UROLOGICAL ONCOLOGY The Efficacy of Transrectal Ultrasound Guided Biopsy Versus Transperineal Template Biopsy of the Prostate in Diagnosing Prostate Cancer in Men with Previous Negative Transrectal Ultrasound Guided Biopsy. Shady Nafie*, Michael Wanis, Masood Khan Purpose: We have previously demonstrated that transperineal template prostate biopsy (TPTPB) has a significant- ly higher cancer detection rate compared to transrectal ultrasound guided (TRUS) biopsy in biopsy naive men with a PSA < 20 ng/mL. We, therefore, performed a prospective study to determine whether TPTPB is still superior to TRUS biopsy in the detection of prostate cancer in men with persistently elevated PSA after one previous negative set of TRUS biopsies. Materials and Methods: 42 patients with a background of one previous negative set of TRUS biopsy, persistent- ly elevated PSA (but < 20 ng/mL) and benign feeling digital rectal examination (DRE) underwent simultaneous standard 12-core TRUS biopsy and 36-core TPTPB under general anaesthesia. We determined the prostate cancer detection rate between the two diagnostic modalities. Results: Mean age was 65 years (range: 50-75), mean prostate volume was 59 cc (range: 21-152), mean PSA is 8.3 ng/L (range: 4.4-19), mean time difference between the study and the previous TRUS biopsy was 33 months (range: 1-150) with mean PSA velocity of 0.7 ng/mL/year (range: 0-8). Out of the 42 patients, 22 (52%) had benign pathology. Of the 20 patients (48%) diagnosed with prostate cancer, 4 (10%) had positive results in both TRUS biopsy and TPTPB, 1 (2%) had positive result in TRUS biopsy with negative TPTPB, while 15 (36%) had negative TRUS biopsy with positive TPTPB. Hence, TRUS biopsy detected cancer in 5/42 (12%) patients versus (19/42) 45% detected by TPTPB (P < 0.01). 13/19 (68%) of cancers detected by TPTPB had Gleason score ≥7. A total of 82/141 (58%) of positive cores was found in the anterior zone. One patient (2%) experienced urosepsis, 2 (5%) temporary urinary retention, 14 (34%) mild haematuria and 13 (32%) haematospermia. Conclusion: TPTPB still shows a significantly higher prostate cancer detection rate compared to TRUS biopsy (12% versus 45%, P < 0.01) in men with a previous set of negative TRUS biopsy, persistently elevated PSA (but < 20 ng/mL) and benign feeling prostate on DRE. Keywords: biopsy; cancer; prostate; transperineal; transrectal; ultrasonography. INTRODUCTION In the absence of a highly specific biomarker, obtain-ing biopsies from the prostate gland remains the gold standard investigation for establishing a diagnosis of prostate cancer (CaP). Over the last three decades, trans- rectal ultrasound guided (TRUS) biopsy of the prostate has been regarded the technique of choice as it is a well tolerated quick procedure that can be carried out under local anaesthesia in the outpatient setting. However, it is associated with a relatively low specificity of around 30% and confers a 5% risk of urosepsis. On the other hand, transperineal template prostate biop- sy (TPTPB) has been previously shown to have a sig- nificantly higher cancer detection rate (CDR) compared with TRUS biopsy (60% versus 32%, respectively) in biopsy-naïve men with an abnormally elevated PSA < 20 ng/mL and a benign feeling prostate on digital rectal examination (DRE).(1) Furthermore, TPTPB was shown to have a CDR of 58% in men with a persistently ele- vated PSA following 2 previous sets of negative TRUS biopsies.(2) In order to determine whether TPTPB would still prove to be superior to TRUS biopsy in detecting CaP in pa- tients with a background of one negative set of TRUS biopsy but still at risk of cancer, we carried out a pro- spective study, directly comparing both biopsy modal- ities by performing simultaneous TPTPB and TRUS biopsies in this group of patients. PATIENTS AND METHODS Study population: Between August 2012 and August 2014, subjects were selected if they had a history of one previous negative TRUS biopsy with benign pathology result, benign feeling prostate on DRE and a persistent- ly elevated serum PSA more than the age specific range but < 20 ng/mL. All of our participants were given a comprehensive information leaflet explaining the na- ture of the study and gave written consent. The research protocol was registered and approved by the National Research Ethics Service (NRES) committee of East Midlands and by the research and development (R&D) department at the University Hospitals of Leicester Department of Urology, University Hospitals of Leicester NHS Trust, LE5 4PW, Leicester, United Kingdom. *Correspondence: Specialty doctor in Urology, Department of Urology, University Hospitals of Leicester NHS Trust, Leicester General Hospital, Gwendolen Road, Leicester, LE5 4PW. Tel: +447772506239& Fax: +44116 273 0639. Email: shady.nafie@me.com. Received September 2016 & Accepted February 2017 Urological Oncology 3008 NHS trust. Procedure: All the patients underwent both biopsies un- der general anaesthetic by the same surgeon (MAK) as a day case under antibiotic cover. Each patient was giv- en a single dose of oral Ciprofloxacin 500 mg at least 30 minutes before anaesthesia. At induction of anaes- thesia, 120 mg of Gentamicin and 1.2 g of Augmentin were administered intravenously unless the patient was penicillin allergic, in which case 400 mg of Teicoplanin was intravenously administered. After placing the patient in the left lateral position, an ultrasound probe (BK Medical Pro-Focus 2202; BK Medical, Mileparken, Denmark) was placed in the rec- tum to visualise the prostate and calculate the prostate volume. Then, 12 TRUS guided core biopsies were tak- en from the right and left peripheral zones as previously described by Presti et al.(3) The ultrasound probe was taken out of the rectum. The patient was subsequently placed in the extended lithotomy position. The perineal area was shaved, the scrotum was secured away from the biopsy area using mepore tape, then the perineum and the genital area were prepped and draped. Thereafter, a 14-Fr urethral catheter was inserted in order to mark the urethra and determine the degree of haematuria at the end of the procedure. TPTPB were then performed as previously described.(4) In short, the ultrasound probe was reinserted in the rectum, A STEPPER (Galil Med- ical; Crawley, Sus- sex, UK) with an articulated arm and a stabilizer was used to fix the ultrasound probe, then a standard 0.5 cm brachytherapy template grid was attached to the STEPPER and positioned over the perineum. With the prostate at its widest in the trans- verse plane, the gland was divided on the ultrasound screen into six sectors (right anterior, left anterior, right mid, left mid, right posterior and left posterior). In each sector six 18-gauge biopsy needles (Pro-Mag™ Biop- sy Needle, 18G x 20cm, MCXS1820AX) were placed into the prostate in the transverse plane view using the brachytherapy template grid. Once all six needles were inserted, the probe was switched to the sagittal plane view and the needles were gently withdrawn, one at a time. In every case, the biopsies were performed in ex- actly the same systematic manner starting with the right anterior sector followed by left anterior and then right mid and so on ending with the left posterior. It was de- cided that TRUS biopsies would be performed before the TPTPB in order not to alter the sensitivity of TRUS biopsies in picking up cancer cells. Evaluations: Histological analysis was undertaken by the same pathologist (JPD), using standard haemo- toxylin and eosin stained, formalin fixed and paraffin embedded sections. Standard 4µm sections were exam- ined over three levels from each core. Where necessary immunoperoxidase to p63, 34betaE12 and AMACR (p504s) antigens were also employed to render a diag- nosis. Statistical analysis: Analysis was carried out using Fisher's exact test to evaluate the association of nominal variables, and Student t-test to evaluate the difference in categorical variables. All calculated values were 2-sid- ed, considering P < 0.05 statistically significant. Power analysis was conducted using a power model based on a one-proportion Z, Chi-squared test within STATIS- TICA (StatSoft, Tulsa, Ohio). This analysis indicated that to obtain a power of 0.9 (using alpha value of 0.05, a TRUS frequency of 0.32 and a TPTPB frequency of 0.6) would require 30 cases. Furthermore, power analy- sis was undertaken for a 2-way 2-proportion Z-test, this analysis indicated that to obtain a power of 0.8 (using the same alpha value and the same frequencies) would require 50 cases. This was based on a null hypothesis that the proportions of positive cases detected were equal. After performing 42 cases, the data was analysed and a large significant difference was determined in CDR between both biopsy modalities. Hence, contin- uing further with the study was felt unethical, as eight further cases would not have altered the overall trend in the study outcome. RESULTS A cohort of 42 men were enrolled in our study, they had a mean age of 65 years (range: 50-75), mean prostate volume of 59 mL (range: 21-152), mean PSA of 8.3 ng/L (range: 4.4-19) and mean PSA density (PSAD) of 0.2 ng/mL/cc (range: 0.07-0.47) at the time of perform- ing the study. At the time of the initial TRUS biopsy, TPTPB after one negative TRUS biopsy-Nafie et al. Table 1. Difference in PSA levels, Prostate volumes and PSAD between initial and study biopsies Mean (± SD) Study Biopsy Initial Biopsies P Value PSA 8.3 (± 3.0) 6.6 (± 2.5) 0.0003 Prostate Volume 59 (± 26.9) 56 (± 23.7) 0.71 PSAD 0.20 (± 0.1) 0.15 (± 0.1) 0.55 Abbreviations: PSA, Prostate Specific Antigen; PSAD, Prostate Specific Antigen Density; SD, Standard Deviation. Pathology Initial TRUS Biopsy Study TRUS Biopsy Study TPTPB Gleason 6 0 (0%) 3 (7%) 6 (14%) Gleason 7 0 (0%) 2 (5%) 13 (31%) Benign 24 (57%) 10 (24%) 5 (12%) Atypia 1 (2%) 8 (19%) 8 (19%) ASAP 7 (17%) 4 (10%) 2 (5%) High PIN 10 (24%) 15 (35%) 8 (19%) Abbreviations: ASAP, Atypical Small Acinar Proliferation; PIN, Prostatic Intraepithelial Neoplasia. Table 2. Pathological findings of initial/new TRUS biopsies and TPTPB Vol 14 No 02 March-April 2017 3009 they had a mean PSA of 6.6 ng/mL (range: 3.1-15) with mean PSA density of 0.14 ng/mL/cc (range: 0.06-0.42). The time interval between the initial TRUS biopsy and the study biopsies ranged from one month up to 150 months, with median of 19 months and mean of 33 months. Mean PSA velocity was 0.65 ng/mL/year (range: 0-3.5). There was a significant difference in PSA levels (P < 0.05) between the time of the initial TRUS biopsy and the study biopsies, but not in PSAD or PSA volumes as shown in Table 1. In total, 22/42 (52%) patients had benign pathology by both TRUS biopsy and TPTPB, while 20/42 (48%) pa- tients had cancer pathology in their biopsies. Of those 20 patients diagnosed with prostate cancer, 15 (36%) had negative TRUS biopsies but positive TPTPB, 4 (10%) had positive biopsies with both TRUS and TPT- PB and 1 (2%) had positive TRUS biopsies but nega- tive TPTPB. Therefore, the overall CDR of TPTPB was 45% (19/42) versus 12% (5/42) for TRUS biopsies (P < .001). Calculated Cohen's Kappa was 0.17 indicating poor concordance between TPTPB and TRUS biopsy results, denoting the genuine difference in the ability of TPTPB to detect prostate cancer compared to TRUS biopsy in this setting. The histopathological findings of the initial TRUS biopsy, the study TRUS biopsy and the TPTPB are all listed in Table 2 and Table 3. Out of the 19 patients who had cancer detected by TPT- PB, 13 (68%) had Gleason score of 7. Furthermore, 11/15 (73%) of cancers that were exclusively detected by TPTPB and missed by TRUS biopsy had Gleason score of 7. A total of 82/141 (58%) of the positive cores detected by TPTPB were found in the anterior sector of the prostate as shown in Figure 1. Only one patient (2%) experienced urosepsis, 2 (5%) had temporary urinary retention, 14 (34%) had mild haematuria and 13 had (32%) haematospermia that re- solved spontaneously within two to three days. DISCUSSION Over the last decade, TPTPB has been recognized as a more clinically efficient diagnostic modality than TRUS biopsy in the initial and repeated biopsy settings. However, few studies have compared the two methods directly in a head-to-head comparison as we performed in this study. Performing both biopsy modalities in each patient provided us with the best control group, as the patients acted as their own controls. In our case, the TRUS biopsy (presenting the conventional practice) was compared to the TPTPB (presenting the newly evaluated practice) in the same Our study further reinforces the superior clinical effi- ciency of TPTPB over TRUS biopsy. TPTPB is par- ticularly indicated when a patient has been subjected to one or more negative sets of TRUS biopsy and a suspicion of prostate cancer remains. Furthermore, a large proportion of cancers detected in the repeat biopsy setting are located anteriorly. Studies have shown that approximately 20% of all prostate cancers are anterior and these cancers are more likely to have extracapsular extension at the time of treatment, potentially resulting in a higher positive surgical margin rate(5). Over the last decade, TPTPB has been recognized as a more clinically efficient diagnostic modality than TRUS biopsy. In 2014, we(1) compared TRUS biopsy and TPTPB in 50 biopsy-naïve men with suspicion of prostate cancer where TRUS and TPTPB were per- formed at the same setting. Overall, 60% were diag- nosed with CaP, with 25% detected by only TPTPB but missed by TRUS biopsy. On the contrary, all cancers detected by TRUS biopsy were also detected by TPT- PB. In 2007 Kawakami et al. published a study of 324 men who underwent 12-core TRUS biopsy followed by 24-core combined TRUS biopsy and TPTPB. 12 men were diagnosed with cancer by the combined technique but missed by TRUS biopsy alone. Subsequent mpMRI showed that 92% of cancers were located anteriorly.(6) In 2015 Ong et al. conducted a study in which TPTPB was performed in 160 biopsy-naïve men with clinical suspicion of CaP underwent 12-core TRUS biopsy and 12-core TPTPB simultaneously. Most cancers detected by TPTPB and missed by TRUS biopsy were located anteriorly, and although most cancers missed by TRUS biopsy were low grade and low volume, some clinically significant cancers were also missed.(7) In 2014, Our clinical group also performed TPTPB in 122 men with two negative sets of TRUS biopsy and persistently elevated PSA. CaP was detected in 58% of these men and 46% of those diagnosed had clinically significant cancer based on criteria of Gleason score of ≥ 7, or more than three positive cores of Gleason 6.(1) A larger study in 2013 by Bittner examined a cohort of 485 men who underwent TPTPB following negative TRUS biopsy due to either persistently elevated PSA, atypical small acinar proliferation (ASAP) or high grade prostat- Table 3. Cancer detection in TRUS biopsy and TPTPB TPTPB (negative Cancer) TPTPB (positive Cancer) TRUS (-ve Cancer) 22 15 TRUS (+ve Cancer) 1 4 Abbreviations: TRUS, Transrectal Ultrasound; TPTPB, Transperineal Template Prostate Biopsy. Figure 1. Site of cancer positive cores detected by TPTPB (n=141) TPTPB after one negative TRUS biopsy-Nafie et al. Urological Oncology 3010 ic intraepithelial neoplasia (PIN). Cancer was detected in 226 men (46.6%), 196 of which were clinically sig- nificant according to the Epstein criteria and most of them were anterior. (8) Results of other published series support the aforementioned findings, demonstrating a higher CDR from TPTPB in the repeat biopsy context (4,7,9–11) as well as superior antero-apical sampling with TRUS biopsy(5,8,10,12). TPTPB is associated with a much lower risk of sepsis compared with TRUS biopsy. A study from Melbourne of 245 patients undergoing TPB showed that there were no readmissions with sepsis post-operatively.(13) Sim- ilarly, in our experience from over 500 patients who have undergone TPTPB we have not had a single case of urospesis (unpublished data). Further published se- ries support this with an overall risk of sepsis follow- ing TPTPB approaching zero in some studies. On the contrary, the risk of sepsis following TRUS is in the region of 5% including infection with multi-resistant organisms.(13) Therefore, TPTPB is particularly favour- able when selecting a procedure for patients who are di- abetic or immunocompromised or those with previous antibiotic resistance.(14) Studies have also shown that TPTPB offers the benefit of mapping of the prostate, thereby decreasing the risk of under-grading patients compared with TRUS biop- sy. A study published in 2015 of 431 patients who un- derwent RP following either TRUS biopsy or TPTPB compared the final Gleason grade with the initial grade on diagnosis.(15) TPTPB was found to be more accurate than TRUS biopsy in predicting final Gleason score. Furthermore, a prospective randomized study compar- ing 12-core TPTPB with 12-core TRUS biopsy in 200 men demonstrated a significantly higher diagnostic ef- ficiency with TPTPB in men with PSA values in the lower end of the pathological range (i.e. 4.1 - 10ng/ml). (16) Finally, TPTPB also has the ability to diagnose CaP in patients who have previously undergone abdomi- no-perineal (AP) resection for rectal cancer.(10) It is well known that more time is required to perform TPTPB, including general anaesthetic time, and that more training is needed for the surgeon. Although its provision is increasing, it is still less widely available than TRUS biopsy.(14) It has also been shown to be more painful than TRB and harbor an increased risk of acute urinary retention in those with larger prostates. More- over, despite the majority of studies showing a higher CDR overall with TPTPB compared with TRUS biop- sy, some studies, although few in number, have shown statistically similar CDRs between the two techniques both in the initial(17,18) and the repeat biopsy setting (19). This could reflect variance in levels of operator expe- rience. Finally, a potential drawback of a higher CDR might be an increased detection of clinically insignifi- cant cancer, which could be cause for concern particu- larly if TPTPB becomes the modality of choice in diag- nosing prostate cancer.(20) This could potentially subject some patients to further unnecessary tests downstream as well as increase financial burden on the healthcare system. There is emerging evidence that multiparametric MRI (mpMRI) may increase the efficiency of TPTPB, whilst reducing the number of biopsies required for a diagno- sis. This could result in reduced pain levels following the procedure as well as a lower risk of urinary reten- tion. However, early studies show that mpMRI may have a false negative rate of up to 20% and may miss some Gleason 3 cancers.(20,21) The significance of the latter is uncertain. The PROMIS trial which is currently taking place consists of a RCT of 714 men and could help answer some critical questions, namely: whether mpMRI could exclude clinically insignificant cancer, thus reducing the number of unnecessary biopsies; and whether prebiopsy MRI increases the detection rate of clinically significant cancer. Finally, it will hopefully determine the sensitivity, specificity, negative predic- tive value and overall cost-effectiveness of mpMRI ver- sus TPB and TRB.(22) In this study we compared TRUS biopsies versus TPT- PB without the advantage of MRI to determine whether we should abandon TRUS biopsies and look specifi- cally for TPTPB. Our results have clearly shown that TPTPB outperformed TRUS biopsies in the diagnostic yield for CaP in men who had previous negative TRUS biopsies and persistently elevated PSA. CONCLUSIONS TPTPB has a significantly higher prostate cancer de- tection rate in comparison to TRUS biopsies in men with persistently abnormally elevated PSA < 20 ng/mL, benign feeling prostate on DRE and one previous set of negative TRUS biopsies. Our findings are consistent with the contemporary literature, which also demon- strates additional advantages in selecting TPTPB, par- ticularly in patients with an inherently higher risk of sepsis as well as those who have undergone previous AP resection. Performing mpMRI may further enhance the CDR from TPTPB by performing TB and SB simul- taneously. However, it is still not widely available and results from the PROMIS trial are awaited to elucidate its role. CONFLICT OF INTEREST The authors report no conflict of interest. REFRENCES 1. Nafie S, Mellon JK, Dormer JP, Khan MA. The role of transperineal template prostate biopsies in prostate cancer diagnosis in biopsy naïve men with PSA less than 20 ng ml(-1.). 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