A Systematic Review of Circulating Tumor Cells in Renal Cell Carcinoma

 Introduction

Renal Cell Carcinoma (RCC) accounts for approximately 85-90% of kidney malignancies with the origination of both the epithelium of proximal convoluted tubules (clear cell RCC) and intercalated cells of the distal nephron (chromophobe RCC) (1). RCC has a high fatality rate among urological malignancies despite tremendous improvement in surgical procedures (2). The clinical manifestations of RCC are indeed confusing owing to the similarity to other conditions' symptoms, which leads to diagnosis in the late stages (3). Given the fact that there is a close reverse association between the time of diagnosis and the overall survival of patients with RCC more than having no appropriate and accurate existing diagnostic tests, some studies were conducted to explore new diagnostic methods (4).

The problems that arise from traditional methods provoke great interest to take into consideration liquid biopsy or as it is called "circulating tumor cells" (CTCs) as either the alternative substitution or augmentation of the use of a biopsy, due to lack of invasive behavior and great potential to surpass the current challenges (5, 6). Widening of indications has underpinned the high capacity of CTCs in the identification of tumors, which are prone to recurrence, or resistance, that can readily aid physicians to be aware of changes occurring during patients' treatment and follow-up course (7). So far, several molecules are of proven value to be measured in body liquids mainly ranging from CTCs to cell-free DNA, that have drawn quite an amount of attention to themselves (8).

In the last decade, with advances in measuring methods that can sense scarce numbers of CTCs, the vast majority of studies were conducted to investigate the feasibility of using CTCs in malignancies. The clinical implications of CTCs in urological malignancies have been an area of plenty of studies' interest. Likewise, the other urological malignancies, the CTCs' count in RCC association with the degree of tumor aggressiveness and a chance of tumor metastasis, has been observed (9).   

Generally, the RCCs' treatments are divided into three major categories: 1, anti-vascular endothelial growth factor (VEGF) agents, and 2, mammalian target of rapamycin (mTOR) agents 3, immunotherapeutic agents that can be administered solitary or in combination with anti-cytotoxic T-lymphocyte antigens. Hence, in view of both CTC's ability to select patients to receive specific treatment and RCC's targeting-based treatment approach, CTC can predict the best treatment for any individual (7, 10).

Even though expanding indications regarding the tremendous applicability of CTCs in RCC have been presented, the issue of suitable CTCs has yet to be addressed completely. Here we designed a systematic review of the studies focusing on CTC in RCC to clear the existing obstacles, which hindered us to bring them into routine practice.

Method

Search strategy

After submitting the protocol on the international prospective register of systematic reviews PROSPERO ID: 158222  the study was run and all relevant literature searches have done from four main databases: MEDLINE (PubMed), Scopus, EMBASE, and Web from January 1^st, 1990, to the last April 2019 with words the key grouping of “Renal Cell Cancers”, “Adenocarcinoma Of Kidney”, “Nephroid Carcinomas”, “Renal Cell Carcinoma”, “Collecting Duct Carcinoma”, “Liquid Biopsies”, “Circulating Neoplastic Cells”, “Circulating Tumor Cells”, and “CTCs”. (Supplementary file 1). To diminish the selection bias, two colleagues (MN and SMKA) separately screened articles by checking titles, abstracts, and access to full-text articles for application. Some additional articles were retrieved from reference lists of selected articles. Grey literature was gotten from the system for information on the grey literature (SIGLE) database (opensigle.inist.fr) and healthcare management information consortium database. ( www.ovid.com/site/catalog/DataBase/99.jsp?top=2&mid=3&bottom=7&subsection=10)  Dissimilarities were solved by agreement and discussion with a third person (FKH). 

Eligibility criteria

All chosen documents were studied by two independent authors and agreeing to their title and abstract were considered as the included one or excluded one. The inclusion criteria were: 1) patients with pathologically confirmed renal cell carcinoma; 2) the control population was exactly specified; 3) all CTCs detection techniques such as cell search system, the NanoVelcro chips, ISET®-Isolation by the size of tumor cells, ScreenCell®, and real-time PCR were selected. Some articles were excluded because they; have no definition of case and controls, were the in vitro or in vivo studies and analyzed CTCs in animals or cell culture or not presented the detection method and indicated which molecular markers is targeted. 

Data extraction and quality assessment

The whole critical information over the CTCs studies in RCC was recorded in the excel file (data extraction file). Unfortunately, because of having no adequate quantity of homogenous studies related to RCC and CTCs, and having the wide methodological heterogeneity and noteworthy variations in study population characteristics doing the additional analysis which lead to the meta-analysis of the data was not applicable. Two main tools of quality assessment were retrieved including quality assessment of diagnostic accuracy assessment (QUADAS) and the Newcastle–Ottawa scale (NOS) assessment tools. All papers that scored 12/16 or more on the QUADOMICS tool together with 6/8 or more on NOS were reflected as the “high quality” ones, versus articles scoring 11/16, 5/8, or less were considered as the “low quality” ones.

Result

The selection algorithm and results of the study selection are presented in Figure 1. A total of 2185 documents were recovered after duplication removal, including 1318 articles from PubMed, 401 from Scopus, 300 from Web of Science, and 190 from Embase. All documents were screened two times by two reviewers through their title and abstract to find the related ones and reach 125 related documents (Figure 1). After deleting the review, in vivo/in vitro studies, and book or conference papers the final 24 articles were a candidate for data extraction and further considerations (Table 1).

Figure 1. Flow diagram of documents selection steps for the current systematic review based on PRISMA

Despite our search strategy covering the time from 1990 to 2019, the first published research related to CTCs detection in RCC was in 2001. After that one study was published in 2004, two in 2005, one in 2008, 0ne in 2009, and other 16 articles were published after 2010. Studies were run mainly in China (7 articles), Germany (4 articles), France, Italy, Canada, Japan, Poland, the USA (2 articles), Austria. Most studies were focused on detection and quantification of CTCs (ISET® - Isolation by size of tumor cells), some others studied the molecular markers and gene expression profile. One study by Amin El-Heliebi just focused on the morphological criteria (15). Some CTCs detection methods were based on the CTC‘s surface marker and gene expression pattern. The most common methods were based on the immunomagnetic separation procedure for the detection of CTCs in the peripheral blood based on the magnetic cell sorting (MACS) technique. The CTC detection methods lacking the enrichment step named direct recognition of CTCs including; line-confocal microscope and SERS. The CellSearch® (Veridex, Raritan, NJ) is the only U.S. food and drug administration (FDA) approved method. However, it cannot isolate CTCs based on phenotype classification and molecular analysis. The CellCollector® (GILUPI GmbH, Potsdam, Germany) is a CE (French phrase “Conformité Européene” which exactly is equal to “European Conformity”) permitted medical method as the first universal CTC separation instrument (35). Structurally it is made of a needle, which is inserted into the patient’s vein for the CTC separation. On the hydrogel coating layer, the anti-EpCAM-antibodies are coated to recognize and separate the EpCAM-positive CTCs. Contrary to the CellSearch®, it does not require costly practical equipment, and the sensitivity and selectivity are advanced because of more blood volume and feasibility of phenotypic characterization of and molecular analysis of the captured CTCs. The enrichment phase states the separation of CTCs from the blood. Subsequently, the CTCs can be identified by fluorescence (e.g. fluorescent microscope, fluorescent spectrophotometer, and flow cytometer), surface-enhanced Raman scattering (SERS), or electrical impedance.

Moreover, CD45-conjugated microbeads, antic-MET Ferrofluid for the immunomagnetic capture of CTCs, EpCAM based enrichment, and real-time polymerase chain reactions were recruited for CTCs quantitation. Several antibodies against CTC molecular markers were targeting for detection including cytokeratin (CK)/CK8/18/19, CD45, CD133, CDW-41,  carbonic anhydrase IX (CAIX), epithelial cell adhesion molecules (EpCAM), cadherin-6, Hypoxia Inducible Factor 1 Subunit Alpha (HIF1A), vascular endothelial growth factor A (VEGFA), vascular endothelial growth factor receptors (VEGFR), and fibroblast growth factor receptors (FGFR) and the presence of N-cadherin. Some detection methods were based on the physical characteristics of CTCs like their size including immunomagnetic circulating tumor cell enrichment, ISET®-Isolation by the size of tumor cells, ScreenCell® filtration, tapered-slit filter (photosensitive polymer-based microfilter), and NanoVelcro chips.

Discussion

CTCs are circulating tumor cells that are released from original or metastatic solid tumors and shed into the bloodstream which can potentially lead to new fatal metastasis. CTCs are real-time representatives of the tumor so they became a hotspot research topic over the last years. CTCs uncovering, as the main liquid biopsy component, can be taken into account of early RCC diagnosis, sooner assessment of cancer recurrence and treatment efficacy, and a special assessment of individual sensitive anti-cancer drugs (personalized medicine). Therefore, CTC detection is a crucial tool in to fight against cancer. Herein we represented the first study on the several CTCs' impacts on both diagnosis and prognosis of RCC. Studies of CTCs in RCC were mostly focused on CTC detection and quantifications based on immunomagnetic cell enrichment of tumor cells through leukocyte-specific reduction. It was shown by Udo Bilkenroth and colleagues that there was a  direct connection between tumor cell number and grading (G2 vs. G3) and an augmented quantity of CK+ patients with progressive tumor stage (11). In 2001, it was MACS reported as an efficient technique to detect CTCs in peripheral blood (11). The same result indicated that immunomagnetic cell enrichment and cytokeratin 8/18 (CK+) targeting as the CTCs marker can be taken into account in CTCs detection in RCC patients (12, 13, 16). The total number of 233 peripheral blood samples from 154 RCC patients were examined for the existence of dispersed tumor cells by automatics technique and immunocytochemical staining of cytokeratin. It was shown that CTCs are correlated to lymph node grade and the existence of synchronous metastases in RCC. Detection of CK+ CTCs in a patient’s blood is suggested as a noteworthy and autonomous prognostic feature for RCC (12).

A recent study by Lucile Broncy, et al., has matched genetic mutation analysis of circulating rare cells (CRC) and tumor sections with CRC cyto-pathological diagnosis. It was done by the blood sample of thirty patients with clear cell RCC tested by ISET® for CRC separation, cytopathology, and single-cell VHL mutations analysis, achieved blindly and matched to VHL mutations to the equivalent tumor tissues and leukocytes (14). The recent study by Tianying Xing and his colleagues represented a new and strange method for CTC detection (34). They recruited the simply equipped silica nano-bio interface chip for CTCs detection in prostate cancer (PCa) and clear cell RCC patients with high productivity. The silica nano-bio interface chip was extraordinarily invented by placing candle soot on a glass slide, tracked by chemical vapor statement, and then by adjusting EpCAM antibodies. These silica nano-bio interface chips presented outstanding capacities to capture PC3PCa cell lines, with typical efficacy of 81.2±1.4%. The solid topographic contact between targeted cells and the nanostructured surface was serious to improving CTCs capturing potential. Tianying Xing and his colleagues additionally checked the peripheral blood samples from 10 preoperative PCa and 7 ccRCC patients. Their results displayed that CTCs were successfully detected and suggested that the nano-bio interface chip will offer the excessive probable potential for the clinical application of CTC (34).

However, some studies debate the morphological characteristics of CTCs and ask the question of whether morphological criteria are sufficient for the identification of circulating tumor cells in RCC (15). The majority of circulating non-hematologic cells (CNHCs)-clusters, putative circulating tumor microemboli (CTMs), recovered by ScreenCell filtration may be of endothelial origin indicate that morphological criteria seem to be insufficient to distinguish malignant from non-malignant cells in renal cancer (15). Circulating tumor cells and "suspicious objects" evaluated through Cell search (R) in metastatic RCC indicated that the low number of CTCs detected through Cell search in renal cell carcinoma may be due to the presence of a CTC population with atypical characteristics and a peculiar gene expression profile, characterized by lack of cytokeratin expression and gain of CD44 (16).

Some advanced molecular techniques like quantitative real-time PCR, RNA in situ hybridization, and copy number variations (CNVs) can solve the weak points of CTCs detection just based on morphological characteristics.  Cadherin-6 mRNA is suggested as the new molecular marker for the detection of circulating renal cancer cells disseminated from conventional RCC (19). The connection of dynamic variations of CTCs and Beclin-1 expression of CTCs with RCC prognosis has been proven (25). The recurrence or metastasis of RCC was dependent on initial CTCs quantitation but may be associated with the variation trend of CTCs, particularly mesenchymal CTCs and Beclin1 positive CTCs (25). Prosperous application in metastatic RCC (mRCC) is very restricted. Extraordinary elasticity and heterogeneity of CTC morphology experiments currently make the enrichment and detection techniques with EpCAM not satisfying in mRCC. The CTC recognition with epithelial, mesenchymal, stem cell-like, or mixed-cell features at several time-points through anti-angiogenic therapy in RCC patients indicated that the amount of N-cadherin-positive or CD133-positive CTC can be associated with lower PFS. However, there was an opposite connection between HIF1A, VEGFA, VEGFR, and FGFR  overexpression and the existence of N-cadherin-positive and CD133-positive (22). CTC Patients with mRCC show different CTC outlines and molecular markers that can indicate differences in therapeutic outcomes (22).

Conclusions

CTCs detection as the main liquid biopsy component has unlimited experimental implications in cancer diagnosis and prognosis, especially in RCC. But traditional techniques still encounter limitations when targeting CTCs with high efficiency and low cost. The CTCs detection and quantitation can represent the tumor and to some extent the tumor stage/ metastasis. Some CTCs molecular markers and gene expression profiling of CTCs can be taken into account for RCC prognosis.

Authors’ contribution

All authors contributed equally.

Acknowledgments

Special thanks to the Urology Research Center (URC), Tehran University of Medical Sciences (TUMS).

Conflict of interest

All authors declare that there is not any kind of conflict of interest.

Funding

There is no funding.

Ethical statement

Not applicable.

Data availability

Data will be provided by the corresponding author on the request.

Abbreviation

CAIX               Carbonic anhydrase IX

CNHCs            Circulating non-hematologic cells

CNVs              Copy number variations

CTCs               Circulating tumor cells

CTMs              Circulating tumor microemboli

CRC                Circulating rare cells

EpCAM           Epithelial cell adhesion molecules

FGFR              Fibroblast growth factor receptors

HIF1A             Hypoxia-inducible factor 1 subunit alpha

MACS             Magnetic cell sorting

mRCC             Metastatic RCC

mTOR             Mammalian target of rapamycin

NOS                Newcastle–Ottawa scale

PCa                  Prostate cancer

PFS                  Progression-free survival

QUADAS        Quality assessment of diagnostic accuracy assessment

RCC                 Renal cell carcinoma

SERS               Surface-enhanced Raman scattering

SIGLE              System for information on grey literature

VEGF               Vascular endothelial growth factor

VEGFA            Vascular endothelial growth factor A

VEGFR            Vascular endothelial growth factor receptors