Gene expression analysis of bone metastasis and circulating tumor cells from metastatic castrate-resistant prostate cancer patients

Abstract

BackgroundCharacterization of genes linked to bone metastasis is critical for identification of novel prognostic or predictive biomarkers and potential therapeutic targets in metastatic castrate-resistant prostate cancer (mCRPC). Although bone marrow core biopsies (BMBx) can be obtained for gene profiling, the procedure itself is invasive and uncommon practice in mCRPC patients. Conversely, circulating tumor cells (CTCs), which are likely to stem from bone metastases, can be isolated from blood. The goals of this exploratory study were to establish a sensitive methodology to analyze gene expression in BMBx and CTCs, and to determine whether the presence or absence of detectable gene expression is concordant in matching samples from mCRPC patients.MethodsThe CellSearch® platform was used to enrich and enumerate CTCs. Low numbers of PC3 prostate cancer (PCa) cells were spiked into normal blood to assess cell recovery rate. RNA extracted from recovered PC3 cells was amplified using an Eberwine-based procedure to obtain antisense mRNA (aRNA), and assess the linearity of the RNA amplification method. In this pilot study, RNAs extracted from CTCs and PCa cells microdissected from formalin-fixed paraffin-embedded BMBx, were amplified to obtain aRNA and assess the expression of eight genes functionally relevant to PCa bone metastasis using RT-PCR.ResultsRNAs were successfully extracted from as few as 1–5 PCa cells in blood samples. The relative expression levels of reference genes were maintained after RNA amplification. The integrity of the amplified RNA was also demonstrated by RT-PCR analysis using primer sets that target the 5′-end, middle, and 3′-end of reference mRNA. We found that in 21 out of 28 comparisons, the presence or absence of detectable gene expression in CTCs and PCa cells microdissected from single bone lesions of the same patients was concordant.ConclusionsThis exploratory analysis suggests that aRNA amplification through in vitro transcription may be useful as a method to detect gene expression in small numbers of CTCs and tumor cells microdissected from bone metastatic lesions. In some cases, gene expression in CTCs and BMBxs was not concordant, raising questions about using CTC gene expression to make clinical decisions.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-016-0829-5) contains supplementary material, which is available to authorized users.

Concise Review: Prostate Cancer Stem Cells: Current Understanding

“…Yes [244,251] Yes [251] Yes, reflection of tumor evolution [160,161,252] Yes (159,135,(151)(152)(153) Yes [243,244,253] ©AlphaMed Press 2018 STEM CELLS example: (a) to predict tumor development; (b) to identify patients with a poor prognosis who can potentially benefit from therapy intensification; (c) to visualize the most critical intratumoral areas containing CSCs that should be taken into account for the local therapy planning (surgery, radiotherapy); (d) to monitor an efficacy of therapeutic approaches administered in cancer patients; (e) to diagnose local or distant tumor recurrences after therapy; and (f ) to develop novel specific anti-CSC therapeutics to effectively destroy the most aggressive and treatment-resistant CSCs. Although a number of strategies for PCSC enrichment and analysis are considered in this review article, there are no specific PCSC markers known up to date which are already implemented in clinical practices.…”

Improving the CellSearch® system

“…The same procedure was also used by others [37,49,77,78]. Cho et al are using a linear T7 preamplification step for the RNA to compare the profile kit CTC mRNA to mRNA from microdissected single bone lesions [79]. They found a high concordance between presence and absence of detectable gene expression in both fractions.…”

The value of biomarkers in bone metastasis

“…A novel line of research investigated the prognostic and predictive role of circulating tumour cells (CTCs) in PC patients. Different methods were used to isolate CTCs from the peripheral blood and the highest efficiency was obtained in the presence of clinically evident BM, suggesting the potential role of CTCs as a prognostic means for patient stratification but not a valid instrument for BM prediction (Cho et al., ; Danila et al., ; Helo et al., ).…”