Circulating Tumor DNA in Patients With Renal Cell Carcinoma. A Systematic Review Of The Literature
Nov 06, 2023
Abstract Context: Over the past decade there has been increasing interest in the potential of liquid biopsies and systematic biomarkers in the diagnosis and management of kidney cancer, as they may provide a tool for early detection of disease and monitoring of treatment response. Objective: To identify and summarize relevant published data on circulating tumor DNA (ctDNA) in patients with renal cell carcinoma (RCC). Evidence acquisition: We performed a systematic review according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement of studies identified in PubMed, MEDLINE, EMBASE, and Cochrane Library up to January 15, 2021. Two reviewers independently screened all articles and performed the data extraction.
Evidence synthesis: Nineteen studies investigating ctDNA in RCC (1237 patients) were included and analyzed in the final review. The study size and design varied widely, and the studies were divided into five groups according to the method used for ctDNA detection. The outcome data included (1) the sensitivity/specificity if available; (2) the method used for ctDNA detection; and (3) the main findings in the studies.
Conclusions: The studies highlight that the level of ctDNA in RCC appears to be low. Studies using multiple methods for ctDNA detection indicate that tumor-guided analysis improves the ctDNA detection rate and suggest that cell-free methylated DNA immunoprecipitation and high-throughput sequencing may be a very sensitive method for ctDNA detection in RCC.
Patient summary: We systematically reviewed the literature to identify all relevant studies investigating circulating tumor DNA in patients with kidney cancer to investigate its use and potential in this highly malignant disease. We found that the level of circulating tumor DNA is low in kidney cancer and that very sensitive methods have to be used for the detection in this disease.
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1. Introduction
Urological malignancies are an increasing healthcare problem worldwide, with more than an estimated 1 million patients newly diagnosed annually [1]. Renal cell carcinoma (RCC) is a highly malignant disease and represents 3% of all malignancies in Western countries, with its incidence increasing annually [2].
Diagnosis and treatment of patients with RCC present many challenges. This is mainly because RCC is characterized by asymptomatic manifestation in the early stages and a poor response to radiotherapy and chemotherapy in metastatic stages [3,4]. Over the past several decades, there has been increasing interest in research on liquid biopsies and circulating cancer biomarkers because they might provide a tool for early detection of disease and for monitoring of treatment response. Ellinger et al [5] reported that the presence of cell-free DNA (cfDNA) in the bloodstream was first detected more than 50 yr ago. cfDNA can easily be obtained from peripheral blood, and it has been shown that it is present in patients with multiple solid malignancies [6]. The presence of cfDNA in the bloodstream of patients and changes in the levels of these circulating nucleic acids are associated with tumor load and tumor progression. However, studies differ as to whether they have been able to show a correlation between cfDNA levels and tumor stage and grade in RCC [7–9]. Circulating tumor DNA (ctDNA) is cfDNA derived from apoptotic or necrotic tumor cells, secretions from macrophages, or circulating tumor cells (CTCs) [10]. Analysis of ctDNA may open the possibility of noninvasive detection of the mutational profile of specific cancer during tumor progression, and studies have shown that the presence of ctDNA correlates with advanced disease and disease progression during treatment [7,11,12]. While several studies on ctDNA in RCC have been performed, there are still no clear recommendations or summaries of the literature.
We report here on a systematic review of all published data on ctDNA in patients with RCC to investigate the use and potential of ctDNA in RCC.
2. Evidence acquisition
2.1. Search strategy
This systematic review was performed according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines [13] (Fig. 1). The review was registered on the PROSPERO platform under identification number CRD42020208730. One author (L.G.) and an expert librarian searched, for which the strategy is available in the Supplementary material. The literature search was performed in the PubMed, MEDLINE, EMBASE, and Cochrane Library databases to identify relevant studies up to January 15, 2021, with sorting and removal of duplicate articles performed in Covidence.
2.2. Screening and selection criteria
Study selection was performed in three sequential steps: articles were first assessed by title, second by abstract, and third by full text. Studies were included if they met the following inclusion criteria: (1) patients diagnosed with any stage of RCC; (2) analysis of DNA in plasma/serum; (3) analysis of mutations at a DNA level or epigenetic changes; and (4) article written in English. Articles regarding malignancies other than RCC, animal and cell-line studies, studies published before 2010, and small pilot studies containing fewer than five patients were excluded.
Two authors (L.G. and K.M.K.) independently screened the titles and abstracts and selected potentially eligible studies. The full text of potentially eligible studies was then screened. The authors discussed any disagreements until a consensus was reached.
2.3. Data extraction
A data extraction sheet was developed and prespecified outcomes of interest were collected. Two reviewers (L.G. and K.M.K.) independently extracted data from the studies included. Disagreements were resolved via consensus.
The trial characteristics extracted included: (1) first author’s name and year of publication; (2) method applied for detection of ctDNA; (3) number of patients/cases; and (4) number of controls. The outcome data included: (1) sensitivity and specificity when available; (2) the method used for ctDNA detection; and (3) key findings in the particular studies.
Quality assessment was not performed according to pre-defined criteria because of the variation in study design and methods in the studies included. The quality of the studies was assessed by one author (K.M.K.) with wide experience with methods used for ctDNA detection.
3. Evidence synthesis
3.1. Studies included
According to our search criteria, 3439 potentially relevant papers were identified and screened in the primary search. All papers were added to EndNote and duplicates were removed, leaving 1670 studies. The titles, abstracts, and full texts were screened manually, and 19 studies met the inclusion criteria [9,14–31]. All studies included were published between 2010 and 2020. They were published in full, but the size and design of the studies varied widely, and therefore they were divided into five groups according to the method used for ctDNA detection: (1) tumor-guided analysis of plasma; (2) targeted sequencing of plasma; (3) global sequencing of plasma; (4) targeted methylation analysis of plasma; and (5) global methylation analysis of plasma.
To cover the use of ctDNA in RCC, we screened the studies in each of the ctDNA detection groups to find the ctDNA detection rate and the sensitivity and specificity if available. Furthermore, we identified studies investigating the concordance between ctDNA samples and tumor tissue from the same patient, and studies investigating the potential of ctDNA for prognosis and disease monitoring. Finally, we summarized the methods used for ctDNA detection and which method to choose based on our findings.
3.2. ctDNA detection rates
3.2.1. Tumor-guided analysis of plasma
Five studies [14–18] applied tumor-guided analysis of plasma (Table 1). This type of analysis is based on the identification of mutations in tumor tissue DNA and subsequent analysis of these mutations in cfDNA. The advantage of this method is that very high technical sensitivity can be achieved when the mutations of the tumor are known. The downside is that the analysis does not identify newly acquired mutations. The five studies included between five and 24 RCC patients and the ctDNA detection rate ranged from 17% to 54%.
In an early ctDNA landmark study, Bettegowda et al [14] investigated the presence of ctDNA in 640 patients with various cancer types, including five patients with metastatic RCC. Detection of ctDNA in plasma was via polymerase chain reaction (PCR)- or sequencing-based analysis of a mutation identified in the tumor. ctDNA was detected in 40% of RCC patients with metastatic disease, which led to the classification of RCC as a low-ctDNA malignancy. Although the classification was based on a very low number of patients, it has been supported by multiple studies performed since then [15–18].
The studies by Corrò et al [15] and Lasseter et al [18] both used targeted deep sequencing of one or a few mutations identified in tumor tissue and had a ctDNA detection rate of 17% and 52%, respectively. The patients in the study by Corrò et al had nonmetastatic disease, whereas the patients in the study by Lasseter et al had metastatic RCC.
The difference in detection rate may result from the different patient cohorts; however, the relatively low detection rates in these studies are surprising, since the tumor-informed strategy has the potential to be one of the most technically sensitive methods. The study by Lasseter et al also showed that sequencing of cfDNA to an extremely high depth (122 035 ) improved ctDNA detection because it increases the detection of variants present at very low frequencies.
Two very recent studies with mixed patient cohorts (nonmetastatic and metastatic RCC) [16,17] applied ctDNA analysis of a large number of variants identified in tumor tissue. In the study by Smith et al [16], 91 patients were investigated using a variety of methods (Tables 1–3). The authors found that a personalized approach targeting an average of 297 tumor-specific variants per patient using custom panel sequencing of mutations identified via exome sequencing of tumor tissue improved ctDNA detection compared to an untargeted approach. Tumor-guided analysis of plasma was performed for 29 patients and identified ctDNA in 54% of the patients for whom the analysis passed quality control. Wan et al [17] applied a similar methodological approach to 24 patients and found that the fraction of ctDNA in RCC patients was approximately 10 4 . This finding highlights that extremely sensitive methods are required for ctDNA analysis in RCC.
3.2.2. Targeted sequencing of plasma
Eight studies [16,18–24] (Table 2) applied targeted panel sequencing of DNA from plasma without prior analysis of tumor tissue DNA. The advantage of this strategy is that it does not require a tissue sample (ie, it is less invasive) and allows for the identification of newly acquired mutations. However, the analysis may be less sensitive than tumor-guided analysis. In general, the studies included a larger number of patients, ranging from 19 to 220, and the detection rate was considerably better, ranging from 19% to 100%. Most of the studies were performed among patients with metastatic disease; only 14 patients in the study by Yamamoto et al [22] and two patients in the study by Smith et al [16] had nonmetastatic RCC (detection rates of 30% and 19%, respectively). The high detection rates may thus reflect a high tumor load in many of these patients. The findings in the study by Maia et al [20] support this hypothesis. The authors found that ctDNA detection correlated with tumor burden; tumor burden was significantly higher in patients with detectable ctDNA than in patients without ctDNA. Furthermore, the number of mutations in ctDNA was also significantly correlated with tumor burden.
Bacon et al [23] found a relatively low detection rate of 33% in comparison to similar studies. This may be because in 40 of 55 patients, plasma samples were collected after surgical removal of the primary tumor. However, in a study by Hahn et al [19] in which 15 of 19 patients also had their primary tumor surgically removed before sample collection, the ctDNA detection rate was 68%. The low detection rate could also be explained by the technical sensitivity of the assay, which did not allow for the detection of variants present at a variant allele frequency (VAF) of <1% [23].
There seems to be a trend towards better detection in studies in which ctDNA analysis was based on a gene panel covering a high number of genes, very high sequencing depths, or a combination of these. For example, the studies by Maia et al [20] and Pal et al [21] both sequenced 73 genes with 15 000 coverage and had detection rates of 53% and 79%, respectively. By comparison, Smith et al [16]sequenced ten genes to a depth of 9688 and Lasseter et al [18] sequenced 27 genes to a depth of 989 and had ctDNA detection rates of 19% and 28%, respectively.
3.2.3. Global sequencing of plasma
Two studies [16,25] that used global sequencing of plasma were identified (Table 3), both of which applied shallow whole-genome sequencing (sWGS). This untargeted method requires no prior knowledge of specific mutations in the tumor and has low sensitivity for single-base mutations but may identify large deletions and duplications. sWGS is relatively inexpensive and easy to perform. Mouliere et al [25] performed sWGS to a depth of 0.4 on 344 samples from 200 patients with various types of cancer including 33 with RCC (unknown stage of disease). ctDNA detection was based on analysis of cfDNA fragment lengths in cancer patients and healthy individuals. They found that in silico enrichment of fragments between 90 and 150 bp in size improved the detection of ctDNA across cancer types. However, in RCC the proportion of fragments <150 bp was very similar to that in healthy individuals, indicating a low amount of ctDNA in RCC. The ctDNA detection rate was 65% for ‘‘low-ctDNA cancers’’ (RCC, pancreatic cancer, and glioma).
Smith et al [16] applied the same in silico size selection of cfDNA fragments as Mouliere et al [25] and found that this improved detection of ctDNA. In a cohort of patients with various stages of RCC and a cohort of mRCC patients, they found that the ctDNA detection rate was 23% (11 of 48) and 33% (14 of 43), respectively, when the untargeted sWGS approach was combined with size selection.
Although there are some advantages of this untargeted and relatively less expensive method, the results suggest that it has relatively low sensitivity for RCC and that optimization is required.
3.2.4. Targeted methylation analysis of plasma
Targeted methylation analysis of plasma using a PCR-based method is very inexpensive and easy to perform. The disadvantage is that very few genes are analyzed and if the patient does not have a mutation in one of these genes, then the ctDNA test result will be negative. We identified five studies [9,28–31] that used this method (Table 4) and included between 27 and 157 patients with various stages of RCC. Four of the studies included a group of healthy controls.
In general, the authors concluded that methylation analysis provided high specificity but low sensitivity in distinguishing cancer patients from healthy controls. Hauser et al [28] and Skrypkina et al [29] performed a combinatorial analysis of multiple genes, which increased the diagnostic sensitivity and specificity. Lin et al [30] and Jung et al [31] investigated the methylation status of PCDH17 and SHOX2, respectively. Lin et al found that the presence of PCDH17 correlated with advanced disease stage, and Jung et al found that patients with SHOX2-positive plasma had a significantly higher risk of death.
PCR-based analysis can be much less expensive to perform than sequencing-based analysis but it only allows for the investigation of a limited number of mutations. Both sensitivity and specificity are often relatively low, as seen in the studies by Hauser et al [28] and de Martino et al [9]. Technically more sensitive methods such as digital droplet PCR might improve sensitivity for patients whose tumor has a mutation in the targeted genomic position [32]. However, it is likely that other methods targeting larger genomic regions are required for ctDNA detection in RCC.
3.2.5. Global methylation analysis
Three studies [18,26,27] investigated global methylation patterns (epigenetic aberrations) in cfDNA (Table 5). Epigenetic alterations are more abundant than genetic alterations in RCC, thereby providing more tumor-specific alterations. Global methylation analysis may thus achieve higher sensitivity. Cell-free methylated DNA immunoprecipitation and high-throughput sequencing (cfMeDIP-seq) is a bisulfite-free technique that can detect whole-genome methylation of cfDNA. The advantage of this technique is that it can enrich genome-wide CrG-methylated cfDNA with a low DNA input (<10 ng) [33]. cfMeDIP-seq was applied in two studies: Nuzzo et al [26] had a ctDNA detection rate of 97% (at the specificity of 100%) in a study of 69 patients with various stages of RCC, and Lasseter et al [18] had a ctDNA detection rate of 100% in a study of 34 patients with mRCC, although at lower specificity of 88%.
In a large study including more than 2000 patients with different types of cancer (including 81 patients with various stages of RCC), Liu et al [27] found that the ctDNA detection rate in RCC was low compared to other types of cancer. The study was conducted in two steps using whole-genome bisulfite sequencing data from 1531 cancer cases and 1521 control samples to build a classifier for ctDNA detection. The authors applied the classifier to a validation cohort of 654 cancer cases and 610 control samples. The detection rate in this study was 21% (specificity of 99.8%) in the training cohort and 12% (specificity of 99.3%) in the validation cohort, and ctDNA detection had increasing sensitivity with increasing stages of the disease.
These studies indicate that cfMeDIP-seq may be a promising strategy for ctDNA detection in RCC, but validation in larger studies is of utmost importance before any clear conclusions can be drawn.
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