Table 2. Clinical relevance of circulating tumor DNA detected in blood (serum/plasma samples) from UCB patients.
| Patients, time point of blood collection | No. of pts. | Methods | Genomic aberrations | Association of ctDNA with tumor/patient characteristics and clinical outcome | Ref. |
|---|---|---|---|---|---|
| MIBC, NT | 17 (248 samples) | SNVs, CNVs mutDNA by Tagged-Amplicon and shallow Whole Genome- Sequencing, UCB-specific sequencing panel, sampling prior to each chemotherapy session | Mutant DNA in 35.3% of pre-NT plasma | Follow up: up to 742 days after NT; mutations found in TURBT samples also found in plasma; pre-NT mutDNA: no correlation to response; less mutant DNA in plasma compared to urine; more mutDNA positive samples in pts. who recurred; most frequent mutated genes: TP53 and PIK3CA | (21) |
| NMIBC, MIBC, before RC | 72; 18 controls | MLPA, serum and plasma; 43 chromosomal regions containing 37 genes | 35/72 (48.6%) CNV, median CNV count: 2, CNVs in CDH1, ZFHX3, RIPK2, PTEN | CNVs in TSG1, RAD21, KIAA0196, ANXA7, TMPRSS2 associated with presence of variant UCB histology; CNVs in miR-15a, CDH1, ZFHX3 associated with presence of incidental prostate cancer; CNVs in KLF5, ZFHX3 and CDH1: CSS | (112) |
| NMIBC, MIBC, RC | 39; 27 | Sequencing of PT for hot spot mutations, plasma, ddPCR, personalized assays | FGFR3, PIK3CA hot spot mutations, present in PT | RFS in MIBC scheduled to RC, positive correlation between ctDNA levels from plasma and urine | (109) |
| Pts. with recurrent or progressive/metastatic disease, at different time points during treatment and follow up | 12 (377 samples) | NGS of PT, plasma, ddPCR, 1 to 6 personalized assays, ddPCR | Genetic aberration present in PT, DEL, INS, INV, ITX, CTX for personalized assays | Follow up: up to 20 years: pts. with progressive disease had higher ctDNA levels than pts. with recurrent tumors; No ctDNA in disease-free pts. | (107) |
| UCB, before treatment | 151, 53 controls | PCDH17, serum, MSP | 79/151 (52.3%) | Methylation associated with higher T stage (T2–T4), G3; independent predictor of OS | (113) |
| UCB | 10 | Mutational analysis of PT, 3 digital methods | Point mutations, rearrangements | 3 pts. with metastatic UCB had ctDNA | (114) |
| UCB | NMIBC: 75; MIBC: 20; TURBT without UCB: 48; Benign disease: 31; Healthy controls: 53 | MSP, serum, digested with Bsh1236I, HpaII, HinP1I to quantify amount of methylated DNA fragments | TIMP3, APC, RARB, TIG1, GSTP1, p14, p16, PTGS2, RASSF1A | <10% methylated DNA, methylation levels at each site and number of methylated genes increased in UCB compared to healthy individuals, methylated DNA not correlated to stage and grade of tumors | (115) |
| UCB and controls | 95 with UCB; 132 without UCB | Real time PCR, serum Short ACTB-106 and large ACTB-384 fragments; ACTB-106/ACTB-384: DNA integrity | UCB pts. increased ACTB-106 levels and lower DNA integrity | No difference in ACTB-106 levels between pts. with and without UCB, no correlation to smoking, pT stage, grade or lymph node metastasis | (116) |
| TCC | 127 pts, 41 healthy controls | CDH13, serum, MSP | 39/127 (30.7%) methylation of CDH13 | Association with advanced tumour stage, poor tumour differentiation (tumour grade), larger tumour size and tumour recurrence, independently with OS | (117) |
| NMIBC, different grades | 42 pts., 36 healthy controls | p16, DAPK, serum, MSP | 17/42 (40.5%) methylation of p16, methylation of DAPK in 27/42 cases (64.3%); in 12 pts. (28.6%) both genes methylated | Higher frequency of DAPK promoter methylation (71.4%) in pts. with lower grade tumors (G1) | (118) |
| UCB, before RC; BPH | 45 UCB; 45 BPH | Serum, real time MSP | APC, DAPK, GSTP1, PTGS2, TIG1, Reprimo Hypermethylation at APC GSTP1 promoters in 59.0% of UCB cases, TIG1 (32.0%), PTGS2 (24.0%), DAPK (2.0%); BPH: also methylated GSTP1, but not of the other genes | Hypermethylation at APC, GSTP1 or TIG1 distinguished UCB from controls with 80% sensitivity and 93% specificity, hypermethylation APC with stage, GSTP1 or GSTP1 or TIG1 with multifocal UCB, APC or APC or TIG1 with surgical margin positivity; APC hypermethylation: increased CS mortality | (119) |
| UCB, before RC; BPH | 45 UCB; 45 BPH | qPCR, serum, 124 bp fragment of PTSG2, mostly apoptotic origin; 271 bp fragment of Reprimo (mostly necrotic origin), AI (apoptotic index ratio 124 bp/271 fragment) | Both fragments increased in UCB pts., AI increase | DNA levels and AI not correlated with clinic-pathological parameters; increased AI correlated to high UCB-specific mortality (multivariate), independent prognostic factor | (120) |
| UCB | 86 pts., 49 controls | p16MSP, serum | 19/86 (22.0%) with aberrant p16 methylation | Associated with tumor presence | (121) |
| UCB | 27 | Plasma, LOH, microsatellite markers (D17S695, D17S654, D13S310, TH2, D9S747, D9S161), p53 and K-ras mutations, MSP: promoter status of p14ARF and p16INK4a | Plasma: 17/27 (63.0%) displayed the same alterations in tumor tissue and plasma; Plasma: 13/15 alterations in PT (87.0%) p14 promoter methylation, 2/5 alterations in PT (40.0%) p16 promoter methylation | Plasma LOH associated with DFS | (122) |
| Superficial UCB | 31 | Serum and plasma; LOH with 2 markers at 17p, TP53 microsatellites and mutations | 2/8 cases: mutation of PT found in blood; 30/31 cases with 17p microsatellite changes (in blood or urine), 52.0% concordance with PT | Mutations more frequent in higher malignant superficial tumors, risk to progress to MIBC; limited value for clinical practice | (123) |
| Conspicuous bladder lesions, before TURBT | 58 | Serum and plasma; LOH with 6 microsatellite markers | Matched microsatellite changes in PT and body fluids (serum, plasma, urine) in 23 cases | Simultaneous and multiple investigations of microsatellite markers of potential clinical relevance | (124) |
| UCB | 18; 2 controls | Serum: LOH, microsatellite analysis; comparison of DNA from sediments with that from supernatants | Higher DNA levels in supernatants than in non-centrifuged plasma samples | Centrifugation of body fluids before DNA extraction ensures high yield and quality of extracted DNA | (125) |
UCB, urothelial carcinoma of the bladder; NMIBC, non-muscle invasive bladder cancer; MIBC, muscle invasive bladder cancer; PT, primary tumor; ctDNA, circulating tumor DNA; NT, neoadjuvant therapy; MPLA, multiplex ligation-dependent probe amplification; ddPCR, digital droplet polymerase chain reaction; SNV, single nucleotide variation; CNV, copy number variation; mutDNA, mutated DNA; LOH, loss of heterozygosity: MSP, methylation-sensitive/specific PCR; BPH, benign prostatic hyperplasia; CS, cancer-specific; APC, adenomatous polyposis coli; DAPK, death-associated protein kinase 1; GSTP1, glutathione S transferase P; PTGS2, prostaglandin-endoperoxide synthase 2; TIG1, Tazarotene-induced gene 1; ACTB, ß-Actin, TP53, protein 53; PIK3CA, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha; PCDH17, protocadherin-17; CDH13, H-Cadherin; DEL, deletions; INS, insertions; INV, inversions; ITX, intrachromosomal translocations; CTX, inter-chromosomal translocations; qPCR, quantitative PCR.