Polymerase Chain Reaction
  • Survival Rate
  • Tissue Kallikreins / genetics
  • Transcription Factors / genetics


  • Biomarkers, Tumor
  • DNA-Binding Proteins
  • FOXA1 protein, human
  • GRHL2 protein, human
  • HOXB13 protein, human
  • Hepatocyte Nuclear Factor 3-alpha
  • Homeodomain Proteins
  • RNA, Messenger
  • Transcription Factors
  • Kallikreins
  • kallikrein-related peptidase 3, human
  • Tissue Kallikreins
  • Prostate-Specific Antigen