Journal of the Scientific Society

REVIEW ARTICLE
Year
: 2021  |  Volume : 48  |  Issue : 3  |  Page : 124--134

Liquid biopsy: A paradigm in diagnostic, predictive, and prognostic marker in urological malignancies


Shreya Chandra1, Shoubhik Chandra2, Shridhar C Ghagane3, Rajendra B Nerli2,  
1 Department of Urology, Division of Urologic-Oncology, KLES Dr. Prabhakar Kore Hospital and Medical Research Centre, Belagavi, Karnataka, India
2 Department of Urology, JN Medical College, KLE Academy of Higher Education and Research, JNMC Campus, Belagavi, Karnataka, India
3 KAHER‟s Dr. Prabhakar Kore Basic & Applied Science Research Center [BSARC], V. K. Institute of Dental Sciences Campus, Belagavi, Karnataka, India

Correspondence Address:
Dr. Rajendra B Nerli
Department of Urology, JN Medical College, KLE Academy of Higher Education and Research, JNMC Campus, Belagavi - 590 010, Karnataka
India

Abstract

Due to the recent rise in the trend of urological malignancies, noninvasive tumor biomarkers are being researched and used for many different clinical settings. Thus, the identification of specific and effective biomarkers in the form of liquid biopsy has become a major focus, mainly due to the necessity of selecting potentially responsive patients and predicting their outcomes. The rationale for using liquid biopsies instead of solid tissue samples is to avoid unnecessary invasive procedures which will provide the same diagnostic information. The main liquids tackled in patients with urological malignancies are blood and urine. In this review, we provide a detailed discussion on the potential clinical utility of diagnostic materials found in these liquids and will focus on cell-free DNA and other circulating molecules, such as circulating tumor cells, RNAs (microRNAs, long noncoding RNAs, and messenger RNAs), cell-free proteins, peptides, and exosomes as cancer biomarkers.



How to cite this article:
Chandra S, Chandra S, Ghagane SC, Nerli RB. Liquid biopsy: A paradigm in diagnostic, predictive, and prognostic marker in urological malignancies.J Sci Soc 2021;48:124-134


How to cite this URL:
Chandra S, Chandra S, Ghagane SC, Nerli RB. Liquid biopsy: A paradigm in diagnostic, predictive, and prognostic marker in urological malignancies. J Sci Soc [serial online] 2021 [cited 2022 Jan 26 ];48:124-134
Available from: https://www.jscisociety.com/text.asp?2021/48/3/124/333854


Full Text



 Background



Urological malignancies account for approximately 14% of all cancers in developed countries,[1] with cancers of prostate cancer antigen (PCA), bladder cancer antigen (BCA), and kidney being the 10 most frequent cancer entities in men. Due to the recent rise in the trend of urological malignancies, liquid biopsy in the form of tumor biomarkers is being used for the diagnosis of disease, identification of prognostic or predictive biomarkers, and for surveillance and monitoring of posttreatment patients. The term liquid biopsy refers to the use of blood and other bodily fluids as a surrogate of tissue samples for diagnostic purposes with a rationale to avoid unnecessary invasive procedures while providing the same diagnostic information.[2] Liquid biopsy also helps in selecting potentially responsive patients and predicting their outcomes. The pathologist has to integrate information from the data obtained from different sources to achieve a final diagnosis.[3]

Precise molecular biomarkers are hence urgently needed in the era of personalized medicine to support clinical decision-making. Excisional biopsies do not truly demonstrate tumor heterogeneity, both within a tumor and between primary tumor and metastases. Moreover, during monitoring/therapy, repeated biopsy is often painful and risky leading to nonfeasibility. In contrast, a liquid biopsy reflects the genetic profile of all tumor subclones present in a patient.[4] It also gives an opportunity to take serial samples to monitor tumoral changes. Meanwhile, it allows the clinician to administer a personalized therapy to the patients.[5] Liquid biopsy is capable of replacing or at least augmenting the use of invasive biopsy which has limited success and associated complications.[6] The main liquids employed in patients with urological malignancies are blood and urine.

In this review, we aim to provide a detailed discussion on the potential clinical utility of diagnostic materials found in these liquids. We will focus on cell-free DNA (cfDNA), circulating tumor cells (CTCs), RNAs (microRNAs [miRNAs], long noncoding RNAs [lncRNAs] and messenger RNAs [mRNAs]), cell-free proteins, peptides, and exosomes as cancer biomarkers for malignancies of kidney, bladder, and prostate.

Tumor cells and their components in body fluids

Tumor cells can reach the bloodstream from their original location through blood and/or lymphatic vessels located in the tumor stroma or by vascular invasion. Via the lymphatics, tumor cells may reach the bloodstream, making CTCs and cfDNA detectable.[3] Tumor itself induces angiogenesis and also forms sinusoidal spaces where tumor cells protrude into the lumen making the tumor cells shed cell components and circulating tumor DNA (ctDNA) directly into the bloodstream.

The genitourinary tumors such as urothelial neoplasms of the renal pelvis, ureters, bladder, and urethra; renal cell carcinomas (RCCs); and prostate cancers shed cells, proteins, and nucleic acids in the urine.[7] The filtering membrane of kidney is permeable only to low-molecular weight DNA fragments, and therefore, the mean size of the cfDNA fragments released in the urine is approximately 50–100 base pairs (70 KDa). This information is relevant to assess the source of urinary-free DNA. The high-molecular weight DNA found in urine speculates DNA derived from whole cells released by the urothelium or by renal tubules.[8] Since the prostate gland drains into the urethra, the genetic material from PCA that has spread within preexisting ducts and acini is easily detected in voided urine.[9] The highest cell yield has been noted in the first voided morning urine but should be processed immediately to avoid the degradation by nucleases. If not processed immediately, urine should be stored at 4°C or frozen at −20°C.[10]

 Cell-free DNA



Although cfDNA was first identified in 1948, it has only recently been investigated as “liquid biopsy” as a cancer biomarker.[11] Tumors release DNA fragments into circulation that contain tumor-specific alterations including point mutations, copy-number variation, and DNA methylation. cfDNA can be derived from both apoptotic and necrotic cells in patients harboring cancer but are also detected in healthy individuals in very low concentrations. Classic methods for cfDNA assessment include polymerase chain reaction (PCR)-based approaches. More recently, digital PCR (dPCR) has emerged as a sensitive tool for the detection of point mutations in cfDNA. Targeted and whole-genome sequencing technologies are also increasingly being applied to cfDNA analysis.

Clinical applications of cell-free DNA

cfDNA has a wide range of diagnostic, prognostic, and predictive applications, as tabulated in [Table 1]. Circulating cfDNA levels, their integrity, methylation, and mutational status are being researched and studies have shown that cfDNA has great potential clinical utility for kidney, bladder, and prostate cancers.{Table 1}

Cell-free DNA levels

Various studies have been conducted which emphasized cfDNA of having promising diagnostic and prognostic applications in cancer. A study found that plasma cfDNA levels were lower in sorafenib-treated RCC patients with remission compared to those who progressed. Higher cfDNA levels during the course of treatment pointed towards a poor prognosis.[12] Another study found elevated plasma cfDNA levels in metastatic RCC compared to localized disease and predicted postoperative recurrence with 91% sensitivity and 100% specificity.[13] Urinary cfDNA levels were significantly elevated in bladder tumors,[14] whereas plasma cfDNA levels were elevated in prostate cancer relative to controls and benign prostatic hyperplasia (BPH) patients,[15],[16] indicating that cfDNA levels served as a diagnostic marker for bladder and prostate cancer.

Cell-free DNA integrity

cfDNA is derived from both apoptotic and necrotic cells in patients harboring cancer. In healthy people, it predominately originates from the apoptotic cells and is highly fragmented. However, DNA from disintegrating/necrotising tumour cells is made up of longer protein fragments.[17] cfDNA integrity evaluates the extent of cfDNA fragmentation and is calculated as the ratio of long-to-short cfDNA fragments derived from necrotic and apoptotic cells (necrotic/apoptotic), respectively.[18] Serum of RCC patients showed elevated cfDNA integrity relative to controls which increased proportionally with higher stage (T3) and larger tumor size (>4 cm).[19] A study used urine cfDNA integrity as a marker for early diagnosis of bladder cancer as its levels remained elevated in BCA patients compared to controls.[20] However, few reports of decreased cfDNA integrity in BCA patients have caused doubts about its diagnostic utility.

cfDNA was reported to be released by apoptotic and nonapoptotic cell death, before and 3 months after PCA diagnosis, whereas it was released only by nonapoptotic cell death 6 months after diagnosis, indicating that cfDNA can be used to follow the evolution of disease.[21] A study showed elevated urine cfDNA integrity in PCA with 79% sensitivity and 84% specificity.[17] However, in another study, PCA patients were found to have contrastingly lower cfDNA integrity in serum relative to BPH and healthy individuals.[22]

To summarize, many studies observed a cancer-associated elevation in cfDNA integrity suggestive of necrotic cell death, but few contradicted this by mentioning the presence of more fragmented cfDNA and hence lower cfDNA integrity in cancer. The presence of more fragmented cfDNA in BCA and PCA was a result of cancer-induced apoptosis of peripheral noncancerous tissues. A study mentioned cfDNA fragmentation to display a stepwise increase with increasing histological grade,[22] again suggesting that high-grade tumors may disrupt peripheral tissues resulting in increased apoptosis.

Circulating tumor DNA

ctDNA is a highly fragmented (180–200 base pairs), reliable, and easily accessible biomarker having a short half-life (ranging from 16 min-13 h) which gets rapidly cleared from circulation following surgery or systemic therapy.[23] Analysis of ctDNA requires highly sensitive techniques.

Epigenetic changes

Epigenetics is defined as “a stable heritable phenotype resulting from changes in a chromosome without alterations in the DNA sequence.”[24] Epigenetic changes include DNA methylation, histone modification, and noncoding RNA-associated gene silencing which initiate and sustain cellular epigenetics and have been identified in virtually all human cancers.[25] DNA hypomethylation activates oncogenes and chromosomal instability, whereas tumor suppressor genes are silenced by DNA hypermethylation. Epigenetic changes occur at the early stages of cancer initiation and accumulate during cancer progression. Thus, the detection of specific epigenetic modifications is being used for the molecular diagnosis of cancer and for prognostic purposes. The identification of epigenetic alterations in urological malignancies is now observed in urine and blood.

Cell-free DNA methylation

DNA methylation is chemically stable and can be accurately and sensitively measured in plasma, serum, and urine.[26] The quantification of methylated DNA fragments using PCR is performed after chemical treatment with bisulfite. Methylated/unmethylated DNA sequences are then discriminated using specific primers methylation-specific PCR (MSP). Enzymatic treatment is also possible, but they may lead to incomplete digestion impairing the specificity.

Cairns[27] analyzed methylation of six tumor suppressor genes in urine and concluded that promoter hypermethylation is diagnostic of early events in organ-confined kidney cancer. Serum cfDNA showing CpG island hypermethylation was frequently observed in patients with RCC with sensitivity of 63% and specificity of 87%.[28] Another study showed serum-containing methylated von Hippel–Lindau (91%) and RASSF1A (93%) DNA notifying of RCC.[29]

Methylation levels of POU4F2 and PCDH17 in urine discriminated BCA from healthy volunteers with 90% sensitivity and 94% specificity.[30] Plasma samples of 27 BCA cases showed methylated DNA sequences in p16INK4a (18%) and p14ARF (56%) having a positive correlation with tumor grade and stage.[31] Urine TWIST1 and NID2 methylation differentiated BCA patients from controls with a combined sensitivity of 90% and 93% specificity.[32] DNA hypermethylation at PCDH10 and PCDH17 correlated with stage and grade in BCA acting as independent predictors of cancer-specific survival patients.[33],[34]

Hypermethylation of genes RASSF1, GSTP1, and RARB2 was found in the serum of PCA patients.[35] Plasma level of methylated GSTP1 DNA showed a reduction following chemotherapy, indicating its potential as a predictive marker for chemotherapy response.[36] A positive correlation of serum GSTP1 hypermethylation and high Gleason score was also established.[37] RASSF1A, RAR-beta2, and GSTP1 hypermethylation were also correlated with the cumulative Gleason score, prostate-specific antigen (PSA), and advanced AJCC tumor stage.[35] Increased plasma cfDNA methylation of SRD5A2 and CYP11A1 in PCA patients with biochemical recurrence following radical prostatectomy indicated aberrant cfDNA methylation as an early predictor for disease recurrence.[38]

Cell-free DNA mutations

Somatic DNA mutations and chromosomal aberrations trigger cancer initiation and progression and when released into circulation, these mutations in cfDNA become detectable in blood and urine. Chromosome 3p microsatellite alterations in plasma DNA and urinary telomerase reverse transcriptase (TERT) promoter mutations were found to correlate with bladder cancer recurrence,[39] while KRAS2 mutation in plasma proved as an early diagnostic marker.[40]

A panel of chromosomal variations detected in serum could discriminate benign prostatic hypertrophy and prostatitis from PCA with 90% accuracy.[41] Newly occurring focal somatic copy-number alteration status (androgen receptor [AR] and MYC) was reported in 40% of patients with progressing metastatic PCA, indicating their value as prognostic biomarkers.[42] High-level AR copy-number gain in plasma served as a prognostic marker in castration-resistant prostate cancer (CRPC) patients.[43] Plasma AR mutations were detected in metastatic (CRPC) who were enzalutamide and abiraterone resistant.[44] Copy-number variation of serum CYP17A1 and AR genes was assessed in metastatic CRPC patients receiving docetaxel-based chemotherapy followed by abiraterone. It was found that patients with copy-number gain had shorter progression-free survival and overall survival compared to patients with no gain, suggesting that AR and CYP17A1 copy-number gain acted as useful markers for abiraterone resistance.[45] Although the diagnostic accuracy of specific cfDNA mutations is high, the detection of rare variants can be challenging as tumor-specific mutations can be as low as 0.01% of total cfDNA.

 Circulating Tumor Cells



CTCs in peripheral blood may occur as one cell/100 million cells that are circulating in the blood originating from the primary tumor or metastatic foci.[46] Thus, highly sensitive and specific analytical tools are needed for their detection, identification, and characterization. CTC counts predict disease burden whereas changes in CTC counts during the course of systemic therapy indicate treatment response.[47]

Patients of RCC with advanced stage and aggressive phenotypes showed elevated blood CTC levels correlating with lymph node status and presence of metastasis.[48] CTC enumeration also correlated with RCC progression and CTC-vimentin expression status.[49]

Circulating urothelial cancer cells served as a marker for metastatic bladder cancer detected by the CELLSEARCH™ assay.[50] High levels of serum and urine CTCs were detected in patients with urothelial carcinoma with 82% sensitivity and 62% specificity.[51] A study found CTC count in peripheral blood to be a predictor of early urothelial carcinoma recurrence and cancer-specific and overall mortality.[52]

The Food and Drug Administration (FDA) has approved the CELLSEARCH® CTC Test to monitor patients with metastatic PCA. This test counts CTCs of epithelial origin (CD45, EPCAM+, and cytokeratins 8, 18+, and/or 19+) in whole blood.[53] CTCs were detected at high frequency in CRPC patients correlating with clinical outcomes.[54] In a phase III clinical study, patients with metastatic CRPC after three cycles of docetaxel–lenalidomide chemotherapy showed elevated CTC levels predicting poor survival.[55] CellSearch™ is currently the only FDA-approved CTC assay for prognostic evaluation of prostate, breast, colon, and lung carcinomas.

Due to variation in sensitivity and specificity, CTCs have not yet been fully accepted into clinical practice to guide treatment decisions. In addition, only a few instruments have reached the certification level for in vitro detection of CTCs. Therefore, researches are being done to analyze CTC content (e.g. miRNAs) as cancer biomarkers.[56]

 Circulating RNAs



Circulating RNAs such as mRNAs, miRNAs, and lncRNAs serve as potential noninvasive cancer biomarkers. Many studies concluded to find altered levels of circulating RNAs in cancer, returning to normal postsurgery suggesting a tumor-associated release of RNA molecules.[57],[58]

microRNAs

miRNAs are short noncoding RNAs, 21-23 nucleotides long that regulate gene expression by pairing to the 3′untranslated region of their target mRNA.[58] Their association with cancer has been well appreciated as they play a key role in tumorigenesis, tumor progression, and metastasis.[59] Circulating miRNAs can be used as noninvasive cancer biomarkers due to their presence in blood, urine, saliva, tears, and cerebrospinal fluid.[60],[61]

Studies reported raised serum levels of miR-210 in RCC patients relative to controls.[62],[63] Raised circulating miR-221 was used as a biomarker for RCC metastasis and elevated miR-221 with miR-222 in plasma was used to distinguish patients harboring RCC from controls.[64] Elevated urine miR-15a levels marked RCC patients, nearly undetectable in oncocytoma.[65] Elevated urinary miR-126 levels were noted in BCA patients[66] with significantly high urinary miR-146a-5p associated with tumor grade and depth of invasion.[58]

Four downregulated miRNAs and six upregulated miRNAs were identified in the sera of patients with PCA.[67] Elevated serum miR-21 levels were noted in patients with hormone-refractory prostate cancer, especially in those resistant to docetaxel-based chemotherapy.[68] In general, miRNAs have shown a great promise as cancer biomarkers. Many consistent reports have identified circulating miR-210 and miR-126 as diagnostic markers of RCC and BCA, respectively, and miR-21 as a prognostic marker for PCA. Although these studies support the use of circulating miRNAs as biomarkers, they have yet to be clinically validated.

long noncoding RNAs

LncRNAs are >200 nucleotides long which regulate gene expression at transcriptional, posttranscriptional, or epigenetic levels.[69] These get altered in cancers and promote tumor formation, progression, and metastasis.[70] A panel of five circulating serum lncRNAs (lncRNALET, PVT1, PANDAR, PTENP1, and linc00963) differentiated benign renal tumors from ccRCC.[71] Urinary sediments with elevated UCA1 levels were identified as a potential diagnostic marker for BCA with 81% sensitivity and 92% specificity.[72] However, elevated blood UCA1 was observed in advanced BCA patients after cisplatin-based combination chemotherapy[73] making circulating UCA1 a promising biomarker for BCA diagnosis and therapeutic monitoring.

PCA3 has been the most credible example because of its specificity as a urine biomarker for prostate cancer. PROGENSA PCA3, an FDA-approved noninvasive urine test, guides the urologist and aids to take a decision for repeat biopsy in men of age 50 years and older, who have had ≥1 previous negative prostate biopsies.[74],[75] Hypermethylation of lncRNA H19 in peripheral blood could distinguish PCA from controls,[76] whereas elevated plasma PCAT18 levels were noted in metastatic PCA.[77] Plasma MALAT1 levels were elevated in PCA and its elevated levels in urine prevented approximately 30%–46% of unnecessary biopsies in patients with serum PSA of 4–10 ng/mL.[78]

Due to their tissue and cancer-specific expression patterns, circulating lncRNAs are now a new class of noninvasive promising cancer biomarkers in the field of clinical diagnostics. The application of cell-free UCA1 and MALAT1 as diagnostic biomarkers for bladder and prostate cancers, respectively, has now been well accepted, but a more detailed awareness of their biology is still required.

Messenger RNAs

Few reports dating back to the 1990s emphasized the association between cancers and circulating mRNAs.[79] They are considered cancer biomarkers as they translate the intracellular proteins and reflect intracellular processes. However, most of the mRNAs get degraded by RNAses leaving only a few stable mRNAs in circulation which get coupled with proteins and/or lipid carriers.[80],[81] Urine CAIX splice variant mRNA showed high diagnostic performance for kidney, prostate, and bladder cancers.[82] B7-H3 mRNA in peripheral blood was significantly elevated in metastatic RCC.[83]

BCA patients showed significantly high urine UBE2C and CK20 mRNA levels.[84] CK20 was ascertained as a potential diagnostic marker for urothelial carcinoma which increased proportionally with tumor grade and stage.[85] Urine hTERT mRNA was found as a potential marker for early diagnosis and prognosis of BCA[86] and also as a marker of poor prognosis in PCA.[87] Whole blood AR-V7 levels served as predictive markers and treatment selection for PCA as they were associated with response to abiraterone treatment in metastatic CRPC.[88] Due to their lack of stability and inter-individual variability, circulating mRNAs have not been fully incorporated into clinical practice.[89] However, many reports have established the potential utility of AR-V7 in prostate cancer and hTERT mRNA in both bladder and prostate cancers. There will an improved diagnostic accuracy if all the circulating molecules are combined into one multi-marker test.

 Circulating Proteins and Peptides



A number of commercially available noninvasive multi-marker tests designate proteomics and peptidomic analyses which have marked a new horizon for cancer biomarker discovery.[90],[91] The prostate health index, a blood-based test, evaluates total PSA, free PSA, and pro-PSA for prostate cancer detection.[92] A multi-marker blood test, the 4KScore, gives an assessment of significant PCA (Gleason >7) before biopsy by combining four kallikreins (total PSA, free PSA, intact PSA, and hKLK2).[93] A urine-based diagnostic test for urothelial carcinoma, the ImmunoCyt™, detects cytoplasmic mucins and high-molecular weight carcinoembryonic antigen.[94] Aura Tek FDP Test™ evaluates the recurrence of bladder tumors by measuring urinary fibrin degradation products.[95]

Patients with high-grade RCC showed an elevated serum Hsp27.[96] Urine AQP1 and PLIN2 were identified as screening biomarkers for clear cell and papillary RCC.[97] A panel of four serum peptides was found to have 100% sensitivity and 93.3% specificity for RCC diagnosis,[98] with 12 urine peptides differentiating malignant from benign renal masses and controls.[99]

Urine levels of APOA1, APOA2, APOB, APOC2, APOC3, and APOE were seen to be elevated in BCA patients.[100],[101] A signature of eight urinary peptides served as a marker for disease progression as they distinguished nonmuscle from muscle-invasive BCA.[102]

Urine β-MSMB was found to be lower in patients with PCA compared to benign prostatic conditions with greater sensitivity when paired with serum PSA.[103] Patients with CRPC showed raised plasma CAV1 and CAV2 compared to controls.[104]

It is ascertained that in spite of all challenges, proteomics shows clinical promise. Many independent studies have emphasized on the utility of circulating AQ1 and APOA1/APOA2 as noninvasive markers for RCC and BCA, respectively. Recently because of the improved sensitivity, specificity, and the clinical success of multi-marker assays, there has been a shift from single to multi-marker assessment.

 Exosomes



Exosomes (30–100 nm) are actively secreted membrane vesicles that are present in nearly all body fluids[105] and influence therapeutic response by aiding in intercellular communication by transferring biologically active molecules.[106] They are stable carriers of various molecules (RNA, DNA, and proteins)[107] whose concentration rises in cancer patients.[108] There has been increasing interest in the application of exosomes as cancer biomarkers with techniques to extract these from plasma which include sequential ultra-centrifugation, ultra-filtration, immune capturing, and more recently field-flow fractionation.[109]

Elevated exosomal lncARSR levels in plasma indicated RCC in patients which also predicted poor response to Sunitinib, decreased postresection, and increased at relapse.[110] A study showed a strong association between exosomal levels of TACSTD2 and bladder cancer.[111] The lncRNAs HOTAIR, HOX-AS-2, ANRIL, and linc-RoR rich in urinary exosomes predicted high-grade muscle-invasive urothelial carcinomas.[112]

ExoDx™ Prostate (IntelliScore) is a recently developed FDA-approved noninvasive urine test that assesses the expression of three exosomal RNAs associated with high-grade PCA and complements serum PSA to distinguish high-grade PCAs (Gleason score ≥7) from low-grade PCAs.[113] Exosomal levels of miR-34a and miR-148a were significantly low in urines of PCA patients v/s BPH patients.[114] Exosomal serum MDR-1, MDR-3, and PABP4 proteins were enriched in docetaxel-resistant CRPC patients relative to docetaxel-sensitive patients.[115]

Exosomes contain “pure” fractions of cancer cell components including nucleic acids, proteins, and metabolites. However, only a few of them have been accepted in clinical practice because of the lack of accurate and precise isolation/detection methods. We speculate that the development of sensitive capture platforms will bring the novel exosomal biomarkers into foreground. A brief tabulated summary of important and accepted circulating biomarkers in urology has been described in [Table 2]. [Table 3] enumerates few tumor markers with their test name that are commercially available specifically for diagnostic and prognostic purposes in urology.{Table 2}{Table 3}

 Challenges Faced by Liquid Biopsy



Despite many advancements in the diagnostic field of liquid biopsy, there are several challenges faced in the preanalytical and analytical phase. Few pros and cons have been mentioned in [Table 4].{Table 4}

♦ The amount of diagnostic material is usually contaminated by normal cell components and nucleic acids making the visual assessment of the enriched diagnostic material impossible. It can only be estimated through characterization for cancer-specific molecular alterations[116]

♦ CTCs while in circulation interact with platelets and fibrin which mask cells from being recognized by natural killer cells and antibodies used for immune-magnetic isolation

♦ Tumor-specific mutations can be as low as 0.01% of total cfDNA[8],[11] which can make the detection of rare tumor variants challenging

♦ The half-life of cfDNA is very short ranging from few minutes to <2 h making the diagnostic window very small[117]

♦ The amount of cancerous cfDNA and CTCs in the blood varies from hour to hour and is regulated by several cancer-related and host-related factors[2]

♦ Urine contains high concentrations of urea which is a powerful inhibitor of the polymerases used in PCR reactions. In addition, in voided urine samples, high concentrations of nucleases like DNAses or RNAses rapidly degrade their targets.

 Conclusion



Cancer biomarker development in the form of liquid biopsy is a rapidly growing field. Implementation of liquid biopsy in urological clinical setting provides comprehensive information on cancer biology and tumor heterogeneity while sparing patients exposure to unnecessary painful needle/surgical procedures. They also help in risk assessment and clinical management of urologic malignancies.

dPCR and targeted resequencing on Next-generation sequencing has a high sensitivity as they can detect DNA mutations as low as 1 mutation/million wild type DNA copies. They can read dozen of genes, amplicons on blood/urine samples of multiple patients in less than two days. In spite of all the developments, further scientific works are needed to validate signatures against intratumoral heterogeneity and to constantly re-evaluate them in the context of evolving “standard” clinicopathological features.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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