In this study, we observed that the concordance rate of somatic mutations between CTC and corresponding primary tumor genomes was relatively low, suggesting that CTC genomes might have undergone distinct evolutionary pathway compared to primary tumors. Supporting this, CTC-specific mutations are distinct from common or primary-specific mutations in terms of sequence compositions (i.e., mutation signatures) and clonality measures such as allele frequencies, suggesting that mutational forces for CTC genomes might be distinct from those associated with primary tumors. In addition, cancer-related genes affected by CTC- and primary-specific mutations were also different from each other.
Research on clinical relevance of ‘liquid biopsy’ has been recently highlighted. CTC may be used as a substitute for tissue biopsy to evaluate drug responsiveness and predict an optimal therapy [22, 23]. CTC-driven genomic or transcriptomic findings may provide valuable information for predicting patients’ prognosis and further guide personalized treatment decision-making in bladder cancer. Until now, primary tumor specimens have served as major cellular sources to obtain genetic information. Some studies have compared genomic or transcriptomic profiles and reported a substantial level of heterogeneity between primary and matched metastatic lesions of given individual [24, 25]. Our study performed analyses of genomic profiles of cultured CTCs and corresponding primary tumor tissue. The current study presented some meaningful results. As demonstrated here, genomic profilings of primary tumors and CTCs are very different in other studies. Reports for colorectal cancer showed similar results. For example, Lyberopoulou et al. [26] have observed 52 patients with colorectal cancer and found discordance between primary tumor and CTCs for KRAS, BRAF, CD133, re3130, and Plastin3 rs6643869. This discordance was confirmed by Kondo et al. [27] and a recent meta-analysis including nine studies and 244 patients [28]. We expect that mutational heterogeneity of CTCs and corresponding primary bladder tumor may recapitulate the relationship between primary and metastatic lesions given that CTCs are major sources for distant metastases. To cope with metastasis and disease progression, genetic information from CTCs might be complementary for longitudinal management of bladder cancer.
To explain the discordance between CTCs and corresponding primary tumor, we need to pay attention to intra-tumor heterogeneity (ITH) and CTCs heterogeneity. Modeling genomic or mutational diversity between regional biopsies in a given individual using a tree structure of tumor growth has been proposed as a ‘trunk-branch model’ [29]. The trunk and branch/leaf in the tree represent founding ubiquitous driver mutations present in every tumor subclone and region and regionally heterogeneous mutations that are not present in every tumor cell or tumor region, respectively [29]. The number of ubiquitous and heterogeneous genetic events in the tumor can depend on the length of the trunk and size of the branch of the phylogenetic trees inferred from mutation profiles and their regional distribution. Thus, as disease progresses, in addition to mutations detected in primary tumors, CTC genomes can acquire mutations in a similar evolutionary process where minority subclones emerge from pre-existing clones. Tumor cells can shed from different and many other tumor sites. Hence, mutation profiles of CTC genomes may be substantially different from those of primary tumors as composite of genetically distinct tumor subject to ITH [30]. Thus, single site tumor biopsy may underestimate the clonal landscape of the overall tumor burden.
Among mutations commonly observed in both CTCs and corresponding primary tumors, KMT2C mutations were detected in five patients. All these five patients (patients TCC 17,28,31,38,45) were shown to be pathologically T2 or higher stage (muscle invasive bladder cancer). KMT2C gene has been frequently observed to be mutated in muscle invasive bladder cancers [19]. On the contrary, KMT2C mutations in CTCs were not observed in patients with T1 or Ta disease. In clinical practice for bladder cancer, T2 disease has a great clinical significance in decision-making for treatment. Although it requires further supporting evidence, the correlation of disease stages and certain mutations such as KMT2C is one of major applications in using CTCs for clinical practice.
To the best of our knowledge, this is the first study to conduct mutation-based comparison between CTCs and primary corresponding tumor mutations using whole-exome sequencing in bladder cancer. Although a number of studies have used high-throughput sequencing to characterize CTCs for bladder cancers [31], their results are only for CTC, not amenable for comparison between CTC and primary tumors. In addition, the present study suggests that the application of sequencing or CTCs could provide a potential clinical role to investigate biologic targets by peripheral blood sampling. Genetic characteristics of CTCs and corresponding primary tumor were shown to differ. Thus, identifying only genetic characteristics of primary tumors is not enough to cope with the disease's progression. Clearly, CTCs are expected to play an important role. The need for genetic analysis using liquid biopsy such as CTCs can also play a role in handling bladder cancer in addition to convenience of sampling in liquid biopsy.
One limitation of this study is an incomplete establishment of CTC culture. Despite the importance of CTCs in prognosis of cancer patients, the clinical utility of CTCs was limited by its rareness in blood (one CTC in a billion normal blood cells) [32]. For this reason, expanding CTCs to large numbers is encouraged for performing CTC genotyping and phenotyping despite there are concerns that in vitro culture of CTCs may change characteristics of bona fide CTCs. It can be questionable whether cultured CTCs originating from primary cancer could retain their original identification. To verify if cultured CTCs could retain their original identification of primary bladder cancer, we have previously verified that cultured CTCs could retain their original identification of primary bladder cancer by applying FISH method although further studies are needed [33].