Circulating cell-free nucleic acids as prognostic and therapy predictive tools for metastatic castrate-resistant prostate cancer

Table 1 Diagnostic and prognostic methods and outcomes of studies investigating cell-free tumor deoxyribonucleic acid in metastatic castrate-resistant prostate cancer by liquid biopsy

Ref.	Methods and patients	Prognostic or predictive outcomes
Ritch et al[12], 2019	Plasma, whole exome sequencing. Intron and exon sequencing and copy numbers of selected genes. Mismatched repair deficiency, MMRd mPCa (mostly mCRPC) patients (n = 433)	cfDNA analysis of hypermutations and MMR gene alterations in MSH2, MSH6, MLH1 marked ctDNA and can identify aggressive disease. Mutations in ctDNA were found in PTEN, RB1, TP53 (and, interestingly, no copy number loss) and in AKT1, PI3KCA, CTNNB1, AR-LBD. Compared with a control cohort, ctDNA hypermutation and MMRd correlated with a poor response to AR inhibition and to a shorter survival
Chapman et al[13], 2019	Plasma, whole-genome sequencing. Somatic gain of function mutations of TP53. mCRPC patients (n = 143)	In cfDNA somatic GOF mutations of TP53 at codons R175, R248, R273, R282 and G245 were positively and significantly associated with abiraterone and/or enzalutamide progression
Gupta et al[14], 2019	Plasma, low pass whole-genome sequencing ULP-WGS. Somatic copy number alteration, mCRPC patients (n = 93)	The SCNA in cfDNA and CTC were mostly concordant (gain in copy number of FOXA1, AR, and MYC, and loss in BRCA1, PTEN, and RB1). Interestingly, some discordant genomic alterations rarely detected in cfDNA (gain in MYC and BRCA2) were associated to a worse outcome, i.e. MYCN copy number gain correlated with a worse outcome in AR-V7 negative patients and BRCA2 copy number gain correlated with a worse outcome in AR-V7 negative patients treated with abiraterone/enzalutamide
Patsch et al[15], 2019	Plasma, qPCR of LINE-1 297 bp, mCRPC patients before and after docetaxel treatment (n = 15)	In cfDNA, LINE-1 quantity decreased after chemotherapy but without statistical significance
Hahn et al[16], 2019	Serum, next generation sequencing, somatic copy number alteration of 69 targeted genes, Metastatic PCa patients (n = 101)	In ctDNA, SCNA significantly correlated with the number of new treatments demonstrating changes of tumor genomic profile after therapies. Interestingly, SCNA did not correlate with time of progression
Qiu et al[17], 2019	Plasma, tissue tumor mutational burden by next generation sequencing, targeted gene panel, mCRPC (n = 20)	The ctDNA was identified by two TMB assays: guardant health and the foundation medicine. Between these two assays used to detect ctDNA the results of SNP, Indel, CNV and fusions were concordant. The study evinced a high correlation between cfDNA TMB and the whole exome sequencing of the corresponding tumor tissues. Additionally, there was a good correlation between cfDNA TMB and tissue TMB having high TMB samples; however, the same was not observed for low/medium TMB samples
Torquato et al[18], 2019	Plasma, AR alterations, whole genome sequencing, Somatic copy number alteration, mCRPC patients (n = 62)	In ctDNA the SCNA significantly associated with worse survival outcomes of mCRPC patients. At multivariable analysis, missense mutations in AR ligand-binding domain correlated with shorter PFS and TP53 loss. PI3KCA copy number gain or pathogenic missense mutations correlated with shorter OS
De Laere et al[19], 2019	Plasma, low pass whole-genome sequencing, and targeted gene-body sequencing. Somatic alterations in AR and TP53. mCRPC patients (n = 168)	The presence of AR and TP53 alterations in ctDNA was used to determine tumor burden. AR and TP53 alterations were associated with a worse PFS. TP53 inactivation was an independent prognostic factor outperforming AR alterations and ARV7 expressions was found be useful to stratify patients’ risk
Sonpavde et al[20], 2019	Plasma, somatic mutations by NGS, mCRPC patients (n = 514)	One or more alterations in ctDNA were found in 94% of patients. Mutations were detected in TP53 (36%), AR (22%), APC (10%), NF1 (9%), EGFR, CTNNB1 and ARID1A (6%), BRCA1, BRCA2, PIK3CA (5%). The increase in copy number was found for AR (30%), MYC (20%), BRAF (18%). At multivariate analysis, increase in MYC copy number was associated with worse OS
Vandekerkhove et al[21], 2019	Plasma, targeted genome, sequencing, somatic copy number alteration, metastatic castrate-sensitive patients (n = 53)	The ctDNA and tissue biopsy showed 80% of concordance for somatic mutations. TP53 was mutated in 50% of patients and truncated mutations in DNA damage repair genes were found in 21%. The cfDNA SCNA was higher in untreated patients than in patients treated with abiraterone acetate, enzalutamide or apalutamide
Mayrhofer et al[22], 2018	Plasma, low pass whole-genome sequencing and hybridization-capture targeted sequencing. Somatic copy number alteration, mPCa patients (n = 217)	The SCNA found in ctDNA was used to determine tumor burden and to compare lines of therapies. AR variants, microsatellite instability and PTEN, RB1 and TP53 inactivation were found in ctDNA demonstrating mirroring of the genetic alterations that mark metastatic cancer
Choudhury et al[23], 2018	Plasma, ultra-low pass whole-genome sequencing. Tumor fractions, TFx, measured by computational tool ichorCNA, CRPC patients (n = 140)	TFx was determined in cfDNA. TFx positively correlated with PSA and alkaline phosphatase and significantly correlated with the presence and numbers of bone metastases
Annala et al[24], 2018	Plasma, whole-exome sequencing and target capture and sequencing of selected genes. Variant allele fractions, mCRPC patients (n = 202)	The alterations in TP53 and AR gene truncations measured in ctDNA correlated to resistance to abiraterone and enzalutamide. A poor clinical outcome was associated with alterations in ctDNA of BRCA2 and ATM genes
Kohli et al[25], 2018	Plasma, dPCR for copy number gain of AR, mCRPC patients (n = 92)	In pre chemotherapy patients, the AR copy number gain found in ctDNA was associated with a worse outcome
Mehra et al[26], 2018	Plasma, cfDNA quantity quant-IT picogreen HS DNA kit and a BioTek microplate spectrophotometer (480ex/520em). mCRPC patients (n = 571)	In multivariable analyses, log10 cfDNA concentration was found to be an independent prognostic variable for rPFS and OS: higher baseline concentrations associated with shorter rPFS and OS following taxane therapy. On the contrary, a decrease in total cfDNA concentration during the first 9 wk of treatment was associated with taxane therapy responsiveness
Seyedolmohadessin et al[27], 2018	Plasma, cfDNA quantity NanoDrop, Localized PCa (n = 50), metastatic PCa (n = 26) and healthy controls (n = 10)	The cfDNA level was significantly higher in metastatic PCa patients (19.62 ng/μL) with respect to localized PCa (15.4 ng/μL) and BPH patients (9.5 ng/μL); healthy controls had the lowest value (8.7 ng/μL)
Belic et al[28], 2018	Plasma, deep AR sequencing, Illumina MiSeq; whole genome sequencing and targeted sequencing. Somatic copy number alteration, mCRPC patients (n = 65)	In ctDNA, AR mutations and copy number alteration were found in most cases. AR amplification and RB1 loss were associated with worse PFS. SCNA was therefore a biomarker for disease progression
Hendriks et al[29], 2017	Plasma, Methylation-specific qPCR and copy number of GSTP1 and APC genes, CRPC patients (n = 47), controls (n = 30)	In CRPC patients the cfDNA quantity was significantly higher than age-matched controls. At baseline, GSTP1 was hypermethylated in patients. Both the copy number of methylated GSTP1 and APC were higher in patients than healthy controls. The increase of cfDNA levels, either each one of the methylated gene copies individually or together (GSTP1 + APC) or together with PSA (GSTP1 + APV + PSA), all correlated with decreased OS
Wyatt et al[30], 2017	Plasma, targeted sequencing, Somatic copy number alteration, mCRPC (n = 45)	The study proved a correspondence between SCNA in ctDNA and matched tissues. Such SCNA genes included AR, BRCA2, ATM, PTEN, PIK3CA, PIK3CB, PIK3R1, TP53, and RB1
Rathkopf et al[31], 2017	Plasma, dPCR of 11 relevant AR-ligand binding domain mutations. Non-metastatic CRPC (n = 51), AAP-näive mCRPC (n = 25), post-AAP (n = 21)	In ctDNA, the AR-LBD mutations were found to be low at baseline (7.5%) and progression (7.3%). The AR-LBD mutations did not correlate with the de novo resistance to apalutamide
Goodall et al[32], 2017	Plasma, Quant-iT, whole exome sequencing and targeted sequencing. Targeted genes, mCRPC patients (n = 49)	At multivariate analysis, the cfDNA concentration was an independent prognostic biomarker: ≥ 50% reduction in cfDNA levels related to longer rPFS and OS. The ctDNA germline and somatic alterations in BRCA2 and PALB2 repair genes were found in ctDNA. All mutations found in the tissue were also detectable in ctDNA
Conteduca et al[33], 2017	Plasma, dPCR. Somatic copy number gain of AR, mCRPC patients (n = 80)	In ctDNA, the AR copy number gain was associated with a worse outcome in patients treated with abiraterone and enzalutamide. Independently from the type of antiandrogen treatment, there was a meaningful correlation among AR gain and TLA/MTV compared to AR non-gained cases (P = 0.001 and P = 0.004, respectively). AR copy number and TLA were associated with a shorter PFS and OS
Annala et al[34], 2017	Plasma, somatic mutations of BRCA2 gene by qPCR, mCRPC germline-mutated patients (n = 11)	In 10 out of 11 germline mutated patients, biallelic gene loss of BRCA2 was found in ctDNA. This information help to guide clinicians to the best therapeutic choice
Conteduca et al[35], 2017	Plasma, dPCR. Copy number gain of AR, CRPC patients (n = 265)	In ctDNA, the AR copy number gain before starting enzalutamide or abiraterone was associated with a decrease in both PFS and OS
Goldstein et al[36], 2017	Plasma, NGS AR sequencing and validation by dPCR, somatic alterations in AR, mCRPC patients (n = 11)	In ctDNA, the AR t (TC > CTC) F877L hotspot was prone to false positive mutations during NGS. Low-abundance mutations need to be verified by highly sensitive PCR, such as dPCR, but amplification conditions must be carefully optimized
Adalsteinsson et al[37], 2017	Plasma WES, metastatic PCA PCa patients (n = 520)	There is a concordance between clonal somatic mutations (88%), copy number alterations (80%), mutational signatures and neoantigens between tumor biopsies and cfDNA form 41 patients with ≥ 10% cfDNA
Wyatt et al[38], 2016	Plasma, AR copy number qPCR and AR deep targeted sequencing, mCRPC patients (n = 65)	In ctDNA, the AR mutation and copy number alterations were found in 48% of baseline patients and in 60% patients at disease progression. The AR copy number gain (two or more AR mutations) and RB1 loss were associated with worse PFS
Salvi et al[39], 2016	Plasma, qPCR. Copy number gain of AR, CRPC patients (n = 59)	In ctDNA, the AR copy number gain was found in 36% of patients. AR copy number gain significantly associated with alkaline phosphatase and lactate dehydrogenase. At multivariate analysis, PSA decreasing ≥ 50% and AR copy number gain were significantly associated with worse OS and PFS
Fawzy et al[40], 2016	Plasma, qPCR of ALU 247bp and ALU115bp, cell-free DNA Integrity, cfDI, metastatic PCa (n = 28), non-metastatic PCa (n = 22), BPH (n = 25), healthy controls (n = 30)	The cfDI levels, measured as ratio ALU247bp/ALU115bp, were significantly higher in metastatic PCa patients vs non-metastatic PCa patients, BPH patients and healthy controls
Azad et al[41], 2015	Plasma, AR qPCR copy number and deep sequencing of AR-LBD, mCRPC (n = 62)	In cfDNA, the AR copy number gain was associated with enzalutamide resistance; also abiraterone resistance was associated to AR mutations but to a lower extent
Deligezer et al[42], 2010	Plasma, qPCR for Sat-2 gene, PCa-localized (n = 22), locally advanced (n = 11), mCRPC (n = 28)	The average quantity of cfDNA measured by amplification of Sat2 gene was not significantly different between patients with localized, locally advanced and metastatic disease
Schwarzenbach et al[43], 2009	Plasma, somatic LOH for D6S1631, D8S286 and D9S171 genes by qPCR, PCa patients (n = 69), metastatic PCa patients (n = 12)	In ctDNA, the somatic LOH significantly correlated with the diagnosis of subgroups made of localized and metastasized prostate cancers. ctDNA LOH significantly associated also with the tumor stage
Bastian et al[44], 2007	Serum, qPCR for GSTP1, MDR1 and EBNRB genes, PCa patients (n = 192)	The levels of cfDNA was found to increase from PCa without recurrence to PCa with recurrence and then to metastatic PCa for all GSTP1, MDR1 or EBNRB genes

MMRd: Mismatch repair deficiency; AAP: Acetate and prednisone treatment; AR: Androgen receptor; ARV7: Androgen receptor variant 7; BPH: Benign prostatic hyperplasia; cfDI: Cell-free DNA integrity; cfDNA: Cell-free circulating DNA; CRPC: Castrate-resistant prostate cancer; CTC: Circulating tumor cells; ctDNA: Circulating tumor DNA; dPCR: Digital PCR; GOF: Gain of function; NGS: Next generation sequencing; PFS: Progression-free survival; LP-WGS: Low pass whole-genome sequencing; LOH: Loss of heterogeneity; OS: Overall survival; PCa: Prostate cancer; mCRPC: Metastatic castrate-resistant prostate cancer; mPCa: Metastatic prostate cancer; PSA: Prostate specific antigen; qPCR: Quantitative PCR; rPFS: Radiographic progression-free survival; SCNA: Somatic copy number alteration; scfDNA: Seminal plasma cfDNA; TGS: Deep targeted sequencing; TFx: Tumor fractions; TMB: Tumor mutation burden; ULP-WGS: Ultra low pass whole-genome sequencing; WES: Whole exome sequencing: LBD: Ligand binding domain.

Table 2 Diagnostic and prognostic outcomes methods of studies investigating circulating tumor ribonucleic acid in prostate cancer by liquid biopsy

Ref.	Methods and patients	Prognostic or predictive outcomes
Joncas et al[48], 2019	Plasma dPCR, AR-7 mRNA, PCa patients (n = 35)	AR-V7 mRNA expression was associated with shorter time to progression (median, 16.0 vs 28.0 mo; P = 0.0499)
Mohammadi Torbati et al[49], 2019	Serum qPCR, miR-20A, miR-26A, PCa patients (n = 40), healthy controls (n = 40)	In PCa samples miR-20A was significantly upregulated compared to healthy controls. On the other hand, there was no significant difference in the levels of pre- and post-operation miR-26A compared to controls
Ishiba et al[50], 2018	Plasma dPCR, PDL-1 mRNA, PCa patients (n = 88)	PD-L1 mRNA was detected and quantified in ctRNA of cancer patients. Interestingly, there was a comparison between expression of PD-L1 protein in tumor tissues and PD-L1 gene expression in plasma of cancer patients
Wang et al[51], 2018	Plasma qPCR, SAP30L-AS1 and SChLAP1 lncRNAs, PCa patients (n = 34), BPH patients (n = 46), Healthy controls (n = 30)	SAP30L-AS1 lncRNAs levels were upregulated in BPH and SChLAP1 lncRNAs levels were significantly higher in PCa than in BPH and healthy controls. The area under the ROC curve indicated that SAP30L-AS1 and SChLAP1 lncRNA had an adequate diagnostic value different from PCa and controls
Zedan et al[52], 2018	Plasma qPCR, miR-93, miR-221, miR-125b, miR-93, PCa patients (n = 149)	Significantly lower levels of miRNA-93 and miRNA-221 in the follow-up of patients vs baseline z = −2.738, P = 0.006, and z = −4.498, P < 0.001, respectively. Similarly, miRNA-125b was significantly lower in the observational cohort (z = −2.656, P = 0.008). There was a correlation between both miRNA-125b and miRNA-221 with risk assessment r = 0.23, P = 0.015 and r = 0.203, P = 0.016, respectively. However, miRNA-93 was significantly correlated with prostatectomy Gleason score (r = 0.276, P = 0.0576)
Farran et al[53], 2018	Plasma qPCR, miRNA signature, PCa patients (n = 114)	Aggressiveness of PCa could be segregated based on circulating miRNA signature consisting of an interaction between a combination of two miRNAs (miR-17/miR-192) and an independent miRNA (miR-181a)
Liu et al[54], 2018	Plasma qPCR, miR-223, miR-24, miR-375, PCa patients (n = 329)	Patients could be significantly reclassified using a 3-miR (miRNA-223, miRNA-24 and miRNA-375) score (training OR 2.72, 95%CI 1.50e 4.94 and validation OR 3.70, 95%CI 1.29e 10.6)
Adalsteinsson et al[37], 2017	Plasma WES, Metastatic PCa patients (n = 520)	There is a concordance between clonal somatic mutations (88%), copy number alterations (80%), mutational signatures and neoantigens between tumor biopsies and cfDNA form 41 patients with ≥ 10% cfDNA
Albitar et al[55], 2017	Urine and plasma qPCR, mRNAs panel, PCa patients (n = 306)	The urine/plasma biomarker test, evaluating the mRNA levels of PCa-specific gene such as PDLIM5, HSPD1, PSA, IMPDH2, PCA3,TMPRSS2, ERG, UAP1, PTEN, AR, the housekeeping B2M and GAPDH genes, accurately predicted high-grade cancer with sensitivity at 92%-97%, while core-biopsy sensitivity was 78%
Endzeliņš et al[56], 2017	Plasma qPCR, miR-375, miR-200-3p, miR-21-5p, miRNA Let-7a-5p, PCa patients (n = 50), BPH patients (n = 22)	miR-375 could be used to differentiate between PCa and BPH patients when analyzed in whole plasma, while miR-200-3p and miR-21-5p performed better in EVs. Let-7a-5p could be used to differentiate PCa patients, with Gleason score ≥ 8 vs ≤ 6
McDonald et al[57], 2017	Plasma qPCR, miRNA panel, PCa patients (n = 134)	miR-381, miR-34a, miR-523, miR-365, miR-122, miR-375, miR-1255b, miR-34b, miR-450b-5p, and miR-639 were the most statistically significant miRNA after adjusting for age (P values ≤ 0.05)
Alhasan et al[58], 2016	Plasma Scano-miR, miRNA panel, very high risk, PCa patients (n = 9), Low risk, PCa patients (n = 9), and healthy controls (n = 10)	miR-200c, miR-605, miR-135a, miR-433, and miR-106a were identified as useful for differentiating indolent and aggressive forms of PCa
Yan et al[59], 2015	Urinary qPCR, TSPAN13 and S100A9 mRNAs, PCa patients (n = 129), BPH patients (n = 105)	qPCR was used to measure urinary nucleic acid levels and tissue mRNA expression. The TSPAN13 and S100A9 mRNA ratio was selected to determine the diagnostic value of urinary nucleic acids in PCa (P = 0.037). It was significantly higher in PCA than in BPH in the mRNA and nucleic acid cohort analyses (P < 0.001 and P = 0.013, respectively). ROC analysis showed that the area under the ROC curve was 0.898 and 0.676 in tissue mRNA cohort and urinary nucleic acid cohort, respectively. This ratio could have a strong potential as a diagnostic PCa marker
Antonarakis et al[60], 2014	Serum qPCR, AR-V7 mRNA, PCa enzalutamide-treated patients (n = 31), PCa abiraterone-treated patients (n = 31)	AR-V7 mRNA detectable (positive) patients receiving enzalutamide had lower PSA response rates compared to AR-V7 mRNA not detectable (negative) patients (0% vs 53%, P = 0.004) and shorter PSA PFS (median, 1.4 mo vs 6.0 mo; P < 0.001), clinical or radiological PFS (median, 2.1 mo vs 6.1 mo; P < 0.001), and OS (median, 5.5 mo vs not reached; P = 0.002). Similarly, AR-V7 mRNA positive patients, receiving abiraterone had lower PSA response rates compared to AR-V7 mRNA negative patients (0% vs 68%, P = 0.004) and shorter PSA PFS (median, 1.3 mo vs not reached; P < 0.001), clinical or radiological PFS (median, 2.3 mo vs not reached; P < 0.001), and OS (median, 10.6 mo vs not reached, P = 0.006)
Korzeniewski et al[61], 2014	Urine qPCR, miR-483-5p, PCa patients (n = 71), healthy controls (n = 18)	miR-483-5p was expressed at higher levels in PCa than in control
Deligezer et al[42], 2010	Plasma qPCR, cBMP6 mRNA, Local PCa patients (n = 22), local advanced PCa patients (n = 11) or mCRPC patients (n = 28)	The levels of cBMP6 mRNA in patients with metastatic disease were higher than those in patients with localized disease (P = 0.001) or in patients with local advanced disease (P = 0.05)
Papadopoulou et al[62], 2006	PBMC and plasma qPCR, PSMA mRNA, newly diagnosed PCa patients (n = 12), under therapy PCa patients (n = 4)	Among the newly diagnosed patients 4/12 (33.3%) had positive mRNA for PSMA in plasma, whereas only 2/12 (16.7%) had positive PSMA mRNA in PBMC. Among under therapy PCa patients, three (15.8%) were positive for PSMA mRNA in plasma, while only one (5.3%) was positive in PBMC. Furthermore, > 60% of PCa had elevated levels of cfDNA

AR: Androgen receptor; AR-V7: Androgen-receptor splice variant 7; BPH: Benign prostatic hyperplasia; cfDNA: Cell free DNA; dPCR: Digital polymerase chain reaction; lncRNAs: Exosomal circulating long non-coding RNAs; mCRPC: Metastatic castrate-resistant prostate cancer; PSA: Prostate specific antigen; WES: Whole exome sequencing; PFS: Progression-free survival; OS: Overall survival; PBMC: Peripheral blood mononuclear cells; PCa: Prostate cancer; qPCR: Quantitative polymerase chain reaction; lncRNA: long non-coding RNA; PSMA: Prostate specific membrane antigen; BMP-6: Bone morphogenetic protein-6.