Background
Method | Platform | Applications | Advantages | Limitations | Reference |
---|---|---|---|---|---|
PCR-based | Nested real-time PCR | • Known point mutations • Methylated genes | • Ease of use • Low cost | • Low sensitivity • Detect limited genomic loci | |
Mutant allele-specific PCR | |||||
Mass spectrometry | |||||
Digital PCR | Droplet digital PCR | • Known point mutations • Methylation | • Very high sensitivity • Quantitative | • Detect limited genomic loci | |
Microfluidic digital PCR | |||||
Targeted Sequencing | Safe-SeqS | • Selected SNVa, SCNAb and rearrangements | • High sensitivity • Low cost | • Less comprehensive than next gen sequencing | |
Tam-Seq | |||||
CAPP-Seq | |||||
Whole genome sequencing | karyotyping | • Genome wide SNV, SCNA and rearrangements | • Detect all genomic loci | • Expensive • Time consuming | |
PAREc
|
Advantages and limitations of cell-free DNA
Clinical applications of cell-free DNA
Clinical application | Cancer type | Sample type | Type of cfDNA analysis | Reference |
---|---|---|---|---|
Predict treatment response | • RCC • Bladder • Prostate | • Plasma • Serum | • cfDNAa level • Methylation • Mutations | |
Predict recurrence | • RCC • Bladder • Prostate | • Plasma • Urine | • Mutations • Methylation • cfDNA % | |
Prognosis | • RCC • Bladder • Prostate | • Plasma • Serum | • ctDNAb level • DNA integrity • Methylation | |
Diagnosis | • RCC • Bladder • Prostate | • Urine • Plasma • Serum | • Alteration in DNA level • cfDNA integrity • Methylation • Mutations |
Measuring cell-free DNA levels
Cell-free DNA integrity
Cell-free DNA methylation
Cell-free DNA mutations
Other circulating molecules and their clinical applications
Molecule | Clinical application | Cancer type | Markers | References |
---|---|---|---|---|
CTCsa
| Diagnostic | Bladder cancer | Cell count | [49] |
Prognostic | RCC, bladder and prostate cancer | Cell count | ||
Predictive (recurrence and treatment response) | Prostate cancer | Cell count | [52] | |
miRNAs | Diagnostic | RCC, bladder and prostate cancer | miR-210, miR-1233, miR-125b, miR-126, let-7e, let-7c, miR-30c, miR-622, and miR-1285 | [63] |
Prognostic | Bladder and prostate cancer | miR-146a-5p | [57] | |
Predictive (treatment response) | Prostate cancer | miR-21 | [68] | |
lncRNAsb
| Diagnostic | RCC, bladder and prostate cancer | PCA3, lncRNA-LET, PVT1, PANDAR, PTENP1, linc00963, UCA1, lncRNA H19 | |
Prognostic | Prostate cancer | PCAT18 | [79] | |
Predictive (treatment response) | Bladder cancer | UCA1 | [76] | |
mRNAsc
| Diagnostic | RCC, bladder and prostate cancer | CAIX, UBE2C | |
Prognostic | RCC, bladder and prostate cancer | B7-H3, CK20, cBMP6 | ||
Predictive (recurrence and treatment response) | Prostate cancer | AR-V7, PSCA, | ||
Proteins | Diagnostic | RCC, bladder and prostate cancer | AQP1, PLIN2, APOA1, APOA 2, APOB, APOC2, APOC3, APOE, β-MSMB | |
Prognostic | RCC and prostate cancer | Hsp27, KNG1, APOD, FG, HP, CAV1, CAV2 | ||
Peptides | Diagnostic | RCC and prostate cancer | - | |
Prognostic | Bladder cancer | - | ||
Exosomes | Diagnostic | RCC and prostate cancer | miR-126-3p, miR-449a, miR-34b-5p, miR-34a, miR-148a | |
Prognostic | Bladder cancer | HOTAIR, HOX-AS-2, ANRIL, linc-RoR | [120] | |
Predictive (recurrence and treatment response) | RCC and prostate cancer | LncARSR, MDR-1, MDR -3, PABP4 |
Circulating tumor cells
Circulating RNAs
miRNAs
Long non-coding RNAs
Messenger RNAs
Circulating proteins and peptides
Exosomes
Challenges facing liquid biopsy
Future perspective
Test name | Molecules assessed | Cancer type | Clinical application | Biological fluid tested | Reference |
---|---|---|---|---|---|
CELLSEARCH® CTC Test | CTCa
| Prostate | Prognostic for patients with metastatic prostate cancer | Blood | [44] |
PROGENSA PCA3 Test | lncRNA PCA3 | Prostate | Diagnostic for prostate cancer patients with previous negative biopsy (Determine need for repeat biopsy) | Urine | [73] |
Prostate Health Index (PHI) | Protein (total PSA, free PSA, and [-2] proPSA) | Prostate | Diagnostic for prostate cancer patients with a PSA between 4 and 10 ng/mL | Blood | [94] |
4KScore | Protein (total PSA, free PSA, intact PSA, and human KLK 2) | Prostate | Prognostic (Assess risk for aggressive prostate cancer) | Blood | [95] |
ImmunoCyt™ Test | Protein (mucins and HMW carcinoembryonic antigen) | Bladder | Diagnostic for G1, G2 and G3 bladder cancer patients with positive urine cytology | Urine | [96] |
Aura Tek FDP Test™ | Protein (fibrin degradation product) | Bladder | Predictive of bladder cancer recurrence | Urine | [97] |
ExoDx™ Prostate (IntelliScore) | Exosomal RNA | Prostate | Prognostic for high-grade prostate cancer at the time of biopsy and at surgery | Urine | [117] |
Conclusions
Key points
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Molecular profiling is becoming the basis for “precision medicine” or individualized treatment.
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Liquid biopsy is a non-invasive tool that can provide a global snapshot of the primary and metastatic tumors.
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Circulating cell-free DNA levels, integrity, methylation and mutational status have promising clinical applications in the field of urological cancer biomarker discovery.
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Circulating tumor cells, circulating RNAs (miRNA, lncRNA and mRNA), cell-free proteins and exosomes obtained through liquid biopsy are promising biomarkers and can provide additional insight into tumor biology.
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A key challenge facing liquid biopsy is understanding where these circulating molecules are coming from, whether they arise from the primary tumor or from the metastatic lesion.
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The shift from a single- to multi-marker view is likely to ensure that the path from discovery to clinical diagnostics continues to be successfully paved.