UROLOGICAL ONCOLOGY Minimal Residual Disease Defines the Risk and Time to Biochemical Failure in Patients with Pt2 and Pt3a Prostate Cancer Treated With Radical Prostatectomy: An Observational Prospective Study Nigel P Murray1,2* , Socrates Aedo1, Cynthia Fuentealba2, Eduardo Reyes3,4, Anibal Salazar2, Marco Antonio Lopez 5, Simona Minzer 6, Shenda Orrego5, Eghon Guzman5. Purpose: To compare Gleason score (GS), pathological stage, minimal residual disease (MRD) and outcome after prostatectomy radical for prostate cancer. Patients and Methods: 290/357 men with GS 6 or 7 and pT2 or pT3a disease treated with radical prostatectomy participated. Blood and bone marrow were obtained one month after surgery. Circulating prostate cells (CPCs) were detected using differential gel centrifugation and immunocytochemistry with anti PSA, micro-metastasis weas detected using immunocytochemistry with anti-PSA. Biochemical failure free survival (BFFS) and restricted mean survival times (RMST) were calculated according to GS and stage. MRD was classified as negative, patients only positive for micro-metastasis and patients positive for CPCs; BFFS and RMST were calculated according to MRD sub-type. Results: GS7 (HR 3.03) and pT3a (HR 3.68) cancers were associated with a higher failure rate, shorter time to fail- ure and associated with CPC positive MRD (p < 0.001), while G6 and pT2 with MRD negative disease (p<0.001). Men with CPC (+) MRD were at high risk of early treatment failure; 15% BFFS at 10 years, RMST 3.0 years. Men positive for only micro-metastasis were at risk of late failure, 50% BFFS at 10 years, RMST 8.0 years compared with MRD negative patients; 80% BFFS at 10 years, RMST 9.0 years. Conclusion: The sub-type of MRD identifies Gleason 6 pT2 patients with a poor prognosis and Gleason 7 pT3a patients with a good prognosis and could be used to classify men according to personal risk characteristics for the use of adjuvant treatment. Keywords: biochemical failure; circulating prostate cells; micro-metastasis; minimal residual disease; prostate cancer INTRODUCTION After radical prostatectomy for prostate cancer, bi-ochemical failure occurs in 15-40% of patients, and is associated with the surgical Gleason score and pathological stage. Extra prostatic extension (EPE) of the tumour is an adverse prognostic risk factor, defining pT2 from pT3a disease(1) and therefore between organ confined and specimen confined disease. It has been suggested that pT3a patients should be classified into focal capsular penetration and non-focal penetration as biochemical failure free survivals are different(2,3); how- ever, all cases of EPE are classified as pT3a disease in the American Joint Committee on Cancer, seventh edi- tion staging manual(4). In both pT2 (organ confined) and pT3a margin negative (specimen confined) all the tumor has been removed at surgery, however there is a difference in prognosis. The simplest explication would be an erroneous patholog- ical classification, which may explain some cases but 1Faculty of Medicine, University Finis Terrae, Santiago, 7501015, Chile. 2Urology Service, Hospital de Carabineros, Santiago 7770199, Chile 3Faculty of Medicine, University Diego Portales, Santiago, 8370179, Chile 4Urology Service, Hospital DIPRECA, Santiago, 7601003, Chile 5Faculty of Medicine, University Mayor, Santiago, 7510041, Chile 6Faculty of Medicine, University de los Andes, Santiago, Chile. *Correspondence: Faculty of Medicine, University Finis Terrae, Santiago, 7501015, Chile. Email: nigelpetermurray@gmail.com. Received February 2019 & Accepted September 2019 not the majority(5). The second possibility is that cancer have disseminated beyond the prostate and thus outside the surgical field at the time of operation. The residual tumor cells that remain after local or systemic therapy in patients with no signs of clinical disease is termed minimal residual disease (MRD). The presence of MRD will depend on the characteristics of the primary tumor and the ability of cancer cells to disseminate, implant and survive in distant tissues. Two types of MRD have been described(6), in patients with circulating prostate cells or tumor cells (CPCs) detected in the blood there is an increased frequency of early treatment failure(7,8). Whereas in patients with tumor cells detected only in bone marrow samples there is an association with late failure(7,9). We present a prospective, observational long-term fol- low up study of the effect of the sub-types of MRD on the outcome of radical prostatectomy monotherapy in Urology Journal/Vol 17 No. 3/ May-June 2020/ pp. 262-270. [DOI: 10.22037/uj.v0i0.5174] men with Gleason 6 and 7 and pT2 and pT3 margin negative prostate cancer. A small group of men with EPE and positive surgical margins who did not undergo adjuvant therapy was used as a control group with ad- verse prognostic features. PATIENTS AND METHODS Study population A single center, prospective observational study of men who underwent radical prostatectomy as mono-therapy for prostate cancer between 2000 and 2008, and the ac- quisition of follow up data was concluded in Decem- ber 2017. Pre-treatment PSA and age at surgery were registered; The pathological study of the surgical piece was performed by dedicated genitourinary pathologists according to the Gleason system (pre-2005) and the pathological stage was defined according to the Partin criteria(10). Extra-capsular extension was defined as a specimen with cancer cells in contact with the prostatic capsule and classified as positive or negative, sub-divi- sion into focal and non-focal capsular penetration was not used. Positive surgical margins were defined as one with cancer cells in contact with the inked surface of the specimen. Patients were classified as pT2 (organ confined), pT3a negative surgical margins (specimen confined) and pT3a positive margin. All men had a nadir PSA post-surgery of < 0.01ng/mL. Exclusion Criteria: Previous treatment or consideration for treatment with androgen blockade or radiothera- py; Infiltration of the seminal vesicles and/or regional lymph nodes with cancer or a positive bone scan; Men with Gleason 8 and 9 cancer. Serial total PSA levels were monitored three monthly for the first year and six monthly thereafter. Biochem- ical failure was defined as a serum PSA > 0.2ng/mL on two separate occasions. The biochemical failure free survival time was defined as the time from surgery to the time of a post-surgery PSA of > 0.20ng/mL or to the time of the last follow up. MRD detection was inde- pendently evaluated with the evaluators being blinded to the clinical details. Procedures a) Detection of secondary circulating prostate cells: one-month post-surgery an 8mL venous blood sample was taken and mononuclear cells were obtained by dif- ferential centrifugation using Histopaque 1,077 (Sig- ma-Aldrich, USA). The cells were used to make slides (silanized, DAKO, USA), air dried for 24 hours and fixed in a solution of 70% ethanol, 5% formaldehyde, and 25% phosphate buffered saline (PBS) pH 7.4. Immunocytochemistry: CPCs were detected using a Table 1. Clinical and pathological findings according pathological stage. Variable pT2 pT3a margin negative pT3a margin positive P-value (two tail) n=192 n=78 n=20 Age, years 65.0 ± 8.2 66.2 ± 9.0 67.0 ± 8.8 03708a mean ± SD PSA, ng/mL 5.21; 1.68 6.37; 5.07 6.66; 6.59 < 0.001a Median; IQR Gleason score 25 (13%) 39 (50%) 12 (60%) < 0.001b greater than 6 n (%) Biochemical failure 49 (26%) 53 (68%) 17 (85%) < 0.001b n (%) Abbreviations: IQR= interquartile range; PSA= serum total prostate specific antigen; a Kruskal-Wallis test; b Pearson's chi-squared test. Figure 1. Circulating tumour cell and leukocyte. Minimal residual disease in prostate cancer-Murray et al. Vol 17 No 03 May-June 2020 263 monoclonal antibody directed against PSA, clone 28A4 (Novocastro Laboratory, UK), and identified using an alkaline phosphatase-anti alkaline phosphatase based system (LSAB2, DAKO, USA), with new fuchsin as the chromogen. Samples positive for PSA staining cells were incubated with anti-CD45 clone 2B11 + PD7/26 (DAKO, USA) and cells identified with a peroxidase based system (LSAB2,DAKO, USA) with DAB (3,3 diaminobenzidine tetrahydrochloride) as the chromo- gen. A CPC was defined as expressing PSA but not CD45 and a leukocyte as expressing CD45 but not PSA (Figure 1) (11) . A test was considered positive when at least 1 cell/8mL of blood was detected. b) Bone marrow biopsy: it has been reported that pros- tate tumor cells detected in bone marrow aspirates are phenotypically different than those prostate cells detected in bone marrow biopsies and may not repre- sent “true” micro-metastasis but rather cells circulating within the bone marrow(12). For this reason, bone mar- row biopsy “touch preps” were used as the sample to test for micro-metastasis. Patients were sedated with intravenous midazolam and a bone marrow biopsy, using local anesthetic, was tak- en from the posterior superior iliac crest one month af- ter surgery. Four ”touch preps” using salinized slides (DAKO, USA) were prepared and processed as de- scribed for CPCs, a micro-metastasis was defined as cells staining positive for PSA and negative for CD45. Evaluations: Patients were divided into three groups: pT2, pT3a (margin negative), pT3a (margin positive) and further subdivided into; Group A negative for both CPCs and micro-metastasis patients (without evidence of MRD); Group B CPC negative, micro-metastasis positive; Group C CPC positive with or without bone marrow micro-metastasis detected. Study end point: The primary study end point was the presence of biochemical failure and secondary end point mean time to failure after primary treatment. Statistical analysis The analysis was performed using the program Sta- ta (Stata/SE 15.0 for Windows, Copyright 1985-2017 StataCorp LLC). Descriptive statistics were used to de- scribe the results. The variables pT2, PT3a margin neg- ative and pT3a margin positive) were compared for age, total serum PSA, pathological Gleason score and MRD (Group A, B and C). The Kruskal–Wallis test was used to test whether samples originate from the same distri- bution. A p value < .05 was taken to signify statistical significance and all tests were two tailed(13). For the whole cohort, a nonparametric survival analysis (13) was performed to establish the survival proportion of Kaplan- Meier (KM) and restricted mean survival time (RMST) for the biochemical failure during the ten-year follow-up period(14). The RMST establishes the expect- Urological Oncology 264 Minimal residual disease in prostate cancer-Murray et al. Table 2. Survival proportion and restricted mean survival Time (RMST) at 10 years for biochemical failure observed from use curves Kaplan-Meier, on 290 Men Treated by Radical Prostatectomy for Prostate Cancer. Variable Survival proportion Kaplan-Meier % (95% CI) RMST Kaplan-Meier a years (95% CI) pT2 Gleason score 6 72.0 9.2 n=192 n=167 (62.5 to 79.6) (8.9 to 9.5) Gleason score 7 26.1 6.5 n=25 (5.2 to 54.4) (5.1 to 8.0) pT3a Gleason score 6 28.1 6.7 Group margin n=39 (10.6 to 48.7) (5.7 to 7.8) negative n=78 Gleason score 7 12.1b 4.0 n=39 (3.5 to 26.2) (3.2 to 4.9) pT3a Gleason score 6 18.8 c 3.7 margin n=8 (1.1 to 53.5) (2.9 to 4.4) positive n=20 Gleason score 7 16.7 d 2.8 n=12 (2.7 to 41.3) (2.1 to 3.5) Abbreviations: %=percentage; CI= confidence interval; a The RMST is the area under the Kaplan-Meier survival curve, determined by the numerical integration; b at 9.08 years last time not censored observed; c at 5.08 years last time not censored observed; d at 4.33 years last time not censored Characteristic pT2 pT3a margin negative pT3a margin positive P-value two tail n=192 n=78 n=20 CPC (-) and mM (-) 114 (60%) 22 (28%) 3 (15%) < 0.001a n (%) CPC (-) and mM (+) 39 (20%) 11 (14%) 4 (20%) .487 n (%) CPC (+) n (%) 39 (20%) 45 (58%) 13 (65%) < 0.001a Abbreviations: CPC, circulating prostate or tumor cells; mM, micro-metastasis a Pearson's chi-square test with Marascuilo procedure for post hoc analysis pT2 versus pT3a margin negative and pT2 versus pT3a margin positive. Table 3. Minimal residual disease according to pathological stage: Classification Criteria Observed survival Predicted RMST Predicted Predicted survival RMST HR Stage MRD Gleason Score Kaplan-Meier FPM Kaplan-Meier a FPM years % (95% CI) % (95% CI) years (95% CI) (95% CI) (95% CI) pT2 n=192 CPC 6 97.8 90.5 9.9 9.7 negative / n=108 (91.3-99.5) (82.1-95.0) 1 mM (9.7-10.0) (9.5-9.9) negative 7 100 b 82.4 5.6 j 9.5 n=6 (65.1-91.7) 1.9 (9.1-9.9) (1.2-3.0) CPC 6 64.1 72.1 9.4 9.2 negative / n=31 (38.7-81.2) (57.5-82.5) 3.3 mM positive (9.0-9.8) (8.7-9.6) (1.4-7.4) 7 75.0 53.2 9.8 8.5 n=8 (12.8-96.1) (29.4-72.2) 6.3 (9.6-10.1) (7.6-8.3) (2.5-15.8) CPC 6 16.6 24.1 positive n=28 (5.3-33.3) (12.0-38.5) 7.0 6.3 9.5 (6.0-7.9) (5.5-7.2) (3.7-24.7) 7 9.01 c 6.4 2.7 4.7 n=11 (0.5-33.3) (0.9-20.0) 18.4 (1.9 to 3.5) (3.5-5.9) (6.5-52.1) pT3a margin CPC 6 88.9 d 81.2 8.5 9.4 negative negative / n=13 (43.30-98.4) (63.4-90.0) 2.1 n=78 mM negative (7.5 to 9.6) (9.0-9.9) (1.3-3.3) 7 45.7 66.9 6.8 9.0 n=9 (6.9-79.5) (41.2-83.3) 4.0 (4.2 to 9.4) (8.3-9.7) (2.4-6.6) CPC 6 33.3 50.7 8.6 8.4 negative / n=11 (7.8-62.3) (27.7-70.0) 6.8 mM positive (7.7-9.5) (7.5-9.2) (2.7-17.2) 7 Not observed 26.9 Not observed 7.3 n=0 (8.6-49.6) 13.1 (5.2-33.2) (6.1-8.5) CPC 6 10.0 5.2 3.9 4.5 positive n=15 (0.8-33.5) (0.9-15.5) 19.8 (2.5-5.3) (3.5-5.6) (7.2-54.5) 7 5.0 0.3 3.4 n=30 (0.5-18.9) (0.1-2.3) 38.2 3.1 (2.6 to 4.2) (2.6-3.7) pT3a (13.8-105.5) margin CPC 6 Not determined 67.0 Not determined 9.0 positive negative / n=1 (36.9-85.1) 4.0 (8.2-9.8) n=20 mM negative (2.1-7.6) CPC 7 50.0 e 46.1 2.6 8.2 negative / n=2 (0,6 to 91.0) (14.1-73.4) 7.7 mM negative (2.0 to 3.3) (6.9-9.5) (3.9-15.2) CPC 6 100% f 27.1 5.8 j 7.3 negative / n=2 (5.8-55.0) 13.0 mM positive (4.7-36.2) (5.9-8.8) 7 50%g 8.0 3.5 6.0 n=2 (0,6-91.0) (0.4-31.7) 25.2 (2.2-4.7) (4.3-7.7) (9-70.6) CPC 6 20.00 h 0.3 3.1 3.1 positive n=5 (0,8-58.2) (0.1-5.1) 38.1 (2.3-4.0) (2.2-4.1) (12.5-115.7) 7 12.5 0.1 2.6 2.3 n=8 (0.7-42.3) (0-0.3) 73.5 (1.7-3.5) (1.8-2.8) (24.0-226.0) Table 4. Survival proportion and restricted mean survival Time (RMST) at 10 years for biochemical failure observed (Kaplan-Meier) and Predicted (Flexible Parameter Model) according to the following classification criteria: a) EPE, b) MRD and c) Gleason score greater than 6; on 290 Men Treated by Radical Prostatectomy for Prostate Cancer Abbreviations: MRD, minimal residual disease, CPC, circulating prostate cells;, mM, micro-metastasis, %,percentage; CI, confidence interval; FPM, flexible parameter model. a The RMST is the area under the Kaplan-Meier survival curve, determined by the numerical integration; b at time 5.58 years not observed events; c 5.76 years last time not censored observed; d at 9.08 years last time not censored observed; e at 2.17 years last time not censored observed; f at time 5.08 years not observed events; g at 4.33 years last time not censored observed; h 3.41 years last time not censored observed; i3.33 years last time not censored observed; J confidence interval not determined, there are no patients with biochemical failure. FPM=flexible parameter model; HR=hazard ratio Minimal residual disease in prostate cancer-Murray et al. Vol 17 No 03 May-June 2020 265 ed time from surgery to biochemical failure during the total observation period(15). Patients were classified ac- cording: a) pathological stage), b) MRD sub-type, and c) Gleason score > 6 and the KM and RMST deter- mined, and the results compared using the log-rank test. A flexible parametric survival model (FP model) was used to predict the survival proportion, RMST and the hazard ratio as there was no compliance with the pro- portional risk assumption (Cox model)(16). The discrimi- nation of a prognostic model reflects its ability to distin- guish between patient outcomes, for which the Harrell’s C discrimination index was used(17). From the FP model for biochemical failure to ten years, the RMST, hazard ratio and survival proportion were established accord- ing to the following classification criteria a) pathologi- cal stage, b) MRD and c) Gleason score > 6. Ethical considerations: The study was approved by the local ethics committee and in complete agreement with the Declaration of Helsinki. All patients provided writ- ten informed consent. RESULTS 357 men underwent radical prostatectomy; 67 fulfilled exclusion criteria leaving 295 men in the study group. The median follow up time was 6.7 years (IQR: 5.9 years; range 1-15 years). The mean age was 65 ± 8.5 years and a median PSA of 6.9 ng/mL (IQR 2.8). Table 1 shows the findings according to pathological stage of the patients. The serum PSA at the time of diagnosis, frequency of Gleason score 7 and frequency of bio- chemical failure were significantly higher with increas- ing pathological stage. Kaplan-Meier survival (KM) curves and RMST time to biochemical failure according to pathological stage and Gleason score: The KM proportion for biochemical failure free surviv- al at ten years of follow-up for the whole cohort was 7.6 years (95% CI: 7.2 to 8.0 years). The biochemical failure free survival and time to failure significantly de- creased with increasing pathological stage and a high- er Gleason score (p < 0.01 log rank test) (Figure 3). Compared to baseline risk of failure (Gleason 6 pT2), univariate hazard ratios (HRs) were: Gleason score 7 HR 3.03 (IC: 1.99 -4.60; p < 0.01), pT3a margin neg- ative HR 3.68 (95% IC: 2.37-5.71; p < 0.01) and pT3a margin positive HR 7.63 (95% IC: 4.03- 14.44; p < 0.01). Multi-variate HR were Gleason score 7 2.12 (95% CI: 1.76-2.57), pT3a margin negative 2.31 (95% CI: 1.94-2.79) and pT3a margin positive 5.32 (95% CI: 4.16-8.73) respectively. There was agreement between the predicted survival (according to the final model of Cox) versus observed survival (model Kaplan-Meier) (Figure 3) with a Har- rell’s C discrimination index of 0.77 (95% IC: 0.74 to 0.81), considered as a good fit. In summary, the results are consistent with the known risk factors for treatment failure, higher Gleason score and pathological stage (organ confined and speci- Figure 2. Bone marrow sample positive and negative for PSA expressing micro-metastasis. Figure 3. Comparing predicted (Cox final model) versus observed survival (Kaplan-Meier Survival) by Gleason score 7 and Pathological Stage, pT2, pT3a margin negative and pT3a margin positive for biochemical failure free progression at 10 years on 290 subjects Treated with Radical Prostatectomy for Prostate Cancer Minimal residual disease in prostate cancer-Murray et al. Urological Oncology 266 Vol 17 No 03 May-June 2020 267 men-confined cancer) and represents a typical prostate cancer population. Kaplan-Meier survival curves and RMST time to bi- ochemical failure according to pathological stage and Gleason score and minimal residual disease: For each pathological stage the minimal residual dis- ease was assessed, (Table 2), as may be predicted, MRD negative patients were significantly more fre- quently found in patients with pT2 disease, those with CPC positive MRD were significantly more frequently found in pT3a disease. However, the frequency of CPC negative MRD was not significantly associated with pathological stage. Classifying the patients according to MRD subtype, and where the number of patients permits this analysis, the presence of CPCs signified a significantly poorer bio- chemical failure free survival and shorter time to fail- ure, and associated with increasing Gleason score and pathological stage. However, patients MRD negative, independent of pathological stage had better biochem- ical failure free survival and longer time to treatment failure, even those patients with pT3a margin positive (Table 3). Patients with micro-metastasis positive MRD (Group B) had a different pattern of failure, al- though with a lower biochemical failure free survival the time to failure was significantly longer than those patients CPC (+). Those patients MRD micro-metasta- sis positive have a four to five years of excellent prog- nosis but afterwards there is increasing late failure, in other words the risk of failure was not constant with time. (Figure 4). The non-parametric comparison of survival by groups: a) pathological stage, b) MRD and c) Gleason score 7 showed differences with statistical significance (p val- ue < 0.01 for log-rank test). For the whole cohort, the Kaplan-Meier survival curves for the three MRD sub- groups were not parallel, which differed from the two survival curves based on Gleason score and pathologi- cal stage alone. Testing for a cohort interaction between Gleason score, pathological stage and MRD category showed a significant difference (p < 0.05), which im- plies that the risk of biochemical failure is not constant, and changes with time. The flexible parametric survival model using the fol- lowing coefficients of variables: a) pT3a margin nega- tive: 0.73 (p-value: 0.003), b) pT3a margin positive:1.39 (p-value < 0.0001) c) CPC negative/micro-metastasis positive: 1.18 ( p-value: 0.005), d) CPC positive: 3.16 (p-value < 0.0001) and e) Gleason score 7: 0.66 (p-val- ue: 0.003). This FP final model considered subjects with: pT2, CPC: negative/micro-metastasis negative and Gleason score 6 as the group basal. There was agreement when comparing the predicted FP model with the observed survival (Kaplan-Meier Sur- vival) with a Harrell’s C discrimination index of 0.91 showing an excellent fit between observed and predict- ed models. (Figure 4). The predicted survival proportions, RMSTs and hazard ratios (group basal: subjects with: pT2, MRD negative and Gleason score 6) for the FP final model accord- ing to pathological stage, MRD and Gleason score are shown in Table 3. As can be seen the HR when using Gleason score and pathological stage alone; HR Gleason 7 3.03, pT3a margin negative 3.68 and pT3a margin positive gives a very different risk classifi- cation. As can be seen from Table 3 patients with pT3a margin negative G6 tumours have a better-predicted outcome than pT2 Gleason 6 patients with only bone marrow micro-metastasis. Similarly, patients with pT3a margin negative G7 tumours and negative for MRD had a better-predicted outcome than pT2 Gleason 6 patients with CPCs detected. Sub-classifying the patients using MRD, Gleason score and pathological stage suggests that not all Gleason 6 or 7 and not all pT2 and pT3a cancers have the same risk of treatment failure. DISCUSSION Classification of patients following radical prosta- tectomy according to the risk of treatment failure is important in the management of prostate cancer. The identification of patients who may or may not benefit from adjuvant therapy, such as radiotherapy or andro- gen deprivation therapy is essential. That Gleason 7 tu- mors or those patients with higher pathological stage cancers had a higher risk of treatment failure, as seen in this study is not surprising. The study has its limita- tions; it was started in 2000 and we maintained the old Gleason 7 score rather than 3 + 4 and 4 +3(18) accepting that some patients classified as Gleason 7 would be classified as Gleason 3 + 4 and that some Gleason 7 would be Gleason 4 +3. Secondly, the small number of patients in some of the subgroups limits the number Figure 4. Kaplan Meier survival curves according to minimal residual disease. Abbreviations: CPC, circulating prostate cells, mM, micro-metastasis, CI, confidence interval. Minimal residual disease in prostate cancer-Murray et al. of conclusions, and this can be seen in the form of the wide confidence intervals. However, the fact that statis- tical significant differences were detected implies real differences between patient populations. A multi-cen- tre study with a much larger number of patients would overcome this limitation and essential before establish- ing concrete conclusions. The few patients with pT3a margin positive cancer were included as a bad prognosis group; only 20/82 (24%) of pT3a margin positive patients did not undergo adjuvant treatment, this group of patients were treated between 2000 and 2004. In the recruitment stage patients with pT3a margin negative disease were observed after rad- ical prostatectomy; studies published covering this era, reported acceptable cancer control and radiation ther- apy did not impact the appearance of metastasis or sur- vival although it did delay time to biochemical failure and improve local control(19). PSA could be considered as a marker for minimal re- sidual disease; post radical prostatectomy a level of over 0.2ng/ml is used to define treatment failure and to consider additional treatment. At these levels the pa- tient normally does not have clinical symptoms, how- ever the PSA level does not determine whether there is local or systemic residual disease. In this context, CPCs do not differentiate between local and systemic disease, whereas micro-metastasis in the bone marrow represent systemic disease. The use of bone marrow biopsies to evaluate the presence of micro-metastasis is more inva- sive than the use of blood tests. However, a three-year annual survey reported only 0.07% of patients reported side effects(20) and significantly less than those occur- ring after prostate biopsy(21). CPC detection is method dependent; methods using an- ti-EpCAM (Epithelial Cell Adhesion Molecule) such as CellSearch® detected CPCs in only 25% of men with lo- calized cancer and failed to distinguish between healthy controls and men with prostate cancer(22). In contrast, using an anti-Ber-4 and telomerase based method; CPCs were detected in 80% of men with localized pros- tate cancer(23). Similarly using a size-based filtration method, CPCs were detected in 34% of men compared with only 18.6% using the CellSearch system24( ). We used a simple differential gel centrifugation method to enrich CPCs and standard immunocytochemistry to de- tect them; the limitation of this method is the lack of external validation. This method used to detect CPCs has been internally validated at our centre, we acknowl- edge that there is variability in inter and intra observer evaluation, however used as a positive/negative test the results show a clinical utility The key points of the results of this study are the fol- lowing: a) stratifying patients according to the sub- types of MRD goes beyond Gleason score and patho- logical stage. Although for the three types of MRD the outcome of Gleason 7 patients is worse than Gleason 6 patients, and similarly patients with pT3a margin neg- ative cancer worse than those with pT2 cancer, not all Gleason 6, 7 and pT2 and pT3a behave in a similar fash- ion. This implies that the worse prognosis for Gleason 7 and pT3a patients in general is due to a higher frequen- cy of MRD CPC positive patients. Independent of the subtype of MRD, Gleason 7 patients had a worse prognosis and shorter time to treatment failure. The implication is that Gleason 7 cancer cells are inherently more aggressive than Gleason 6 tumour cells. However independent of the mechanism of tu- mour dissemination, there is a subgroup of Gleason 7 patients MRD negative with an excellent prognosis. More recently, a 30 gene mRNA expression signature improved predictions of indolent and lethal outcome of men with Gleason 7 prostate cancer, independent of whether the Gleason score was 3 + 4 or 4 + 3, for both types there were indolent and lethal variants(25). The dif- fering sub-types of MRD represent different biological potentials of cancer cells and may help to differentiate between indolent and lethal forms of cancer, even in patients with the same Gleason score and pathological stage. Morphological analysis of the cancer does not as- sess the biological potential of the tumour. b) the time kinetics of treatment failure differs between Gleason 6 and Gleason 7 tumours. In Gleason 7 the risk of early failure is significantly higher than in Gleason 6 cancer. However, by ten years post prostatectomy the risk of future failure had decreased to be the same as MRD negative patients. In contrast with Gleason 6 can- cer there was a constant failure risk. This suggests that the biological characteristics and behaviour of Gleason 6 and 7 tumour cells are different. This pattern has been reported previously, patients with adverse pathological findings at surgery, Gleason score ≥ 7, higher pre-sur- gery PSA levels had a high initial risk of failure which rapidly decreased to almost zero, while those with low Gleason scores and T2 disease had fairly constant pro- gression rates for up to ten years(26). Patients CPC positive had a significantly higher bio- chemical failure rate and shorter time to failure sug- gesting a more aggressive form of MRD. Although this simple system of MRD classification allows risk stratification of prostate cancer patients, the future mo- lecular characterisation of these tumour cells may allow for individualized treatments that are more effective, potentially reveal targets to prevent relapse and avoid overtreatment of patients with indolent MRD. There is a clinical need to delineate the patients with indolent MRD as they present a different biological and thus clinical process, which may require different treatment strategies. CONCLUSIONS Within the limitations of the study, the results suggest that the differences in treatment failure between Gleason 6 and Gleason 7 and pT2 and pT3a cancer patients can be explained by the phenotypic characters of the tumour cells, which give rise to differing patterns of MRD and in the different clinical patterns of relapse. Patients MRD negative or an “indolent” pattern may thus avoid overtreatment whereas those with CPC positive disease and a high risk of early relapse may benefit from early adjuvant treatment. This would need to be confirmed with larger scale randomized long-term trials. ACKNOWLEDGEMENTS The authors wish to thank Mrs Ana Maria Palazuelos for her help in redacting the manuscript. The study was supported by a Hospital de Carabineros de Chile research grant. CONFLICT OF INTEREST Dr Murray reports having received consultancy fees from Viatar CTC Solutions. Minimal residual disease in prostate cancer-Murray et al. Urological Oncology 268 Vol 17 No 03 May-June 2020 269 REFERENCES 1. Mottet N, Bellmunt J, Bolla M, et al. EAU- ESTRO-SIOG Guidelines on prostate cancer. Part 1: screening, diagnosis and local treatment with curative intent. Eur Urol 2017: 71: 618- 629 2. Maubon T, Branger N, Bastide C, et al. 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