Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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

Author(s): Danila DC, Anand A, Schultz N, Heller G, Wan M, Sung CC, Dai C, Khanin R, Fleisher M, Lilja H, Scher HI

Publication: Eur Urol, 2014, Vol. 65, Page 1191-7

PubMed ID: 23954088 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study sought to determine whether levels of specific transcripts in peripheral blood are correlated to the number of circulating tumor cells (CTCs) determined using the CellSearch System, and whether transcript detection alone or in combination with CTC count is predicative of prostate cancer patient prognosis. Peripheral blood was collected from 97 men with metastatic castration-resistant prostate cancer and 51 healthy volunteers in CellSave tubes and PAXgene tubes. Blood (7.5 mL/individual) collected in CellSave tubes was processed for CTC enumeration using the CellSearch system. Blood (2.5 mL) collected in PAXgene tubes were stored at -80°C until analysis by real-time RT-PCR for five prostate cancer-related genes (KLK3, KLK2, HOXB13, GRHL2, and FOXA1) for specimens that had an RNA intergrity number (RIN) that was ≥6. Intra-assay coefficient variability was performed in triplicate using six replicates and inter-assay variability was assessed in triplicate by three different technologists.

   Summary of Findings:

   At least two of the five genes targeted were detected by real-time RT-PCR in 53% of patients diagnosed with metastatic prostate cancer (51/97). Five or more CTCs per 7.5 ml of blood were detected by CellSearch in 46% of metastatic prostate patients (45/97) and 89% (40/45) of those patients also had detectable levels of two or more genes targeted by real-time RT-PCR. Of the 45 patients with ≥5 CTCs/7.5ml blood (associated with poor prognosis), KLK3 alone was detected in two patients and FOXA1 alone was detected in one patient. None of the 5 transcripts were detected for one patient with 26 CTCs/7.5ml and a serum PSA <0.05ng/ml. Importantly, 21% (11 patients) of the 52 patients with ≤4 CTCs/7.5 of blood (associated with a favorable prognosis) had detectable levels of two or more of the five genes targeted by real-time RT-PCR. For the real-time RT-PCR assay, intra- and inter-assay variability were below the acceptable threshold (CV< 15%) for all five targeted genes (CV range of .27–2.98%, P<.0001 for all).

   Biospecimens

   • Bodily Fluid - Blood

   Preservative Types

   • PAXgene
   • Other Preservative

   Diagnoses:

   • Normal
   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   RNA	Real-time qRT-PCR
   Cell count/volume	Fluorescent microscopy

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Real-time qRT-PCR Specific	Targeted nucleic acid	KLK3 KLK2 HOXB13 GRHL2 FOXA1
   Biospecimen Aliquots and Components	Cell capture method	CellSearch System for CTC Enumeration

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