Circulating Endothelial Progenitor Cells in Castration Resistant Prostate Cancer: A Randomized, Controlled, Biomarker Study

<div><p>Background</p><p>Endothelial progenitor cells (CEPs) and circulating endothelial cells (CECs) are potential biomarkers of response to anti-angiogenic treatment regimens. In the current study, we investigated the effect of docetaxel and sunitinib on CEP/CEC kinetics and clinical response in castration resistant prostate cancer (CRPC) patients.</p><p>Patients and methods</p><p>Chemonaive patients with CRPC were enrolled in this study to receive either sunitinib (37.5 mg/d), in combination with docetaxel (75 mg/m²) or docetaxel alone. CEP and CEC kinetics were analyzed for every cycle. The primary objective was to compare CEP/CEC pharmacodynamics between both treatment arms. We also investigated if CEC/CEP spikes, induced by MTD docetaxel, are suppressed by sunitinib in patients treated with docetaxel/sunitinib relative to docetaxel monotherapy.</p><p>Results</p><p>A total of 27 patients were enrolled. We observed a significant increase of CEP/CEC (total/viable) counts over time within each cycle (coefficients 0.29233, 0.22092 and 0.26089, respectively; p<0.001). However, no differences between the treatment groups, in terms of CEP and CEC kinetics, were detected. In the docetaxel monotherapy arm 4 (30%) patients responded to therapy with a 50% PSA decline, while 9 (64%) patients showed a PSA decline in the combination group (n.s.). The median PFS in the docetaxel monotherapy group was 3.1 months (2.6–3.6 months, 95% CI) and 6.2 months (4.9–7.4 months, 95% CI; p = 0.062) in the combination arm. Sunitinib/docetaxel was reasonably well tolerated and toxicity manageable.</p><p>Conclusion</p><p>In summary, no significant differences in CEC and CEP kinetics between the treatment arms were observed, although a highly significant increase of CEPs/CECs within each cycle over time was detected. These results mirror the challenge we have to face when employing anti-angiogenic strategies in CRPC. Additional preclinical research is needed to elucidate the underlying molecular mechanisms. However, docetaxel/sunitinib therapy resulted in a better response in terms of PSA decline and a trend towards improved PFS.</p><p>Trial Registery</p><p>clinicaltrialsregister.eu <a href="https://www.clinicaltrialsregister.eu/ctr-search/search?query=2007-003705-27" target="_blank">EudraCT 2007-003705-27</a></p></div

PFS between both treatment arms.

<p>Kaplan-Meier curves depicting progression free survival between sunitinib/docetaxel arm (orange) and docetaxel monotherapy arm (blue). Black bars represent censored patients.</p

Bone scan results.

<p>Bone scan results.</p

Patient characteristics.

<p>Patient characteristics.</p

PSA response.

<p>Waterfall plot of PSA response to docetaxel (blue) and sunitinib/docetaxel (yellow) in CRPC patients.</p

CEP and CEC kinetics.

<p>Representative example of flow cytometry dot plots chosen for CEP measurements. The left panel shows CD146 positive endothelial cells of which a small number were CD133 positive accounting for CEPs as indicated by the red arrow (right panel) (a). Regression analysis employing a linear mixed model of total CEC (b), viable CEC (c) and CEP (d) counts on a logarithmic scale of docetaxel (blue) and docetaxel/sunitinib treated patients. Each dot represents a single patient. X-axis represents cycles and time points; Y-axis represents CEP and CEC numbers on a logarithmic scale.</p

Consort flow diagram.

<p>Consort flow diagram.</p

Primary melanoma cells express ErbB3 and ErbB4 as well as the EPO-R.

<p><i>A</i>, Freshly isolated primary melanoma cells (patient #3) were stained with fluorochrome-conjugated monoclonal antibodies (mAb) against CD31, CD45, ErbB3, IGF-1-R, and CD146. Melanoma cells were gated as CD31−/CD45− cells and defined as CD146+ cells. Dot plots show expression of ErbB3 (middle panel) and IGF-1-R (right panel) on CD146+ melanoma cells. The isotype control is also shown (left panel). <i>B</i>, Melanoma cells of patient #10 were stained with mAb against CD31, CD45 and CD146 as well as EPO-R and CD24. The dot plot in the right panel shows co-expression of EPO-R and CD24 in a distinct subpopulation of (CD146+) melanoma cells. <i>C</i>, Xenotransplanted EPO-R+ melanoma cells of patient #6 were stained with biotinylated recombinant human EPO, an isotype-matched mouse IgG2b-PE antibody (left panel) and a mAb directed against the EPO-R (right panel). <i>D</i>, Melanoma cells of patient #10 were stained with mAb against CD146, an isotype-matched control antibody (left panel) and an antibody against ErbB4 (right panel). Dot plots show expression of ErbB4 in a distinct subpopulation of melanoma cells.</p

Expression of cytokine- and growth factor receptors on melanoma cells.

<p>Score of reactivity of melanoma cells with antibodies: −,<5% positive cells; −/+, weakly expressed on a subset; +/−, 6–20%; +, 21–60%, and ++, 61–100% of cells positive.</p>a<p>Number of donors (n) analyzed.</p>b<p>Cells were obtained from tumors grown in NSG mice injected with unfractionated patient-derived melanoma cells.</p><p>Abbreviations: VEGF-R2, vascular endothelial growth factor receptor-2; EPO-R, erythropoietin receptor; IGF-I-R, insulin-like growth factor I receptor; G-CSF-R, granulocyte colony-stimulating factor receptor; GM-CSF-R, granulocyte/macrophage colony-stimulating factor receptor; M-CSF-R, macrophage colony-stimulating factor receptor; SCF, stem cell factor; IL-3-RA, interleukin-3 receptor alpha chain; FLT-3, FMS-like tyrosine kinase 3; n.t., not tested.</p

IC₅₀ values (µM) for the inhibition of the cell survival (MTT assay) in human melanoma cells by targeted drugs.

<p>Melanoma cells were cultured in complete medium at 37°C in the absence or presence of various concentrations of targeted drugs for 48 hours. Then, cell viability was examined by MTT assay as described in the text.</p>*<p>xenotransplanted patient-derived melanoma cells from patient #6 and #7; n.t., not tested.</p