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Tumor vascularity in prostate cancer: an update on circulating endothelial cells and platelets as noninvasive biomarkers In order to individually tailor prostate cancer (PCa) treatment, clinicians need better tools to predict prognosis and treatment response. Given the relationship between angiogenesis and cancer progression, circulating endothelial cells (CECs) and their progenitors have logically been proposed as potential biomarkers. The utility of their baseline levels and kinetics has been investigated for years. However, owing to a lack of standardization and validation of CEC and circulating endothelial progenitors enumeration protocols, results have been inconsistent in prostate and other cancers. Similarly, platelets play a significant part in cancer progression, yet the role of platelet-related biomarkers in PCa is unclear. While there have been a number of theoretically interesting platelet-related markers proposed, limited research has been conducted in PCa patients. Currently, CECs and platelets do not have a clear role as biomarkers in routine PCa care. Given the theoretical merits of these cells, prospective trials are warranted. KEYWORDS: angiogenesis n biomarker n cancer n circulating endothelial cell n platelet n progenitor n prostate
Prostate cancer (PCa) remains the most com monly diagnosed cancer in men, accounting for 28% of new cancer diagnoses in the USA. Although PCa death rates are declining, it remains the second leading cause of cancer death in men, contributing 10% of all male cancer deaths and an estimated 29,720 deaths in the USA alone [1]. Clinicians need to be able to accurately pre dict PCa prognosis and treatment response in order to further reduce PCa-specific mortality while avoiding overtreatment. Identification of when to intervene, and in which patients, is one of the great challenges of PCa management. PCa is now commonly detected at an early stage [2]. For many patients the risk of disease progression is low, and they will be more likely to die from non-PCa causes [3]. Currently, PSA, Gleason score and clinical stage are the main parameters used to predict risk and inform treatment deci sions. Identifying which cancers will progress remains challenging. Given the important role of tumor vascular ity (angiogenesis) in cancer, much research has gone into the investigation of surrogate mark ers of angiogenesis and tumor vascularity as biomarkers of prognosis and treatment response.
dormancy to outgrowing vascularized tumor occurs when the balance tips in favor of angio genesis. This is referred to as the ‘angiogenic switch’ and is controlled by both antiangiogenic and proangiogenic regulators [6]. Neovasculature can arise from the sprouting of new vessels from existing ones (angiogenesis), or de novo vessel formation from circulating endothelial precursor cells (vasculogenesis) [4]. Tumor vessels are characteristically hetero geneous, in contrast to normal mature blood vessels [7].
Angiogenesis Tumors require an increased blood supply for growth. Inducing angiogenesis is one of the hallmarks of cancer [4] and a critical mechanism behind tumor dormancy [5]. The transition from
PCa vascularity Tumor vascularity in PCa has been linked to dis ease aggressiveness. Microvessel density (MVD) has been used as a histological marker of cancer vasculature. However, correlation of clinico pathological parameters with MVD has been variable across published studies. Tumor vascu larity also has implications for antiangiogenesis treatment effect; highly vascularized tumors are more responsive [8]. MVD can be calculated using analysis of vas cular ‘hot spots’, random area selection, larger representative areas of the specimen or even whole-specimen analysis. These differing meth ods have been held responsible for the variability of results of MVD in cancer research. Several investigators have attempted to reduce bias by using automated analysis [9–11]. Aggressive prostate tumors are seen to form vessels primitive in morphology and function.
10.2217/BMM.13.100 © 2013 Future Medicine Ltd
Biomarkers Med. (2013) 7(6), 879–891
Fairleigh Reeves*1, Nikhil Sapre1, Niall Corcoran1,2 & Christopher Hovens1,2 Department of Urology & Surgery, University of Melbourne, Level 3 Centre, Royal Melbourne Hospital, Parkville, VIC 3050, Australia 2 Australian Prostate Cancer Research Center, Epworth Hospital, Richmond, VIC, Australia *Author for correspondence: Tel.: +61 393 427 294 Fax: +61 393 428 928 [email protected] 1
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Poorly differentiated tumors have greater MVD, irregularity of vessel lumen and smaller vessels. In addition, tumors exhibiting the smallest ves sel diameter or the most irregularly shaped ves sels have been associated with the development of lethal disease [12]. In a recent study of PCa xenograft tumors, MVD correlated with nuclear pleomorphism, mitotic count and vascular inva sion. In human specimens in the same study, no association was seen with vascular invasion. However, the sample size of ten was probably insufficient to detect any correlation [13]. Tumors in different zones of the prostate may behave differently with respect to microvascular parameters. Peripheral zone PCa has been shown in several studies to have an increased vascular ity compared with transition zone tumors [14] or benign prostatic tissue [10,11,14]. However, this finding of increased MVD is not consis tent across all studies [9]. By contrast, transition zone tumors display a large variability in micro vascular parameters. They can be both hypoor hyper-vascularized compared with normal transition zone tissue [11]. Despite these differences, several studies have failed to find any significant association between MVD and other clinicopathological parameters, such as Gleason score, tumor grade or PSA [11,15]. Also, in a study using a tissue microarray from 3261 radical prostatectomy specimens, MVD failed to provide an independent prognostic fac tor when combined with standard predictors in a multivariable analysis [14]. There is no consensus about the utility of MVD as a prognostic marker in PCa. It also requires histological specimens, ideally at pros tatectomy, as the significance of MVD measured in biopsy specimens is unclear [14]. Therefore, MVD has a limited application in the clinical setting.
Circulating endothelial cells & circulating endothelial progenitors Circulating endothelial cells (CECs) and circu lating endothelial progenitors (CEPs) comprise subsets of cells that are functionally and pheno typically different. Both reflect angiogenesis and have been heralded as promising noninvasive biomarkers for the prediction of prognosis and evaluation of treatment response in cancer. CECs are mature, terminally differentiated cells that are shed from the vessel wall into the circulation in response to injury or as a result of endothelial dysfunction. Increased levels of CECs have been reported in a wide range of 880
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pathological processes that involve vascular injury, including cardiovascular disease, stroke, diabetes, chronic renal failure and vasculitis [16]. A proportion of CECs are apoptotic or necrotic and some studies have described subpopula tions of CECs as resting or activated. Interac tion with apoptotic or necrotic CECs induces a proinflammatory response in healthy endothe lial cells [17], which may alter the homeostasis of the vessel wall. However, the precise role of CECs in malignancy is unclear. At this stage, CECs are best understood as a product of vas cular turnover and, as such, a surrogate marker of angiogenesis. There has been much interest in endothelial progenitor cells (EPCs) since they were first iso lated in peripheral blood by Asahara and col leagues in 1997 [18]. Unlike CECs, CEPs are mobilized from bone marrow (BM). A popula tion of CEPs have clonogenic and proliferative potential (endothelial colony-forming cell) [19]. CEPs are relatively rare in healthy individuals. The role of CEPs in promoting angiogenesis in tumor growth is contentious. Within the vessel wall, BM-derived EPCs are thought to merge and differentiate into endothelial cells [20]. How ever, there are variable reports regarding the extent of CEP-derived vessels in cancer. Nolan and colleagues propose that the heterogeneity of results can be attributed in part to the changing relative contribution of CEP-derived vessels dur ing different stages of tumor development [20]. Using genetically marked BM progenitor cells they demonstrated that early tumors showed a marked recruitment of EPCs in the tumor periphery, followed by endothelial cell luminal incorporation into a subset of sprouting neoves sels. In established tumors, host-derived vessels diluted the EPCs. The authors suggested that this temporal course of EPC involvement is the reason why other studies have previously reported a low contribution of EPCs to tumor neovascularization. Another study reported that 12% of neovessels in metastatic disease showed luminal incorporation by BM-derived endothe lial cells [21]. More recently, Wickersheim and colleagues reported that although local VEGF production induces massive infiltration of BMderived EPCs, confocal microscopy and 3D reconstruction showed that these cells failed to incorporate into the vessel wall to any significant extent. As such, they concluded that vasculariza tion in tumor progression occurs primarily via angiogenesis rather than vasculogenesis [22]. Sim ilarly, Purhonen and colleagues demonstrated a lack of BM-derived EPC incorporation after future science group
Tumor vascularity in prostate cancer: circulating endothelial cells & platelets as noninvasive biomarkers
VEGF stimulation [23]. However, this study has been heavily criticized for its methodology [24]. BM-derived EPCs have also been shown to be involved in rebound revascularisation after some treatments. Certain chemotherapy drugs given at or near maximal tolerated dose (particu larly paclitaxel, 5-FU and docetaxel) induce a rapid mobilization of BM-derived CEPs, evident within 24 h of a single bolus injection. Treat ing with targeted antiangiogenic drugs prior to administration of these chemotherapy agents prevented acute mobilization and enhanced treatment efficacy [25]. CEPs have also been implicated in metasta sis development. Gao and colleagues describe BM-derived EPCs as critical regulators of the angiogenic switch responsible for mediating progression of micrometastases to lethal macro metastases. In preclinical lung metastasis models, micrometastases were largely avascular whereas macrometastases demonstrated neovessel infil tration. Blocking EPC mobilization resulted in reduced vessel density in metastatic lesions and impaired formation of lethal macrometastases [21].
Enumeration of CECs & CEPs Enumeration of CECs and CEPs is challeng ing for a number of reasons. The critical bar rier is that no one marker can uniquely identify CECs and CEPs, and there is no agreement on which combination of markers can iden tify them reliably (Table 1). Most commonly, CECs and CEPs are enumerated using flow cytometry [26] or immunomagnetic separation [27,28]. Multiparametric flow cytometry allows simultaneous identification of different anti gens. However, there is currently no consensus definition for CEC or CEP phenotypes. CECs have been characterized by a lack of leukocyte (i.e., CD45) and progenitor cell mark ers, and expression of endothelial markers such as vWF, CD31 or CD146 [27]. Other endothe lial markers that have been used include CD34 [29] and CD309 (VEGFR2/KDR) [30]. Unfor tunately, more recent evidence suggests that CD146 is not uniquely expressed by endothelial cells [31] and that CD309 is not a reliable marker in CEP enumeration [32,33]. Activated CECs have been identified by the expression of CD106 [34] or CD105 (Endoglin) [19,35,36]. DNA staining has been used to enumer ate viable, apoptotic and dead CECs [37]. Pro genitor markers used to identify CEPs include CD117 [37] in mice and CD133 [38] in humans. However, in humans, CD133 is also expressed by other hematopoietic stem cells [39]. future science group
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There is concern that current methods to identify CECs and CEPs may in fact capture a broader cell population. In recognition of this, Strijbos and colleagues set out to confirm the endothelial nature of cells meeting their immunophenotypic criteria for CECs (low-tointermediate forward light scatter, low sideways light scatter, CD31+, CD45-). They found that the cells thought to be CECs failed to express the endothelial marker CD146. Upon electron microscopy they also lacked endothelial cellspecific Weibel–Palade bodies, instead display ing morphological characteristics consistent with large platelets [30]. More recently, upon further analysis of CD31+ CD45- cells (thought to be CECs), Wong and colleagues revealed positive expression of platelet-specific markers (CD41a and CD42b). Wong and colleagues reported that these cells are more likely a subtype of plate lets rather than CECs [40]. For this reason, the use of a cell viability stain and DNA-specific staining has been advocated to discriminate DNA-containing CECs from DNA-free CECderived macroparticles and platelets [41,42]. Another study revealed that cells thought to be EPCs were in fact primitive hematogenous progenitors [43]. Despite this, subsequent stud ies have been published using this phenotypic definition for EPCs [44]. Another problem is the lack of validated pro tocols to identify CECs/CEPs. Different flow cytometry practices and different antibodies are used across studies. Although enumeration methods are often based on previously published protocols [30,37,40], this review identified no recent studies in PCa in which the CEC or CEP enu meration protocol had been formally validated in independent cohorts. Even in nonprostate cohorts, few studies have been validated. The use of diverse nonvalidated enumeration protocols, with different flow cytometry practices and anti bodies, probably contributes to the variability of reported results. Individual protocols are generally reported to be reproducible. Mancuso and colleagues veri fied the reproducibility of their CEC enumera tion protocol with nine rounds of intrareader and inter-reader variability studies [42]. In addi tion, they confirmed the cells to be endothelial in nature by the presence of Weibel–Palade bod ies upon electron microscopy. However, the pro tocol was not validated in an independent cohort [42]. Despite reassuring single-study validation, cross-study correlation of different methods is moderate to poor [26]. Therefore, one must refrain from comparing results from studies with www.futuremedicine.com
881
882 -
Mancuso et al. (2009)
Biomarkers Med. (2013) 7(6)
+
+
+ Intermediate
-
+
+
+
+
+
CD105
CD106
-
CD117
+
-
-
CD133
-
+
+
+
+
+
+
+
+
+
+
+
+
CD146
+
+
-
CD309 (VEGFR2/KDR)
Flk1+
Other
7AAD +/-
DAPI +
[40]
[55]
[51]
[40]
[37]
[35]
[34]
[48]
[50]
[30]
[28]
[42]
[47] +
[49] +
Syto16 7AAD +/-
DAPI
DAPI
[38]
[52]
[40]
[59]
[55]
+
7AAD -
DAPI
+
Nuclear Ref. stains
Blank cells represent not applicable. † Activated CEC. +: Positive for the specified marker in the studies described in the table; -: Negative for the specified marker in the studies described in the table; 7AAD: 7-aminoactinomycin-D; CEC: Circulating endothelial cell; CEP: Circulating endothelial progenitor; DAPI: 6-diamidino-2-phenylindole.
Wong et al. (2012)
Blann et al. (2011)
Human prostate cancer
CEPs
Namdarian et al. (2010) -
-
Wong et al. (2012)
Animal nonprostate cancer
-
-
-
-
-
Li et al. (2008)
Animal prostate cancer
+
+
Ronzoni et al. (2010)
Wang et al. (2013)
+
Ramcharan et al. (2013)
†
+
-
Strijbos et al. (2007)
Rigolin et al. (2010)
-
Simkens et al. (2010)
+
-
-
Kondo et al. (2012)
+
-
+
+
Kawaishi et al. (2009)
Goon et al. (2009)
Brunner et al. (2008)
Human nonprostate cancer
-
-
CD45
Wong et al. (2012)
+
CD34
-
+
CD31
Strijbos et al. (2010)
Blann et al. (2011)
Human prostate cancer
CECs
Study (year)
Table 1. Phenotypic definitions used to identify circulating endothelial cells and circulating endothelial progenitors.
Review Reeves, Sapre, Corcoran & Hovens
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CD31
future science group
+ +
Rigolin et al. (2010)
Ronzoni et al. (2010)
www.futuremedicine.com
+
CD117
CD133
+
+
+
+
+
+
+
+
CD146
+
+
+
+
CD309 (VEGFR2/KDR)
Flk1+
CD3CD19 ‑ CD33-
Other
Syto16
7AAD -
+
Nuclear stains
Blank cells represent not applicable. † Activated CEC. +: Positive for the specified marker in the studies described in the table; -: Negative for the specified marker in the studies described in the table; 7AAD: 7-aminoactinomycin-D; CEC: Circulating endothelial cell; CEP: Circulating endothelial progenitor; DAPI: 6-diamidino-2-phenylindole.
Namdarian et al. (2010) Intermediate
-
Wong et al. (2012)
Animal nonprostate cancer
-
-
-
-
-
Li et al. (2008)
+
+
Ramcharan et al. (2013)
Animal prostate cancer
+
Case et al. (2007)
Mancuso et al. (2009)
+
CD106
+
CD105
Goon et al. (2009) -
CD45
+
+
CD34
Brunner et al. (2008)
Human nonprostate cancer
CEPs (cont.)
Study (year)
Table 1. Phenotypic definitions used to identify circulating endothelial cells and circulating endothelial progenitors (cont.).
[51]
[40]
[37]
[34]
[50]
[48]
[43]
[42]
[38]
[52]
Ref.
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differing methodology unless there is evidence to support their validity. Another difficulty with studies using CECs/CEPs is the need for fresh samples. In particular, CEP enumeration needs to occur soon after collection as counts decrease after storage for 24 h [33]. The implication of this is that these cells can only be effectively evalu ated in prospective studies, rather than utiliz ing established tissue banks. This is particularly time consuming if investigators are attempting to compare CEC/CEP levels with long-term clinical outcomes in order to evaluate prognos tic ability. Mancuso and colleagues evaluated the reliability of frozen sample analysis of CEC count through the analysis of fresh and frozen samples evaluated at different time points. The coefficient of variation for variability over time in fresh samples (analyzed 0–72 h after collection) was 17 ± 7% and frozen samples (thawed and analyzed after 0–14 days frozen storage) was 26 ± 16%. The issue of reproducibility of CEC/CEP analysis from specimens in long-term storage was also investigated. In samples that were sent inter nationally and assessed after more than 6 months of storage, the coefficient of variation associated with duplicate reading was 22 ± 12% [42]. Hence, the technical limitations of performing prospec tive trials continue to hamper the field, preclud ing a rigorous assessment of the roles of these cells in tumor outcome prognosis. There are many issues with current enumera tion techniques. The main factor limiting the progression of quality research in this area is the lack of consensus definitions of CEC/CEP phenotypes. Validated protocols need to be estab lished that reliably identify these cells without erroneously including a broader cell population. Even if this is achieved, researchers will still need to contend with the challenge of requiring fresh samples for analysis.
CEC & CEP counts in PCa & other malignancies Very few studies have evaluated CEC and CEP in PCa in the last 5 years. Given the relationship between various vascular parameters and tumor aggressiveness or lethality in PCa, the develop ment of noninvasive markers of angiogenesis is of great relevance. The lack of research in this area probably reflects the methodological challenges of enumeration of these cells rather than a lack of scientific interest. CECs/CEPs have been investigated in a wide range of different cancers, and other reviews have considered CECs/CEPs in this context [45,46]. 884
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This review does not aim to give a thorough insight into CECs/CEPs in all types of cancer, and instead draws on a selection of recent studies to illustrate the heterogeneity of evidence and give further insight into the understanding of the controversies and challenges of CEC/CEP use as biomarkers. Importantly, when consid ering CECs/CEPs in other cancers, it is diffi cult to make simplistic comparisons with PCa. Although angiogenesis is a feature common to many tumor types, PCa has other features that make it unique, including hormone dependence. PCa is also generally slow growing, and as it has a reasonably good progression marker in PSA, it may be monitored for long periods without aggressive treatment. These differences may translate into differences in angiogenic require ments and consequently patterns of CEC/CEP levels. Baseline CEC levels in malignancy Baseline CEC levels have been shown to be elevated in patients with a variety of cancers in comparison with healthy controls, including pancreatic cancer [47], colorectal cancer (CRC) [48] [34], breast cancer [38], non-small-cell lung cancer (NSCLC) [49] and chronic lymphocytic leukemia (CLL) [50]. More specifically, activated CECs were increased in patients with NSCLC compared with healthy volunteers [35]. Simi larly, in human renal cell carcinoma xenograft models, CEC levels were significantly elevated in tumor-bearing mice compared with healthy controls [51]. In patients with primary breast cancer, CEC level correlated with tumor invasiveness and size, and positively predicted Nottingham Prognostic Index scores [38]. In primary CRC, a stepwise increase in CEC and EPC levels significantly cor related with increasing Dukes’ stage. However, upon multiple regression analysis, they failed to remain significant, and there was no signifi cant correlation between CEC or CEP level and American Joint Committee on Cancer (AJCC) stage [48]. Another study in CRC showed a posi tive correlation between ‘total’ and ‘resting’ CEC levels and the number of sites of metastasis [34]. No correlation between CEC level and dis ease stage was seen in CLL [50] or primary head and neck squamous cell carcinoma (SCC) [52]. In patients with head and neck SCC, there was no difference in CEC levels between patients and control [52]. Lower levels of mature CECs have been reported in pediatric patients with malignant solid tumors compared with controls [53]. future science group
Tumor vascularity in prostate cancer: circulating endothelial cells & platelets as noninvasive biomarkers
Although CEC levels are reported to be higher in various malignancies (compared with healthy controls), this is not consistently reported across all studies.
Baseline CEC & CEP levels in PCa The significance of CEC and CEPs in PCa has been questioned owing to the lack of correla tion between their levels and accepted clinical markers. Blann and colleges reported no significant difference in CECs and CEPs in patients with biopsy-proven PCa, benign prostate disease or no prostate disease. Also, there was no signifi cant correlation between CEC or CEP level and PSA, Gleason score or other plasma markers (vWf and soluble E‑selectin) [55]. In the preclinical arm of the study by Wong and colleagues, levels of CD31+ CD45 - cells (thought to be CECs) were significantly elevated in tumor-bearing mice. These correlated with tumor size, MVD and volume. With respect to CEPs, there was no significant difference in CEP levels in any of the cell lines in the xenograft models compared with controls. In the clinical arm of the study, which evaluated CECs/CEPs in patients with localized PCa undergoing prosta tectomy, there was no correlation between CEC number and stage, Gleason score, extraprostatic extension or preoperative PSA [40]. There is currently no evidence to support the use of CEC/CEP enumeration in diagnosis of PCa.
shorter time to first treatment and were more likely to have a worse treatment response [50]. Similarly, elevated baseline CEC levels in advanced primary NSCLC [36] and pancreatic cancer [47] was associated with a poorer overall survival (OS). By contrast, other studies reported favor able outcomes in patients with higher CEC counts. In patients with advanced breast can cer treated with metronomic chemotherapy plus bevacizumab, higher baseline CEC levels were associated with increased time to progres sion. At the time of progression, the number of CECs was significantly reduced compared with baseline. The authors hypothesized that this may represent an shift to an alternative type of vascularization [56]. In NSCLC there was no significant correlation between base line CEC count and estimated tumor volume. However, NSCLC patients with a higher base line CEC count showed longer progressionfree survival (PFS) and improved clinical benefit from carboplatin plus paclitaxel-based chemotherapy [49]. Some studies failed to find an association between CEC level and survival. CEC baseline level in a study of various malignancies treated with chemotherapy was not associated with PFS or OS [57]. Similarly, CEC level failed to corre late with response to radiotherapy or survival in patients with head and neck SCC [52]. Varying reports have been published in CRC. One study in CRC failed to find a correlation between baseline CEC and PFS or OS [28], while another study of metastatic CRC patients found that those with the lowest ‘total’ and ‘resting’ CEC levels at baseline showed a longer PFS [34]. Their conflicting results have been attributed to their differing techniques for CEC enumera tion [28]. In a trial of CRC patients treated with FOLOX4 plus bevacizumab, those with 65 or more CECs at baseline had significantly shorter median PFS and OS. Interestingly, there was no similar correlation seen in those treated with FOLFOX4 alone [58].
Baseline CEC levels as predictor of treatment response & survival in malignancy Although some studies have described the value of baseline CEC levels in predicting treatment outcomes and prognosis, the evidence remains unclear. In some studies, higher CEC levels were asso ciated with more aggressive disease. In patients with CLL, those with higher CEC levels had a
Baseline CEC levels as a predictor of treatment response & survival in PCa There is currently very limited research pertain ing to CECs/CEPs as predictors of treatment response and survival in patients with PCa. In the clinical arm of Wong and colleagues’ study, men who experienced treatment failure within the first year had significantly higher CD31+ CD45- levels. The authors hypothesized that higher CD31+ CD45- levels may be a marker
Baseline CEP levels in malignancy Similarly, conflicting evidence exists regarding CEP levels in patients with cancer. CEPs have been shown to be in higher numbers in patients with primary gynecological cancer (ovarian or cervical) [54], head and neck SCC [52], CRC [48] and CLL [50], compared with controls. Con versely, no correlation was found with baseline CEPs and metastatic CRC [34]. Circulating pro genitor cells were lower in patients with breast cancer than matched controls [38].
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of micrometastatic disease in localized PCa, and that in patients with early cancer recurrence, cancer had already spread prior to surgery. Fur ther characterization of these cells revealed that they represented a subpopulation of circulating platelets rather than CECs, highlighting the challenges in interpreting evidence in this area [40]. With respect to survival in patients with castration-resistant PCa, baseline CEC level is not associated with OS [59].
CEC & CEP kinetics in cancer treatment Shifting CEC and CEP levels in response to cancer treatment are presumably due to mobi lization of either angiogenic or vasculogenesis processes induced by acute cytotoxic stress, although definitive data pertaining to the mechanism remain lacking. CEC kinetics in other malignancies There have been a number of clinical studies in cancer that showed no significant change in preand post-treatment CEC levels. These include studies in patients with advanced NSCLC (receiving first-line chemotherapy) [36], patients with primary head and neck SCC (receiving surgery, chemoradiation or radiotherapy alone) [52], pancreatic cancer (treated with gemcitabine chemotherapy for locally advanced disease, metastases or recurrence) [47] and various pedi atric malignant solid tumors [53]. Furthermore, in pancreatic cancer, the CEC count change from baseline was not associated with tumor response [47]. In a few studies, CEC levels are reported to change in response to treatment, but there is no consistent evidence regarding how CEC kinetics relate to prognosis. Breast cancer patients treated with first-line chemotherapy plus bevacizumab had a signifi cant increase in CECs following treatment. This increase was associated with improved time to progression when a threshold of 20 CECs per 4 ml was used [60]. In a study that included patients with vari ous malignancies treated with (neo)adjuvant chemotherapy or chemotherapy for metastatic disease, increases in CECs and EPCs were seen after the first cycle of chemotherapy. In some patients this was evident as early as 4 h after treatment. A large increase of CEC levels 7 days after chemotherapy was significantly associated with poor PFS, and at day 21 a large change in CEC level correlated with both PFS and OS. Upon univariate analysis, EPC levels were not 886
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significantly associated with PFS or OS at days 7 and 21 [57]. In 473 patients with advanced CRC treated with first-line chemotherapy and bevacizumab, there was a significant increase in CEC after 1–2 weeks of treatment; however, changes in CEC levels did not predict PFS or OS at any time point [28]. CECs were reported to decrease in NSCLC patients following carboplatin plus paclitaxel-based chemotherapy [49]. In another study in patients with NSCLC, inverse correlations existed between changes in activated CECs following treatment and PFS [35]. CEP kinetics in other malignancies In a study of various malignancies treated with chemotherapy, only those treated with taxanebased chemotherapy showed an immediate increase in EPCs. After 21 days, EPC levels were consistently increased irrespective of chemo therapy type. EPC levels were not significantly associated with PFS or OS at day 7 or 21 [57]. In a small study of cancer patients receiving an antivascular agent plus cisplatin, CEP lev els increased following treatment. The authors hypothesized that vascular trauma as a result of treatment may cause upregulation of angiogenic factors that promote mobilization of BM-derived EPCs [61]. By contrast, the number of CEPs declined fol lowing chemoradiation or surgical treatment of ovarian or cervical cancers [54]. CEC & CEP kinetics in PCa treatment CECs have been reported to increase in response to treatment in both xenographt [37] and clinical studies [59,62] of PCa. Li and colleagues compared viable CECs, dead/apopototic CECs and CEP response to thalidomide, docetaxel or a combina tion of the two drugs in a human PCa xenograft model [37]. Apoptotic/dead CECs were measured at baseline as well as 4 and 17 days into treatment. Increased levels of apoptotic/dead CECs were observed after treatment with docetaxel. Addition of thalidomide further increased apoptotic/dead CEC levels. Interestingly, the increase in CECs, both viable and apoptotic/dead, was greatest at 4 days compared with 17. To ensure that the CEC variation did not reflect nonspecific cyto toxicity of the chemotherapy agents used, Li et al. measured CEC levels in response to cisplatin (a chemotherapy drug with no antiangiogenic activ ity), which revealed no variation. There was no statistically significant change in CEP levels in response to treatment. future science group
Tumor vascularity in prostate cancer: circulating endothelial cells & platelets as noninvasive biomarkers
Strijbos and colleagues studied CECs, circu lating tumor cells, TF‑1 and ET‑1 as prognostic indicators in 162 patients with castration-resis tant PCa treated with a docetaxel-containing chemotherapy regimen [59]. They found no sig nificant association between the markers assessed. However, a significant increase in CEC levels was seen 2–5 weeks after initiation of docetaxel. A 3.8-fold or higher increase in CEC from baseline at 2–5 weeks was associated with a significantly worse OS. There was no significant increase found at the second follow-up measurement at 6–8 weeks. This is in keeping with Simkens and colleagues’ study in advanced CRC [28], which showed no further increase in CEC after that observed at 1–2 weeks after treatment. It is hypothesized that the rise in CECs is due to vascular damage inflicted by docetaxel, and that in patients with a poorer OS it may be due to continued endothelial shedding from vessels in tumors progressing during treatment. Strijbos also highlighted the need for prospective studies to confirm these findings, as the study was only exploratory [30]. In a Phase II trial of bevacizumab, thalidomide, docetazel and prednisolone in patients with meta static castration-resistant PCa, circulating apop totic endothelial cells (CAECs) were a secondary outcome measure [62]. Levels were measured at baseline and six weeks. A strong inverse correla tion between relative change in PSA over 6 weeks and the absolute difference in CAECs was dem onstrated. Patients with a 75% or greater decline in PSA had a significant increase in CAEC levels compared with those with a
Tumor vascularity in prostate cancer: an update on circulating endothelial cells and platelets as noninvasive biomarkers In order to individually tailor prostate cancer (PCa) treatment, clinicians need better tools to predict prognosis and treatment response. Given the relationship between angiogenesis and cancer progression, circulating endothelial cells (CECs) and their progenitors have logically been proposed as potential biomarkers. The utility of their baseline levels and kinetics has been investigated for years. However, owing to a lack of standardization and validation of CEC and circulating endothelial progenitors enumeration protocols, results have been inconsistent in prostate and other cancers. Similarly, platelets play a significant part in cancer progression, yet the role of platelet-related biomarkers in PCa is unclear. While there have been a number of theoretically interesting platelet-related markers proposed, limited research has been conducted in PCa patients. Currently, CECs and platelets do not have a clear role as biomarkers in routine PCa care. Given the theoretical merits of these cells, prospective trials are warranted. KEYWORDS: angiogenesis n biomarker n cancer n circulating endothelial cell n platelet n progenitor n prostate
Prostate cancer (PCa) remains the most com monly diagnosed cancer in men, accounting for 28% of new cancer diagnoses in the USA. Although PCa death rates are declining, it remains the second leading cause of cancer death in men, contributing 10% of all male cancer deaths and an estimated 29,720 deaths in the USA alone [1]. Clinicians need to be able to accurately pre dict PCa prognosis and treatment response in order to further reduce PCa-specific mortality while avoiding overtreatment. Identification of when to intervene, and in which patients, is one of the great challenges of PCa management. PCa is now commonly detected at an early stage [2]. For many patients the risk of disease progression is low, and they will be more likely to die from non-PCa causes [3]. Currently, PSA, Gleason score and clinical stage are the main parameters used to predict risk and inform treatment deci sions. Identifying which cancers will progress remains challenging. Given the important role of tumor vascular ity (angiogenesis) in cancer, much research has gone into the investigation of surrogate mark ers of angiogenesis and tumor vascularity as biomarkers of prognosis and treatment response.
dormancy to outgrowing vascularized tumor occurs when the balance tips in favor of angio genesis. This is referred to as the ‘angiogenic switch’ and is controlled by both antiangiogenic and proangiogenic regulators [6]. Neovasculature can arise from the sprouting of new vessels from existing ones (angiogenesis), or de novo vessel formation from circulating endothelial precursor cells (vasculogenesis) [4]. Tumor vessels are characteristically hetero geneous, in contrast to normal mature blood vessels [7].
Angiogenesis Tumors require an increased blood supply for growth. Inducing angiogenesis is one of the hallmarks of cancer [4] and a critical mechanism behind tumor dormancy [5]. The transition from
PCa vascularity Tumor vascularity in PCa has been linked to dis ease aggressiveness. Microvessel density (MVD) has been used as a histological marker of cancer vasculature. However, correlation of clinico pathological parameters with MVD has been variable across published studies. Tumor vascu larity also has implications for antiangiogenesis treatment effect; highly vascularized tumors are more responsive [8]. MVD can be calculated using analysis of vas cular ‘hot spots’, random area selection, larger representative areas of the specimen or even whole-specimen analysis. These differing meth ods have been held responsible for the variability of results of MVD in cancer research. Several investigators have attempted to reduce bias by using automated analysis [9–11]. Aggressive prostate tumors are seen to form vessels primitive in morphology and function.
10.2217/BMM.13.100 © 2013 Future Medicine Ltd
Biomarkers Med. (2013) 7(6), 879–891
Fairleigh Reeves*1, Nikhil Sapre1, Niall Corcoran1,2 & Christopher Hovens1,2 Department of Urology & Surgery, University of Melbourne, Level 3 Centre, Royal Melbourne Hospital, Parkville, VIC 3050, Australia 2 Australian Prostate Cancer Research Center, Epworth Hospital, Richmond, VIC, Australia *Author for correspondence: Tel.: +61 393 427 294 Fax: +61 393 428 928 [email protected] 1
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Poorly differentiated tumors have greater MVD, irregularity of vessel lumen and smaller vessels. In addition, tumors exhibiting the smallest ves sel diameter or the most irregularly shaped ves sels have been associated with the development of lethal disease [12]. In a recent study of PCa xenograft tumors, MVD correlated with nuclear pleomorphism, mitotic count and vascular inva sion. In human specimens in the same study, no association was seen with vascular invasion. However, the sample size of ten was probably insufficient to detect any correlation [13]. Tumors in different zones of the prostate may behave differently with respect to microvascular parameters. Peripheral zone PCa has been shown in several studies to have an increased vascular ity compared with transition zone tumors [14] or benign prostatic tissue [10,11,14]. However, this finding of increased MVD is not consis tent across all studies [9]. By contrast, transition zone tumors display a large variability in micro vascular parameters. They can be both hypoor hyper-vascularized compared with normal transition zone tissue [11]. Despite these differences, several studies have failed to find any significant association between MVD and other clinicopathological parameters, such as Gleason score, tumor grade or PSA [11,15]. Also, in a study using a tissue microarray from 3261 radical prostatectomy specimens, MVD failed to provide an independent prognostic fac tor when combined with standard predictors in a multivariable analysis [14]. There is no consensus about the utility of MVD as a prognostic marker in PCa. It also requires histological specimens, ideally at pros tatectomy, as the significance of MVD measured in biopsy specimens is unclear [14]. Therefore, MVD has a limited application in the clinical setting.
Circulating endothelial cells & circulating endothelial progenitors Circulating endothelial cells (CECs) and circu lating endothelial progenitors (CEPs) comprise subsets of cells that are functionally and pheno typically different. Both reflect angiogenesis and have been heralded as promising noninvasive biomarkers for the prediction of prognosis and evaluation of treatment response in cancer. CECs are mature, terminally differentiated cells that are shed from the vessel wall into the circulation in response to injury or as a result of endothelial dysfunction. Increased levels of CECs have been reported in a wide range of 880
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pathological processes that involve vascular injury, including cardiovascular disease, stroke, diabetes, chronic renal failure and vasculitis [16]. A proportion of CECs are apoptotic or necrotic and some studies have described subpopula tions of CECs as resting or activated. Interac tion with apoptotic or necrotic CECs induces a proinflammatory response in healthy endothe lial cells [17], which may alter the homeostasis of the vessel wall. However, the precise role of CECs in malignancy is unclear. At this stage, CECs are best understood as a product of vas cular turnover and, as such, a surrogate marker of angiogenesis. There has been much interest in endothelial progenitor cells (EPCs) since they were first iso lated in peripheral blood by Asahara and col leagues in 1997 [18]. Unlike CECs, CEPs are mobilized from bone marrow (BM). A popula tion of CEPs have clonogenic and proliferative potential (endothelial colony-forming cell) [19]. CEPs are relatively rare in healthy individuals. The role of CEPs in promoting angiogenesis in tumor growth is contentious. Within the vessel wall, BM-derived EPCs are thought to merge and differentiate into endothelial cells [20]. How ever, there are variable reports regarding the extent of CEP-derived vessels in cancer. Nolan and colleagues propose that the heterogeneity of results can be attributed in part to the changing relative contribution of CEP-derived vessels dur ing different stages of tumor development [20]. Using genetically marked BM progenitor cells they demonstrated that early tumors showed a marked recruitment of EPCs in the tumor periphery, followed by endothelial cell luminal incorporation into a subset of sprouting neoves sels. In established tumors, host-derived vessels diluted the EPCs. The authors suggested that this temporal course of EPC involvement is the reason why other studies have previously reported a low contribution of EPCs to tumor neovascularization. Another study reported that 12% of neovessels in metastatic disease showed luminal incorporation by BM-derived endothe lial cells [21]. More recently, Wickersheim and colleagues reported that although local VEGF production induces massive infiltration of BMderived EPCs, confocal microscopy and 3D reconstruction showed that these cells failed to incorporate into the vessel wall to any significant extent. As such, they concluded that vasculariza tion in tumor progression occurs primarily via angiogenesis rather than vasculogenesis [22]. Sim ilarly, Purhonen and colleagues demonstrated a lack of BM-derived EPC incorporation after future science group
Tumor vascularity in prostate cancer: circulating endothelial cells & platelets as noninvasive biomarkers
VEGF stimulation [23]. However, this study has been heavily criticized for its methodology [24]. BM-derived EPCs have also been shown to be involved in rebound revascularisation after some treatments. Certain chemotherapy drugs given at or near maximal tolerated dose (particu larly paclitaxel, 5-FU and docetaxel) induce a rapid mobilization of BM-derived CEPs, evident within 24 h of a single bolus injection. Treat ing with targeted antiangiogenic drugs prior to administration of these chemotherapy agents prevented acute mobilization and enhanced treatment efficacy [25]. CEPs have also been implicated in metasta sis development. Gao and colleagues describe BM-derived EPCs as critical regulators of the angiogenic switch responsible for mediating progression of micrometastases to lethal macro metastases. In preclinical lung metastasis models, micrometastases were largely avascular whereas macrometastases demonstrated neovessel infil tration. Blocking EPC mobilization resulted in reduced vessel density in metastatic lesions and impaired formation of lethal macrometastases [21].
Enumeration of CECs & CEPs Enumeration of CECs and CEPs is challeng ing for a number of reasons. The critical bar rier is that no one marker can uniquely identify CECs and CEPs, and there is no agreement on which combination of markers can iden tify them reliably (Table 1). Most commonly, CECs and CEPs are enumerated using flow cytometry [26] or immunomagnetic separation [27,28]. Multiparametric flow cytometry allows simultaneous identification of different anti gens. However, there is currently no consensus definition for CEC or CEP phenotypes. CECs have been characterized by a lack of leukocyte (i.e., CD45) and progenitor cell mark ers, and expression of endothelial markers such as vWF, CD31 or CD146 [27]. Other endothe lial markers that have been used include CD34 [29] and CD309 (VEGFR2/KDR) [30]. Unfor tunately, more recent evidence suggests that CD146 is not uniquely expressed by endothelial cells [31] and that CD309 is not a reliable marker in CEP enumeration [32,33]. Activated CECs have been identified by the expression of CD106 [34] or CD105 (Endoglin) [19,35,36]. DNA staining has been used to enumer ate viable, apoptotic and dead CECs [37]. Pro genitor markers used to identify CEPs include CD117 [37] in mice and CD133 [38] in humans. However, in humans, CD133 is also expressed by other hematopoietic stem cells [39]. future science group
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There is concern that current methods to identify CECs and CEPs may in fact capture a broader cell population. In recognition of this, Strijbos and colleagues set out to confirm the endothelial nature of cells meeting their immunophenotypic criteria for CECs (low-tointermediate forward light scatter, low sideways light scatter, CD31+, CD45-). They found that the cells thought to be CECs failed to express the endothelial marker CD146. Upon electron microscopy they also lacked endothelial cellspecific Weibel–Palade bodies, instead display ing morphological characteristics consistent with large platelets [30]. More recently, upon further analysis of CD31+ CD45- cells (thought to be CECs), Wong and colleagues revealed positive expression of platelet-specific markers (CD41a and CD42b). Wong and colleagues reported that these cells are more likely a subtype of plate lets rather than CECs [40]. For this reason, the use of a cell viability stain and DNA-specific staining has been advocated to discriminate DNA-containing CECs from DNA-free CECderived macroparticles and platelets [41,42]. Another study revealed that cells thought to be EPCs were in fact primitive hematogenous progenitors [43]. Despite this, subsequent stud ies have been published using this phenotypic definition for EPCs [44]. Another problem is the lack of validated pro tocols to identify CECs/CEPs. Different flow cytometry practices and different antibodies are used across studies. Although enumeration methods are often based on previously published protocols [30,37,40], this review identified no recent studies in PCa in which the CEC or CEP enu meration protocol had been formally validated in independent cohorts. Even in nonprostate cohorts, few studies have been validated. The use of diverse nonvalidated enumeration protocols, with different flow cytometry practices and anti bodies, probably contributes to the variability of reported results. Individual protocols are generally reported to be reproducible. Mancuso and colleagues veri fied the reproducibility of their CEC enumera tion protocol with nine rounds of intrareader and inter-reader variability studies [42]. In addi tion, they confirmed the cells to be endothelial in nature by the presence of Weibel–Palade bod ies upon electron microscopy. However, the pro tocol was not validated in an independent cohort [42]. Despite reassuring single-study validation, cross-study correlation of different methods is moderate to poor [26]. Therefore, one must refrain from comparing results from studies with www.futuremedicine.com
881
882 -
Mancuso et al. (2009)
Biomarkers Med. (2013) 7(6)
+
+
+ Intermediate
-
+
+
+
+
+
CD105
CD106
-
CD117
+
-
-
CD133
-
+
+
+
+
+
+
+
+
+
+
+
+
CD146
+
+
-
CD309 (VEGFR2/KDR)
Flk1+
Other
7AAD +/-
DAPI +
[40]
[55]
[51]
[40]
[37]
[35]
[34]
[48]
[50]
[30]
[28]
[42]
[47] +
[49] +
Syto16 7AAD +/-
DAPI
DAPI
[38]
[52]
[40]
[59]
[55]
+
7AAD -
DAPI
+
Nuclear Ref. stains
Blank cells represent not applicable. † Activated CEC. +: Positive for the specified marker in the studies described in the table; -: Negative for the specified marker in the studies described in the table; 7AAD: 7-aminoactinomycin-D; CEC: Circulating endothelial cell; CEP: Circulating endothelial progenitor; DAPI: 6-diamidino-2-phenylindole.
Wong et al. (2012)
Blann et al. (2011)
Human prostate cancer
CEPs
Namdarian et al. (2010) -
-
Wong et al. (2012)
Animal nonprostate cancer
-
-
-
-
-
Li et al. (2008)
Animal prostate cancer
+
+
Ronzoni et al. (2010)
Wang et al. (2013)
+
Ramcharan et al. (2013)
†
+
-
Strijbos et al. (2007)
Rigolin et al. (2010)
-
Simkens et al. (2010)
+
-
-
Kondo et al. (2012)
+
-
+
+
Kawaishi et al. (2009)
Goon et al. (2009)
Brunner et al. (2008)
Human nonprostate cancer
-
-
CD45
Wong et al. (2012)
+
CD34
-
+
CD31
Strijbos et al. (2010)
Blann et al. (2011)
Human prostate cancer
CECs
Study (year)
Table 1. Phenotypic definitions used to identify circulating endothelial cells and circulating endothelial progenitors.
Review Reeves, Sapre, Corcoran & Hovens
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CD31
future science group
+ +
Rigolin et al. (2010)
Ronzoni et al. (2010)
www.futuremedicine.com
+
CD117
CD133
+
+
+
+
+
+
+
+
CD146
+
+
+
+
CD309 (VEGFR2/KDR)
Flk1+
CD3CD19 ‑ CD33-
Other
Syto16
7AAD -
+
Nuclear stains
Blank cells represent not applicable. † Activated CEC. +: Positive for the specified marker in the studies described in the table; -: Negative for the specified marker in the studies described in the table; 7AAD: 7-aminoactinomycin-D; CEC: Circulating endothelial cell; CEP: Circulating endothelial progenitor; DAPI: 6-diamidino-2-phenylindole.
Namdarian et al. (2010) Intermediate
-
Wong et al. (2012)
Animal nonprostate cancer
-
-
-
-
-
Li et al. (2008)
+
+
Ramcharan et al. (2013)
Animal prostate cancer
+
Case et al. (2007)
Mancuso et al. (2009)
+
CD106
+
CD105
Goon et al. (2009) -
CD45
+
+
CD34
Brunner et al. (2008)
Human nonprostate cancer
CEPs (cont.)
Study (year)
Table 1. Phenotypic definitions used to identify circulating endothelial cells and circulating endothelial progenitors (cont.).
[51]
[40]
[37]
[34]
[50]
[48]
[43]
[42]
[38]
[52]
Ref.
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differing methodology unless there is evidence to support their validity. Another difficulty with studies using CECs/CEPs is the need for fresh samples. In particular, CEP enumeration needs to occur soon after collection as counts decrease after storage for 24 h [33]. The implication of this is that these cells can only be effectively evalu ated in prospective studies, rather than utiliz ing established tissue banks. This is particularly time consuming if investigators are attempting to compare CEC/CEP levels with long-term clinical outcomes in order to evaluate prognos tic ability. Mancuso and colleagues evaluated the reliability of frozen sample analysis of CEC count through the analysis of fresh and frozen samples evaluated at different time points. The coefficient of variation for variability over time in fresh samples (analyzed 0–72 h after collection) was 17 ± 7% and frozen samples (thawed and analyzed after 0–14 days frozen storage) was 26 ± 16%. The issue of reproducibility of CEC/CEP analysis from specimens in long-term storage was also investigated. In samples that were sent inter nationally and assessed after more than 6 months of storage, the coefficient of variation associated with duplicate reading was 22 ± 12% [42]. Hence, the technical limitations of performing prospec tive trials continue to hamper the field, preclud ing a rigorous assessment of the roles of these cells in tumor outcome prognosis. There are many issues with current enumera tion techniques. The main factor limiting the progression of quality research in this area is the lack of consensus definitions of CEC/CEP phenotypes. Validated protocols need to be estab lished that reliably identify these cells without erroneously including a broader cell population. Even if this is achieved, researchers will still need to contend with the challenge of requiring fresh samples for analysis.
CEC & CEP counts in PCa & other malignancies Very few studies have evaluated CEC and CEP in PCa in the last 5 years. Given the relationship between various vascular parameters and tumor aggressiveness or lethality in PCa, the develop ment of noninvasive markers of angiogenesis is of great relevance. The lack of research in this area probably reflects the methodological challenges of enumeration of these cells rather than a lack of scientific interest. CECs/CEPs have been investigated in a wide range of different cancers, and other reviews have considered CECs/CEPs in this context [45,46]. 884
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This review does not aim to give a thorough insight into CECs/CEPs in all types of cancer, and instead draws on a selection of recent studies to illustrate the heterogeneity of evidence and give further insight into the understanding of the controversies and challenges of CEC/CEP use as biomarkers. Importantly, when consid ering CECs/CEPs in other cancers, it is diffi cult to make simplistic comparisons with PCa. Although angiogenesis is a feature common to many tumor types, PCa has other features that make it unique, including hormone dependence. PCa is also generally slow growing, and as it has a reasonably good progression marker in PSA, it may be monitored for long periods without aggressive treatment. These differences may translate into differences in angiogenic require ments and consequently patterns of CEC/CEP levels. Baseline CEC levels in malignancy Baseline CEC levels have been shown to be elevated in patients with a variety of cancers in comparison with healthy controls, including pancreatic cancer [47], colorectal cancer (CRC) [48] [34], breast cancer [38], non-small-cell lung cancer (NSCLC) [49] and chronic lymphocytic leukemia (CLL) [50]. More specifically, activated CECs were increased in patients with NSCLC compared with healthy volunteers [35]. Simi larly, in human renal cell carcinoma xenograft models, CEC levels were significantly elevated in tumor-bearing mice compared with healthy controls [51]. In patients with primary breast cancer, CEC level correlated with tumor invasiveness and size, and positively predicted Nottingham Prognostic Index scores [38]. In primary CRC, a stepwise increase in CEC and EPC levels significantly cor related with increasing Dukes’ stage. However, upon multiple regression analysis, they failed to remain significant, and there was no signifi cant correlation between CEC or CEP level and American Joint Committee on Cancer (AJCC) stage [48]. Another study in CRC showed a posi tive correlation between ‘total’ and ‘resting’ CEC levels and the number of sites of metastasis [34]. No correlation between CEC level and dis ease stage was seen in CLL [50] or primary head and neck squamous cell carcinoma (SCC) [52]. In patients with head and neck SCC, there was no difference in CEC levels between patients and control [52]. Lower levels of mature CECs have been reported in pediatric patients with malignant solid tumors compared with controls [53]. future science group
Tumor vascularity in prostate cancer: circulating endothelial cells & platelets as noninvasive biomarkers
Although CEC levels are reported to be higher in various malignancies (compared with healthy controls), this is not consistently reported across all studies.
Baseline CEC & CEP levels in PCa The significance of CEC and CEPs in PCa has been questioned owing to the lack of correla tion between their levels and accepted clinical markers. Blann and colleges reported no significant difference in CECs and CEPs in patients with biopsy-proven PCa, benign prostate disease or no prostate disease. Also, there was no signifi cant correlation between CEC or CEP level and PSA, Gleason score or other plasma markers (vWf and soluble E‑selectin) [55]. In the preclinical arm of the study by Wong and colleagues, levels of CD31+ CD45 - cells (thought to be CECs) were significantly elevated in tumor-bearing mice. These correlated with tumor size, MVD and volume. With respect to CEPs, there was no significant difference in CEP levels in any of the cell lines in the xenograft models compared with controls. In the clinical arm of the study, which evaluated CECs/CEPs in patients with localized PCa undergoing prosta tectomy, there was no correlation between CEC number and stage, Gleason score, extraprostatic extension or preoperative PSA [40]. There is currently no evidence to support the use of CEC/CEP enumeration in diagnosis of PCa.
shorter time to first treatment and were more likely to have a worse treatment response [50]. Similarly, elevated baseline CEC levels in advanced primary NSCLC [36] and pancreatic cancer [47] was associated with a poorer overall survival (OS). By contrast, other studies reported favor able outcomes in patients with higher CEC counts. In patients with advanced breast can cer treated with metronomic chemotherapy plus bevacizumab, higher baseline CEC levels were associated with increased time to progres sion. At the time of progression, the number of CECs was significantly reduced compared with baseline. The authors hypothesized that this may represent an shift to an alternative type of vascularization [56]. In NSCLC there was no significant correlation between base line CEC count and estimated tumor volume. However, NSCLC patients with a higher base line CEC count showed longer progressionfree survival (PFS) and improved clinical benefit from carboplatin plus paclitaxel-based chemotherapy [49]. Some studies failed to find an association between CEC level and survival. CEC baseline level in a study of various malignancies treated with chemotherapy was not associated with PFS or OS [57]. Similarly, CEC level failed to corre late with response to radiotherapy or survival in patients with head and neck SCC [52]. Varying reports have been published in CRC. One study in CRC failed to find a correlation between baseline CEC and PFS or OS [28], while another study of metastatic CRC patients found that those with the lowest ‘total’ and ‘resting’ CEC levels at baseline showed a longer PFS [34]. Their conflicting results have been attributed to their differing techniques for CEC enumera tion [28]. In a trial of CRC patients treated with FOLOX4 plus bevacizumab, those with 65 or more CECs at baseline had significantly shorter median PFS and OS. Interestingly, there was no similar correlation seen in those treated with FOLFOX4 alone [58].
Baseline CEC levels as predictor of treatment response & survival in malignancy Although some studies have described the value of baseline CEC levels in predicting treatment outcomes and prognosis, the evidence remains unclear. In some studies, higher CEC levels were asso ciated with more aggressive disease. In patients with CLL, those with higher CEC levels had a
Baseline CEC levels as a predictor of treatment response & survival in PCa There is currently very limited research pertain ing to CECs/CEPs as predictors of treatment response and survival in patients with PCa. In the clinical arm of Wong and colleagues’ study, men who experienced treatment failure within the first year had significantly higher CD31+ CD45- levels. The authors hypothesized that higher CD31+ CD45- levels may be a marker
Baseline CEP levels in malignancy Similarly, conflicting evidence exists regarding CEP levels in patients with cancer. CEPs have been shown to be in higher numbers in patients with primary gynecological cancer (ovarian or cervical) [54], head and neck SCC [52], CRC [48] and CLL [50], compared with controls. Con versely, no correlation was found with baseline CEPs and metastatic CRC [34]. Circulating pro genitor cells were lower in patients with breast cancer than matched controls [38].
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of micrometastatic disease in localized PCa, and that in patients with early cancer recurrence, cancer had already spread prior to surgery. Fur ther characterization of these cells revealed that they represented a subpopulation of circulating platelets rather than CECs, highlighting the challenges in interpreting evidence in this area [40]. With respect to survival in patients with castration-resistant PCa, baseline CEC level is not associated with OS [59].
CEC & CEP kinetics in cancer treatment Shifting CEC and CEP levels in response to cancer treatment are presumably due to mobi lization of either angiogenic or vasculogenesis processes induced by acute cytotoxic stress, although definitive data pertaining to the mechanism remain lacking. CEC kinetics in other malignancies There have been a number of clinical studies in cancer that showed no significant change in preand post-treatment CEC levels. These include studies in patients with advanced NSCLC (receiving first-line chemotherapy) [36], patients with primary head and neck SCC (receiving surgery, chemoradiation or radiotherapy alone) [52], pancreatic cancer (treated with gemcitabine chemotherapy for locally advanced disease, metastases or recurrence) [47] and various pedi atric malignant solid tumors [53]. Furthermore, in pancreatic cancer, the CEC count change from baseline was not associated with tumor response [47]. In a few studies, CEC levels are reported to change in response to treatment, but there is no consistent evidence regarding how CEC kinetics relate to prognosis. Breast cancer patients treated with first-line chemotherapy plus bevacizumab had a signifi cant increase in CECs following treatment. This increase was associated with improved time to progression when a threshold of 20 CECs per 4 ml was used [60]. In a study that included patients with vari ous malignancies treated with (neo)adjuvant chemotherapy or chemotherapy for metastatic disease, increases in CECs and EPCs were seen after the first cycle of chemotherapy. In some patients this was evident as early as 4 h after treatment. A large increase of CEC levels 7 days after chemotherapy was significantly associated with poor PFS, and at day 21 a large change in CEC level correlated with both PFS and OS. Upon univariate analysis, EPC levels were not 886
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significantly associated with PFS or OS at days 7 and 21 [57]. In 473 patients with advanced CRC treated with first-line chemotherapy and bevacizumab, there was a significant increase in CEC after 1–2 weeks of treatment; however, changes in CEC levels did not predict PFS or OS at any time point [28]. CECs were reported to decrease in NSCLC patients following carboplatin plus paclitaxel-based chemotherapy [49]. In another study in patients with NSCLC, inverse correlations existed between changes in activated CECs following treatment and PFS [35]. CEP kinetics in other malignancies In a study of various malignancies treated with chemotherapy, only those treated with taxanebased chemotherapy showed an immediate increase in EPCs. After 21 days, EPC levels were consistently increased irrespective of chemo therapy type. EPC levels were not significantly associated with PFS or OS at day 7 or 21 [57]. In a small study of cancer patients receiving an antivascular agent plus cisplatin, CEP lev els increased following treatment. The authors hypothesized that vascular trauma as a result of treatment may cause upregulation of angiogenic factors that promote mobilization of BM-derived EPCs [61]. By contrast, the number of CEPs declined fol lowing chemoradiation or surgical treatment of ovarian or cervical cancers [54]. CEC & CEP kinetics in PCa treatment CECs have been reported to increase in response to treatment in both xenographt [37] and clinical studies [59,62] of PCa. Li and colleagues compared viable CECs, dead/apopototic CECs and CEP response to thalidomide, docetaxel or a combina tion of the two drugs in a human PCa xenograft model [37]. Apoptotic/dead CECs were measured at baseline as well as 4 and 17 days into treatment. Increased levels of apoptotic/dead CECs were observed after treatment with docetaxel. Addition of thalidomide further increased apoptotic/dead CEC levels. Interestingly, the increase in CECs, both viable and apoptotic/dead, was greatest at 4 days compared with 17. To ensure that the CEC variation did not reflect nonspecific cyto toxicity of the chemotherapy agents used, Li et al. measured CEC levels in response to cisplatin (a chemotherapy drug with no antiangiogenic activ ity), which revealed no variation. There was no statistically significant change in CEP levels in response to treatment. future science group
Tumor vascularity in prostate cancer: circulating endothelial cells & platelets as noninvasive biomarkers
Strijbos and colleagues studied CECs, circu lating tumor cells, TF‑1 and ET‑1 as prognostic indicators in 162 patients with castration-resis tant PCa treated with a docetaxel-containing chemotherapy regimen [59]. They found no sig nificant association between the markers assessed. However, a significant increase in CEC levels was seen 2–5 weeks after initiation of docetaxel. A 3.8-fold or higher increase in CEC from baseline at 2–5 weeks was associated with a significantly worse OS. There was no significant increase found at the second follow-up measurement at 6–8 weeks. This is in keeping with Simkens and colleagues’ study in advanced CRC [28], which showed no further increase in CEC after that observed at 1–2 weeks after treatment. It is hypothesized that the rise in CECs is due to vascular damage inflicted by docetaxel, and that in patients with a poorer OS it may be due to continued endothelial shedding from vessels in tumors progressing during treatment. Strijbos also highlighted the need for prospective studies to confirm these findings, as the study was only exploratory [30]. In a Phase II trial of bevacizumab, thalidomide, docetazel and prednisolone in patients with meta static castration-resistant PCa, circulating apop totic endothelial cells (CAECs) were a secondary outcome measure [62]. Levels were measured at baseline and six weeks. A strong inverse correla tion between relative change in PSA over 6 weeks and the absolute difference in CAECs was dem onstrated. Patients with a 75% or greater decline in PSA had a significant increase in CAEC levels compared with those with a
