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

Navid Sobhani,Texas Medical Centre,Baylor College of Medicine,Alkek Building,Houston,TX 77030,United States

Marianna Sirico,Daniele Generali,Multidisciplinary Operative Unit of Mammary Pathology and Translational Research,ASST of Cremona,Cremona 26100,Italy

Daniele Generali,Fabrizio Zanconati,Department of Medical,Surgical and Health Sciences,University of Trieste,Cattinara Academic Hospital,Trieste 34149,Italy

Bruna Scaggiante,Department of Life Sciences,University of Trieste,Trieste 34127,Italy

Abstract

Key words:Metastatic castrate-resistant prostate cancer;Circulating free deoxyribonucleic acid;Cell-free tumor deoxyribonucleic acid;Circulating free ribonucleic acid;Liquid biopsy;Prostate cancer

INTRODUCTION

Prostate cancer is the most frequently occurring type of cancer among men in over one-half (105 of 185) of world’s countries,and it is the leading cause of cancer-related deaths among men in 46 countries[1].Although inhibition of androgen receptor (AR)signaling with antiandrogens and conventional chemotherapeutic molecules in metastatic castrate-resistant prostate cancer (mCRPC) patients has increased the 5-year survival rate to 29%,it still remains difficult to cure[2].In mCRPC,cancer cells adapt to live with very low levels of androgens.Such cells try to use different strategies to independently promote the AR pathway.

After developing the first antiandrogens (flutamide,nilumatide and bicalutamide),there was an urgent need for impeding any agonist activity against wild-type AR.There was a necessity to inhibit wild-type AR recruitment of co-activators and to block the consequent AR binding to deoxyribonucleic acid (DNA),which is a transcription factor for androgen-dependent genes that leads to tumor proliferation[3].This brought the development of second-generation antiandrogens,which showed a significant improvement in clinical treatment.In fact,in 2012 the Food and Drug Administration approved enzalutamide and abiraterone acetate as second-generation antiandrogens for the treatment of mCRPC,while other second-generation antiandrogens are at different stages of pre-clinical and clinical development[4].Although these two drugs act on different levels of the AR pathway,the cross-resistance is a common event;resistance to abiraterone and enzalutamide was found to be associated with splicing variants of AR without the ligand-binding domain.Such splice variants encode for a receptor incapable of binding to ligands.It is constitutively active as a transcription factor and capable of promoting the activation of target genes.Because enzalutamide exerts its antitumor activity by binding to the ligand-binding domain of the receptor,the splice variants cannot be targeted by the drug,which generates drug-resistance.Moreover,as the receptor is constitutively activated,it will not be influenced by abiraterone,which inhibits the synthesis of androgens.In this scenario,androgens are not required to activate the pathway,as it is already active[5].Interestingly,20%-40% of mCRPC patients not previously treated with chemotherapy are intrinsically resistant to abiraterone acetate or enzalutamide[6].

Thus mCRPC cells evolving into an AR-independent type escape targeted therapies against androgens.Sometimes they can acquire characteristics of neuroendocrine prostate cancer,which is a transformation that occurs in the clinical context of a moderate increment of blood prostate specific antigen (PSA) levels[7].It is clear that mCRPC progression can have different therapeutic options,but the monitoring of mCRPC is an urgent task for patients’ surveillance to anticipate relapse of the disease or to evaluate the efficacy of the therapies.In this respect,we will discuss the emerging role of liquid biopsy for metastatic castrate-resistant prostate cancer patients with a focus on nucleic acids to predict disease progression or therapy efficacy.

LIQUID BIOPSY IN METASTATIC CASTRATE-RESISTANT PROSTATE CANCER

In contrast to traditional tissue biopsy,liquid biopsy involves the analysis of different biological fluids.As technologies advance,the amplification and detection methods have become more and more efficient and sensitive in the target identification,using minute quantities of nucleic acids.As a proof of principle,liquid biopsy can detect various tumor-specific macromolecules to profile and stratify the pathology.Longitudinal monitoring by liquid biopsy could give critical support to clinical management of patients because specific molecular changes are correlated with tumor progression and response to therapies.

Di Nunnoet al[8]summarized in six points the advantages of liquid biopsy as an innovative approach to prostate cancer:(1) Low invasiveness;(2) Early detection of most aggressive tumors;(3) Early diagnosis of residual tumors or micrometastasis after surgery;(4) Monitoring patient response and/or progression of the disease after therapy,particularly for mCRPC;(5) Prognostication of the response to therapy;and(6) possibility to delineate an accurate genetic profile of the disease directing the analysis towards key mutations correlated with tumor resistance[8].

Many molecular alterations (mutations,amplifications,deletions,alterations in the expression of specific genes or in non-coding RNAs transcription levels) have been correlated with resistance or sensibility against specific treatments.Targets in liquid biopsy can be circulating cell-free nucleic acids.Circulating cell-free tumor DNA(ctDNA) analysis includes detection of circulating free DNA (cfDNA) levels,circulating cell-free DNA integrity (cfDI),methylation rate,mutations and/or aberrations in the copy number of specific genes.For example,amplification of AR and/or the detection of certain driver mutations,e.g.deletion of PTEN,can be useful to anticipate an unfavorable clinical outcome[9].Of interest are the AR mutations,particularly of certain splicing variants (AR-V7),correlating with resistance against second-generation hormone treatments,such as abiraterone acetate and enzalutamide.Moreover,inherited gene mutations that have been found significantly correlated with the prognosis of the disease are eligible for liquid biopsy analysis[10].

Other valuable emerging tumor biomarkers in liquid biopsy are represented by different classes of RNAs,including microRNAs (miRNAs),long non-coding RNA and messenger RNA (mRNA).

ctDNA analysis in mCRPC patients

The total cfDNA concentration was increased in cancer patients and may be a useful tool[11].However,more information for patient management is derived from the analysis of ctDNA.Table 1 summarizes the main studies investigating cfDNA in mCRPC patients by liquid biopsy.A strategy is to calculate the circulating cfDI,which is the evaluation of the cfDNA fragmentations of repetitive elements such as ALU and LINE-1.It has been observed that in tumors the cfDI value can increase or decrease.The concentration and integrity of circulating cfDNA have been proposed as a clinical tool for the diagnosis of prostate cancer.Khaniet al[45]proved in Iranian patients that the values of cfDI were significantly higher in patients with prostate cancervsthose with benign hyperplasia and healthy individuals.They measured a region of ALU repetitive elements with shorter fragments of 115 bp nested in longer 247 bp ones by quantitative polymerase chain reaction (qPCR);the ratio of 247 bp over the 115 bp gave the integrity DNA index.Additionally,both the DNA concentration and cfDI were found to be elevated in the metastatic conditions[45].These results were confirmed by Arko-Bohamet al[46].Interestingly,cfDI of ALU247/115 had already been found to be higher in metastatic prostate cancer patientsversusnon metastatic ones[40].

In the amount of purified cfDNA,ctDNA can be analyzed by searching gene aberrations such as copy number variations or mutations in target genes.The analysis of genetic alterations of AR,identified through ctDNA in mCRPC patients,that circulating molecules have a significant potential to guide the use of therapies against this receptor.Furthermore,the monitoring of ctDNA levels could result in a powerful way to trace response of tumors and detect the uprising of resistant subclones[6].In addition to alterations of the AR pathway,other modifications inTP53,WNT,PI3Kand cell cycle pathway (RB1andCCND1) genes are important for prognosis and drug-development[7].It should be noted that the deletion ofPTENis found in 40% of patients with mCRPC and loss-of-function mutations in genes of the DNA repair pathway (homologous recombination and mismatch) are expressed in approximately 20% of metastatic prostate cancer[7].TP53gene is very frequently mutated in mCRPC.In particular,TP53gain-of-function mutations have been associated with cancer cell survival and chemoresistance.Interestingly,TP53mutations conferring gain of function were related to disease progression and drug resistance after abiraterone or enzalutamide treatments[13].

Table1 Diagnostic and prognostic methods and outcomes of studies investigating cell-free tumor deoxyribonucleic acid in metastatic castrate-resistant prostate cancer by liquid biopsy

sequencing of selected genes.Variant allele fractions,mCRPC patients (n = 202)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-n?ive 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

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.

Different studies offer a comparison between ctDNA and the corresponding metastatic tissue.For example,Wyattet al[30]sequenced 72 clinically relevant genes in 45 cfDNA samples and corresponding tissues obtained during biopsy.The comparison of data concerning alterations in ctDNA with those of the tissues has been demonstrated that for the majority of patients an assay of ctDNA could be enough to identify all the alterations in a metastatic tissue.The authors suggested that with appropriate validation methods,it could be possible to develop DNA-based biomarkers useful in identifying ctDNA for the management of patients with mCRPC.An important advantage of ctDNA is the ability to integrate somatic information from metastatic sites to discover mutation heterogeneity of the tumor,which should be taken into consideration to monitor the tumor stage and its progression.This information could add knowledge to the pathological analysis of the prostate tissue biopsy,which by itself may be not completely representative of the heterogeneous behavior of tumors[30].

ctRNA in prostate cancer patients

The necessity of a multi-parameter approach has been highlighted in different studies.Indeed,important additional knowledge could arise from a simultaneous investigation of molecules using liquid biopsy (i.e.cfDNA) and DNA extracted from circulating tumor cells and cfRNA,with the purpose of building a complete molecular profile[47]to integrate with the clinical data.

RNA evaluation in liquid biopsy represents the next frontier in integrated molecular medicine.Table 2 summarizes the main clinical investigations on ctRNA.Most studies involved prostate cancer patients at diagnosis.Pioneer exploration of RNA molecules in liquid biopsy has involved the analysis of prostate specific membrane antigen mRNA,but only a limited number of newly diagnosed patients were positive[62],whereas the bone morphogenetic protein-6 mRNA,whose upregulation is strongly associated with bone metastasis,was found to be a biomarker for the metastatic disease[42].

Circulating mRNAs are generally degraded by RNases.However,circulating mRNAs are capable of forming complexes with transporters,which are proteins and/or lipids.When they form such complexes,they turn into a relatively stable structure in the blood circulation,thus becoming potentially useful biomarkers[63].Currently,androgen receptor variants are generally responsible for AR activity,survival and progression of prostate cancer[64].TheAR-V7is the only one of the androgen receptor variants observed as a protein,and it is therefore properly defined[3].Different studies in the literature have shown thatAR-V7is the main AR variant[65].TheAR-V7product is a truncated AR protein,which lacks the C-terminal ligand-binding domain but retains the transactivating N-terminal domain.Consequently,this protein is not capable of binding to ligands but remains a constitutively active transcription factor promoting the activation of target genes promoting cancer progression[65-67].Antonarakiset al[60]evinced thatAR-V7mRNA is expressed at high levels in circulating tumor cells[60]and is associated with abiraterone and enzalutamide resistance in mCRPC patients[68,69].Indeed,Joncaset al[48]recently found blood levels ofAR-V7mRNA,which was shown to be correlated with response and resistance to abiraterone in mCRPC patients,demonstrating its potential as predictive biomarker[48].Particularly worthy of note,upregulation of the programmed death-ligandPD-L1mRNA causes cancer cells to able to evade the host immune system[50].

In most patients,altered levels of miRNAs have been found,such as miR-21,miR-221,miR-1290 and miR-375.Such differential expression compared to healthy controls have been associated with different prognostic outcomes in mCRPC patients.For example,a potential diagnostic and prognostic role played by miR-141 has been suggested.miR-141 levels have been found to be progressively increased from hypertrophy of the prostate to prostate cancer and to the metastatic disease[70].Interestingly,the quantification of miR-141 in the liquid biopsy by droplet digital PCR has been described[71].However,a recent study showed that miR-18a has the highest potency to discriminate between healthy individuals and cancer patients,whereas miR-221 discriminated between patients with localized disease from those with metastasis.miR-141 did not show the same potency[72].

FUTURE PERSPECTIVES

Liquid biopsy represents an attractive field of research for many types of cancer,including prostate adenocarcinoma.In particular,liquid biopsy has been proven to provide support in therapeutic planning for patients with mCRPC,allowing detection of molecular changes in cell-free nucleic acids (i.e.DNA and RNA) that are associated with tumor progression and response or resistance to different drugs.The low invasiveness is particularly relevant because of the possibility of repeating the analyses frequently over time,allowing longitudinal monitoring of patients.In the near future,liquid biopsy will lead to a deeper understanding of the metastatic evolution of prostate cancer with the possibility of developing new targeted therapies in the perspective of an even more personalized oncology.

The analysis of ctDNA appears to be the most promising tool to monitor cancer diseases.In fact,ctDNA is the only target recommended by the Food and Drug Administration and the European Medicines Agency for cancer diagnosis and to monitor the efficacy of treatments.For prostate cancer,ctDNA is a very interesting biomarker for the anticipation of progression-free survival and overall survival in response to therapies and for improving the clinical management of patients avoiding overtreatments.The high concordance between ctDNA genomic alterations and those found in tumor tissue biopsies strongly supports the potential of liquid biopsy to integrate clinical data and improve patient management.The next generation sequencing of cfDNA has demonstrated the potential for the follow-up of the mutational changes of the tumor by being able to identify all its heterogeneity and to anticipate drug resistance.Moreover,many affordable high-throughput technologies,e.g.digital PCR,are now available to precisely detect the copy number variations of selected target genes (e.g.AR,TP53,BRCA2,PIK3CA) that are relevant for the progression of the disease and in response to therapies.

Table2 Diagnostic and prognostic outcomes methods of studies investigating circulating tumor ribonucleic acid in prostate cancer by liquid biopsy

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.

The potential usefulness of cfRNAs in mCRPC is emerging,especially as additional markers for aggressiveness and metastasis.Many of the studies in cfRNA involved miRNA analysis,but more recently even other classes of non-coding RNA have been explored,such as long non-coding RNA.In the clinic the potential use of cfRNA analysis could implement information about the staging of the disease,but it might be useful to discriminate indolentvsaggressive prostate cancer.

Liquid biopsy data offers robust evidence to consider cell-free nucleic acid analysis useful to improve the clinical management of mCRPC patients.In this new approach the use of PSA as a biomarker must be considered.PSA is the biomarker approved for men by the Food and Drug Administration in 1986.From then on,it has been widely used to predict incidence and recurrence of prostate cancer,despite its poor specificity.However,in mCRPC the PSA seems to be more specific as a biomarker than in the onset of prostate cancer;its increase is related to cancer progression[73].The significance of PSA measurements in mCRPC is still interesting for the scientific community.For example,Aggarwalet al[74]recently demonstrated that low PSA secretion levels can stratify mCRPC patients with treatment-emergent small-cell neuroendocrine prostate cancer.In fact,low PSA secretors showed high treatment-emergent small-cell neuroendocrine prostate cancer,RB1andTP53loss and low AR transcription.In addition,overall survival and progression-free survival were shorter in the low PSA secretor group[74].In a retrospective study,Buttiglieroet al[75]showed that early PSA drop was related to a better overall survival and progression-free survival in mCRPC patients treated with abiraterone or enzalutamide (docetaxel-na?ve or post-docetaxel setting).Finally,a mathematical model of PSA dynamics has been proposed to predict individual response to intermittent androgen deprivation therapy[76].

CONCLUSION

In our opinion,PSA can play an important role as a biomarker for the management of mCRPC patients.However,PSA measurements could maintain some limitations due to the high individual variability.Liquid biopsy on circulating cell-free nucleic acid offers the same low invasiveness but important molecular details on each specific tumor heterogeneity evolution.In conclusion,liquid biopsy on circulating cell-free nucleic acid along with PSA measurements and other clinical data can assure the best treatment decision-making for mCRPC patients.

ACKNOWLEDGEMENTS

The authors thank dr.Giorgia Ficco for the English language editing of the manuscript.