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

ABSTRACT: Characterization 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.The 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.RNAs 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.This 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.

SUBMITTER: Cho WJ

PROVIDER: S-EPMC4791970 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

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

Cho Won Jin WJ Oliveira Daniel S M DS Najy Abdo J AJ Mainetti Leandro E LE Aoun Hussein D HD Cher Michael L ML Heath Elisabeth E Kim Hyeong-Reh C HR Bonfil R Daniel RD

Journal of translational medicine 20160315

<h4>Background</h4>Characterization 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 fr ...[more]

PMID: 26975354

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Project description:BackgroundMetastatic prostate cancer is a clonally heterogeneous disease state characterized by progressive somatic perturbations. The aim of this study was to identify cell free DNA- (cfDNA-) based alterations and their associations with outcomes in progressive metastatic prostate cancer.MethodsIn this longitudinal prospective cohort study plasma cfDNA/circulating tumor DNA (ctDNA) was analyzed before, during, and after androgen deprivation therapy (ADT) in 4 independent patient groups ranging from untreated metastatic hormone sensitive prostate cancer (mHSPC) to metastatic castrate resistant prostate cancer (mCRPC). Next generation sequencing was performed on ctDNA and germline DNA to characterize alterations and associations with clinical outcomes were determined for each group.FindingscfDNA yields were different in progressive mHSPC and mCRPC states (P < .001). In mHSPC, a higher than median ctDNA fraction was predictive of shorter time to ADT failure (HR, 2.29 [95% CI, 1.13-4.65]; Log-Rank P = .02). cfDNA, ctDNA taken with volume of metastatic disease in mHSPC and with alkaline phosphatase levels prognosticated survival better than clinical factors alone in mHSPC and mCRPC states (Log Rank P = 0.03). ctDNA-based AR, APC mutations were increased in mCRPC compared to mHSPC (P < ·05).TP53 mutations, RB1 loss, and AR gene amplifications correlated with poorer survival in mCRPC. Mutations in multiple DNA repair genes (ATM, BRCA1, BRCA2, CHEK2) were associated with time to ADT treatment failure and survival in mHSPC.InterpretationctDNA fraction can further refine clinical prognostic factors in metastatic prostate cancer. Somatic ctDNA alterations have potential prognostic, predictive, and therapeutic implications in metastatic prostate cancer management.FundingSeveral funding sources have supported this study. A full list is provided in the Acknowledgments. No funding was received from Predicine, Inc. during the conduct of the study.

Project description:BACKGROUND:High circulating tumor cell (CTC) counts are associated with poor prognosis in advanced prostate cancer, and recently CTC number was suggested to be a surrogate for survival in metastatic castrate-resistant prostate cancer (mCRPC). Ki67 and vimentin are well-characterised markers of tumour cell proliferation and the epithelial-mesenchymal transition (EMT), respectively. Here we asked if the expression of vimentin and Ki67 in CTCs offered prognostic or predictive information in mCRPC. METHODS:In two separate patient cohorts, anti-vimentin or anti-Ki67 antibodies were added to the free channel in the CellSearch® system for analysis of peripheral blood samples. For each cohort, association of CTC number with clinical characteristics were assessed using Fisher's exact, Mann-Whitney and chi-squared tests. Kaplan-Meier method and log-rank tests were used to analyse overall survival (OS) of vimentin-expressing and Ki67-expressing CTC patient cohorts. RESULTS:In this retrospective analysis, CTC vimentin expression was analysed in 142 blood samples from 93 patients, and CTC Ki67 expression was analysed in 90 blood samples from 51 patients. In the vimentin cohort, 80/93 (86 %) of baseline samples from patients were CTC-positive overall (?1 total CTC per 7.5 mls blood), and 30/93 (32.3 %) vimentin CTC-positive (?1 vimentin-positive CTC per 7.5 mls blood). 41/51 (80.4 %) of baseline samples from patients in the Ki67 cohort were CTC-positive overall, and 23/51 (45.1 %) Ki67 CTC-positive (?1 Ki67-positive CTC per 7.5 mls blood). There was no significant difference in baseline PSA in patients with vimentin-positive CTC at baseline versus those with no vimentin-positive CTC at baseline (p = 0.33). A significant reduction in OS was shown in patients with vimentin-positive CTC compared to those without vimentin-positive CTC (median 305 days vs 453 days, p = 0.0293). There was no significant difference in baseline PSA in patients with Ki67-positive CTC at baseline versus those without Ki67-positive CTC (p = 0.228), but OS was significantly reduced in the Ki67-positive CTC group (median 512 days vs 751 days, p = 0.0091). No changes in relative proportion of vimentin- or Ki67-positive CTCs were observed in post-treatment samples compared to baseline. CONCLUSIONS:Analysis of vimentin and Ki67 expression can straightforwardly be assessed in CTCs from patients with mCRPC. Poorer survival outcomes were observed in vimentin- and Ki67-positive CTC patients. TRANSLATIONAL STUDY PROTOCOLS:CEC-CTC (IDRCB2008-AOO585-50) and Petrus ( NCT01786031 ).

Dataset Information

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

Publications

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

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