Nowadays, prostate cancer is known as the most prevalent cancer and had the second-highest mortality rate following lung cancer in men (1). While substantial advancements in the field of prostate cancer diagnosis and treatments have been gained, the outcomes of current treatment have not been satisfactory enough. The majority of localized prostate tumors respond to radical prostatectomy well enough, but when the tumor becomes metastatic, the main treatment is androgen-deprivation therapy (ADT). Although the tumor responds to ADT immediately, the tumor fails to respond to that eventually. It has been shown that there is no chance of curing metastatic prostate cancer. The unknown mechanism behind the tumor progression and metastasis is the main cause of fail in prostate cancer treatment (1-5).
Liquid biopsy has been introduced as a desirable method for detecting biomarkers. The current method for diagnosis of malignancies is an invasive biopsy, which contains a variety of complications and the failure rate of invasive biopsy is not low. There are several reasonable reasons in support of the fact that liquid biopsy is a far better method for cancer diagnosis in comparison to invasive biopsy. The foremost benefit of liquid biopsy versus invasive biopsy is relatively less invasivity and thereby, a considerably lower rate of morbidity. Moreover, liquid biopsy can pertain more times than invasive biopsy, which provides us a good view of the malignancy and during the treatment, the resistance to the treatment can be identified. Liquid biopsy is capable of estimating the levels of some tumoral biomarkers including, but not limited to cell-free DNA (cfDNA), different RNAs, circulating tumor cells (CTCs), and extracellular vesicles (6, 7) (Figure1).
Various aspects of liquid biopsy in urological malignancies exclusively prostate cancer have been investigated so far. Herein, we made a narrative review of knowledge that has been achieved with regards to liquid biopsy in prostate cancer.
Figure 1. Schematic presentation of body fluids as the liquid biopsy sources
Although cfDNA has been introduced in 1948, utilizing it as a biomarker for cancer detection has drawn great amounts of attention in two recent decades. It can be released by the primary tumor or metastatic sites and more importantly, relatively low levels of cfDNA can be detected in individuals without any diseases (8). Circulating tumor DNA is a potential biomarker with a high capability to be detected easily, nonetheless, it has a severe limitation that restricted the wide usage that. It has been shown that it remains in the blood for the duration between 16 minutes until 13 hours after surgery or systematic treatment. One of the main obstacles that have been addressed is the method of detecting cfDNA (9). With evolving new techniques such as whole-genome sequencing and digital PCR, nowadays chasing cfDNA in body fluids is more accessible than before (8).
We found out that lots of studies have been conducted concerning cfDNA in prostate cancer. It is of importance to note that the main focus of previous studies was on detecting cfDNA in blood plasma, but, recent publications show that the other body fluids, for instance, semen and urine have been investigated in the recent literature (10) (Figure2). Variation in the cDNA's source and also, in the method of cDNA's detection are the two most important reasons behind differences in the cfDNA values in different studies (11).
Figure 2. Tumor released its cells (CTCs), DNA fragments (cfDNA), and proteins to the blood
Detecting cfDNA in blood plasma has been the mainstay of cfDNA detection and the majority of studies used blood plasma as a source of cfDNA. Blood sampling despite a very simple method, which provides an opportunity to scan the course of patients’ treatment, is not sensitive enough, which may lead to confusion. It has been demonstrated that low values of cfDNA in blood plasma can be detected in patients with benign prostate hyperplasia (BPH) (12-14) and even healthy subjects (15, 16). The detection sensitivity of cfDNA can be identified by two main factors: the reference gene and the method of measurement (12, 17). Since the levels of cfDNA in blood samples is not sufficient enough, it seems that quantitative PCR is the best option for measuring cfDNA in a blood sample (18-21). The study that was performed by Gordian et al demonstrated that for patients who have a prostate-specific antigen (PSA) between 4 and 10, patients with cfDNA values higher than 180 ng/ml have a high risk of prostate cancer, and finally, they suggested that measuring blood plasma cfDNA value will be beneficial to prevent unnecessary biopsy (19).
Several studies have been performed to compare the values of cfDNA in patients with prostate cancer in comparison to patients with BPH and healthy subjects (20, 22). Khani et al illustrated the significant associations between cfDNA level and integrity and prostate cancer. Moreover, they claimed that the values of blood cfDNA and its integrity were higher in patients with BPH than healthy subjects, however, these were not statically significant (20).
Recently, cfDNA is mainly measured by three methods: 1. spectrophotometric 2. fluorometry 3. quantitative PCR. First Muller et al in 2006 sought to estimate the levels of blood plasma cfDNA level by microsatellite analysis. They elucidated that prostate cancer patients have higher values of cfDNA than BPH patients and additionally, loos of heterogenicity were significantly higher in prostate cancer patients than BPH patients (34% vs 22%). Schwarzenbach et al tried to measured cfDNA levels in the bone marrow of prostate cancer patients. They used a new method to detect cfDNA of blood and bone marrow, called methylation-specific multiplex ligation-dependent probe amplification, which has the potential to detect genetic and epigenetic aberrations of about 37 tumor suppressor genes. At last, they matched 13 samples of blood and bone marrow and found out that the incidence of genetic changes in blood samples (25) was higher than in bone marrow aspirates (11).
Measuring urine cfDNA using this method has been investigated by Casadio et al. They measured urine cfDNA and its integrity in 29 prostate cancer patients and 25 healthy individuals. Sensitivity and specificity cfDNA integrity was estimated to be about 79% and 84%, respectively with considering the cut-off value of 0.04ng/μL, and therefore, they concluded that urine cfDNA integrity can be used for early diagnosis of prostate cancer in clinical practices (23).
For the first time, Jung et al used fluorometry on 59 healthy individuals, 34 patients with BPH, and 91 prostate cancer patients to measure cfDNA. They found out that patients with localized prostate cancer had the same levels of cfDNA as healthy individuals had. On the other hand, the values of cfDNA in patients with metastasis, lymph node involvement, and even BPH were much higher than the normal range. The authors proposed that the value of plasma cfDNA is a potent biomarker for predicting prostate cancer patients (13).
Fluorometry as a method for measuring cfDNA levels in urine samples has been evaluated by Xia et al. They assessed before and after treatment copy number variations of urine cfDNA in patients who had undergone docetaxel and androgen deprivation treatment. A significant decrease in copy number variations in 34 genomes occurred after the treatment in comparison with before the treatment (24).
Ponti et al have focused on the detection of cfDNA in semen. They demonstrated in 2018 that cfDNA levels of semen in patients with prostate cancer are significantly higher than in patients with BPH and healthy subjects (25). They designed another study and reached the same findings. They also claimed that the prevalence of cfDNA fragments, which are longer than 1000 base pairs, in prostate cancer patients are significantly higher than BPH patients (26).
In 2004, Allen and her colleges designed a study to investigate cfDNA in prostate cancer using quantitative PCR. They measured blood plasma β‐globin of 37 prostate cancer, intraepithelial neoplasia, and BPH patients in three different intervals including once before prostate biopsy and 60 minutes, and two weeks later the prostate biopsy. Prostate cancer and intraepithelial neoplasia patients had significantly higher values of cfDNA in comparison to the other group (27). The association between blood plasma cfDNA and the stage of prostate cancer has been investigated. In patients without prostate cancer metastasis or lymph node involvements, blood plasma cfDNA has a significant association with stage three of prostate cancer, thereby, it can be considered as a marker for prostate cancer monitoring (28). Several studies have been conducted to distinguish between benign and malignant lesions with DNA integrity, which is measured by quantitative PCR (18, 29). Feng et al measured blood plasma cfDNA and its integrity of 96 prostate cancer patients and 112 BPH patients, whose PSA was higher than 4 ng/ml. The cfDNA distinguishes between BPH and prostate cancer with the sensitivity and specificity of 73.2% and 72.7%, respectively, and DNA integrity with higher sensitivity (81.7%) and specificity (78.8%) can distinguish (18).
Circulating tumor cells
Stem cells are capable of self-renewing, hence they can survive throughout the host lifetime. This feature constitutes the rationale for stem cells to be the source of cancers. In the early 1960s, some studies showed that cancers are comprised of cells, which are vary in self-renewal capacity. So, they concluded that the origination of cancers may be several cells, named CTCs (30-33). Prostate cancer is composed of different cells and the histo-structure of prostate cancer in various stages is different as well. In early prostate cancers, both differentiated and undifferentiated cells can be observed, but the majority of cells are differentiated. In contrast to undifferentiated areas, cells, which are located in differentiated areas are positive for PSA and androgen receptors (AR). For late stages of prostate cancers, undifferentiated areas are more prominent than differentiated areas (34-36). Metastatic lesions of prostate cancer have been assessed and interestingly, they have consisted of different cells similarly. Apart from heterogenicity in prostate cancer structure, heterogenicity in mutations and chromosol rearrangement has been detected (37, 38).
CTCs can be detected through several methods, however, the main focus of most of the studies was toward reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC) (39, 40). Several extracellular markers, including, but not limited to, CD117/c-kit, CD133, CD44, α2β1 integrin/ α6 integrin have been evaluated.
In 2014, the predictive ability of some markers especially CD117/c-kit was assessed by Ker et al. blood serum concentrations of markers before radical prostatectomy and 1-3 months after the surgery were calculated. Among investigated markers, the serum values of only CD117/c-kit decreased significantly following the surgery. The authors reached positive correlations between CD117 and tumor progression and PSA values. At last, they clarified that if prostate tumors are enriched with CD117, they become more aggressive (41).
It has been proposed that CD117 may cooperate with BRCA2 in bone metastasis of prostate cancer patients. It is of proven value that BRCA2 loss led to tumor progression and metastasis in prostate cancer patients. Mainetti et al illustrated expression of CD117 with loss of BRCA2 triggers prostate cancer cells to migrated into the other side of the body especially bone (42-44).
CD133 expression was measured in 17 healthy subjects,17 patients with high-grade intraepithelial neoplasia, and 65 patients with prostate cancer. It was expressed in 67 prostate cancer patients, 5 of a healthy subject, and none of the high-grade intraepithelial neoplasia patients (45). In another study, the expression of four markers in particular CD1333 was estimated in 38 prostate cancer patients. CD133 was expressed with the normal range or slightly higher than the normal range. Also, it has a positive association with Gleason score (46). Fan et al determined to evaluate the resistance of cells with expression of CD1333 to cisplatin and demonstrated that these cells are more resistant to cisplatin (47).
The role of CD44, which is a multipotential protein, in the proliferation of prostate cancer has been illustrated. Patrawala et al demonstrated that prostate tumors that are positive for CD44 when compared to those which are negative for CD44 are more tended to progress and be metastatic. Additionally, they claimed that prostate tumors positive and negative for CD44 and AR, respectively, are potent to differentiate into tumors positive for both CD44 and AR (48). For prostate cancer to be metastatic, Matrigel through epithelial-mesenchymal transition should be invaded, which has been attributed to prostate cancers positive for CD44, thereby the existence of CD44 for tumor metastasis is required (49). Moreover, prostate cancer positive for CD44 through the Wnt pathway can lead to failure to respond to chemotherapy in patients with castration resistance prostate cancer (50).
Expression of α2 integrin and α6‐integrin, stem cell markers, in 461 prostate cancer patients have been assessed. α2 integrin and α6‐integrin were expressed in 94.7% and 28.4% of the patients. Significant relationships between α2-integrin and α6‐integrin expression and PSA greater and lower than 10ng/ml, respectively, have been detected. Furthermore, α6‐integrin associated significantly with Gleason scoreα6‐integrin maintains a predictive value to diagnosis a local recurrence of the tumor (51). Assessing the expression of these two markers and c-Met in the bone marrow of patients with prostate cancer revealed significant associations between expression greater than 0.1% of the three markers and tumor metastasis, stages, and death related to the tumor (52).
Lines of research stated that a variety of RNAs maintain a mandatory role in protein production, which is a complex process. Ample evidence is existing in support of the fact that non-coding RNAs (ncRNAs), which are produced as a result of continuous genome transcription, are implicated in several diseases in particular cancers (53-55). Non-coding RNAs are consist of two major subgroups short non-coding RNAs and long non-coding RNAs (LncRNAs). Different categorizations of LncRNAs currently exist. For instance, one of the well-known categorizations of LncRNAs is based on the location of LncRNAs on the genome (56).
Prostate cancer antigen 3 (PCA3) is a LncRNA that only express in prostate glands and has been known as a marker that controls the survival of prostate cancer cells through moderating AR signaling. The most important PCA3 advantageous over PSA is the situation, for example, BPH that impacts the values of PSA are not able to change the values of PCA3 (57-59). Groskopf and his colleges measured urine values of PCA3 and PSA of 52 healthy subjects (age predictive ability of LncRNAs in prostate cancer was evaluated since the Gleason score contains several limitations (61). The values of SChLAP1 in patients who experienced radical prostatectomy, which is a LncRNAs, increases as prostate cancer progresses, and more importantly, for patients with high concentrations of SChLAP1 before the surgery, worsen outcomes following the surgery can be expected (62). The expression of PCAT14, another LncRNA, in 585 prostate cancer patients was investigated. Negative associations between LncRNA expression and Gleason score, metastasis, cancer-related death, and biochemical recurrence were achieved in two different studies (63, 64). Prostate cancer tissues contain higher values of urothelial carcinoma-associated 1 (UCA1) than normal prostate tissues. The inverse association between the level of UCA1 and the prognosis of patients with prostate cancer was observed. Likely, KLF4, which had a significant relationship with UCA1, expressed in higher concentrations in patients with prostate cancer than healthy subjects (65). The prostate tumor which is positive for UCA1 in comparison with negative ones is more resistant to radiotherapy (66).
Exosomes are a well-validated component of normal and tumoral cells component. The main roles of exosomes are communication between cells and transmission of materials, for instance, DNAs, RNAs, and proteins necessitates for cells ‘survival (67). According to several years of experience, they are responsible for prostate cancer metastasis, resistance to chemotherapy, and development. As mentioned above, they can be secreted from tumoral cells, and these exosomes through impacting recipient cells, provide situations, which are desirable for prostate cancer development and metastasis (68, 69). The mechanism responsible for inducing prostate cancer progression and metastasis was explained by Beheshti and his co-workers. They pointed out that prostate cancer exosomes cause apoptosis reduction, enhancement in the progression of prostate cancer cells, and transmission of proteins with the ability to prevent apoptosis (70). In another study, it was proposed prostate cancer exosomes through an interruption in immune system function may lead to tumor progression and metastasis (71). Prostate cancer exosomes are capable of transferring αvβ6 integrin, a marker that only exists on prostate tumoral cells, to cells negative for this integrin, which leads to enhancement in cell adhesion and migration (72). Exosomes are potent in influencing epithelial-mesenchymal transition, which is an inseparable part of tumor metastasis.
Exosomes can enhance prostate cancer resistance to drugs and chemotherapy. In the comprehensive review that was performed by Min et al, drug efflux, comprising genes with drug resistance capacity, and cross-talk between pathways of cellular signaling are proposed as the mechanisms that cause drug resistance in patients with different cancers exclusively prostate cancer (73). Exosomes involve in bone metastasis of prostate cancer. Exosomes, which are derived from prostate cancer, can prohibit osteoclast function and differentiation and in contrast, stimulate the function of osteoblast. Hence, they have a major role in osteoblastic bone metastasis (74).
Former research expressed that microRNAs (miRNAs), a well-known ncRNA (75), may participate in tumor growth, progression, differentiation, and apoptosis (76). The main functions of miRNAs are attributed to two subgroups of miRNAs including micro-miRNAs and tumor-suppressor miRNAs. miR-21 has been proven to act as an ono-miRNAs in prostate and bladder malignancies. (77) miR-125 is the other example of onco-miRNAs in prostate cancer (78), however, for breast cancer patients, it acts as a tumor suppressor (79).
Some studies have addressed the predictive value of exosomal miRNAs (80-82). Li et al designed a study to investigate different aspects of miR-141. First, they showed that prostate cancer patients had higher values of miR-141 than healthy individuals and even BPH patients. Values of miR-141 in metastatic patients were also higher versus patients with localized prostate cancer. They found out that this marker can distinguish between localized and metastatic prostate cancers with a sensitivity of 80% and specificity of 87.1% (82).
Survival of castration-resistant prostate cancer patients can be predicted by using the combination of miR-1290 and miR-375. 20 months of follow-up showed that patients who have higher values of miR-1290 and miR-375 in comparison to patients whose values of those are within the normal range have significantly higher mortality rates (83).
Liquid biopsy which primarily includes cell-free DNA, circulating tumor cells, RNAs, and exosomes in can change the prostate cancer diagnosis for the better.
All authors contributed equally. All authors reviewed and approved the final version of the manuscript.
Special thanks to the Department of Urology, Isfahan University of Medical Sciences, Isfahan, Iran.
Conflict of interest
The authors declare that there is no conflict of interest.
The authors received no financial support for this research.
Data will be provided by the corresponding author on request.
ADT Androgen deprivation therapy
cfDNA Cell-free DNA
CTC Circulating tumor cell
PSA Prostate-specific antigen
UCA1 Urothelial carcinoma-associated 1