Prostate Cancer and Prostatic Disease (2014) 17, 206–211 & 2014 Macmillan Publishers Limited All rights reserved 1365-7852/14 www.nature.com/pcan ORIGINAL ARTICLE Prostate cancer diagnosis through electronic nose in the urine headspace setting: a pilot study AD Asimakopoulos1, D Del Fabbro2, R Miano2, M Santonico3, R Capuano4, G Pennazza3, A D’Amico4 and E Finazzi-Agro` 2 BACKGROUND: To evaluate the efficacy of prostate cancer (PCa) detection by the electronic nose (EN) on human urine samples. METHODS: Urine samples were obtained from candidates of prostate biopsy (PB). Exclusion criteria were a history of urothelial carcinoma or other malignant disease, urine infection, fasting for o12 h before PB or ingestion of alcohol or foods that might alter the urine smell in the last 24 h. The initial part of the voided urine and the midstream were collected separately in two sterile containers. Both samples were analyzed by the EN immediately after the collection. All patients underwent a standard transperineal, transrectal-ultrasound-guided PB. The pathological results were compared with the outcomes of the EN. Sensitivity and specificity of EN were assessed. RESULTS: Forty-one men were included in the study. Fourteen out of the 41 patients were positive for PCa. Midstream urine did not correlate significantly neither with a positive nor with a negative PB. Instead, significantly different results on the initial part of the urine stream between positive and negative PBs were obtained. The EN correctly recognized 10 out of the 14 cases (that is, sensitivity 71.4% (confidence interval (CI) 42–92%)) of PCa while four were false negatives. Moreover, the device recognized as negative 25 out of the 27 (that is, specificity 92.6% (CI 76–99%)) samples of negative PBs, with only two false positives. CONCLUSIONS: We believe this is the first demonstration of an olfactory imprinting of the initial part of the urine stream in patients with PCa that was revealed by an EN, with high specificity. Prostate Cancer and Prostatic Disease (2014) 17, 206–211; doi:10.1038/pcan.2014.11; published online 1 April 2014 INTRODUCTION MATERIALS AND METHODS Prostate cancer (PCa) is the most frequent noncutaneous Urine samples were obtained from Caucasian patients recruited in our malignancy in men.1 Although PSA blood testing remains the center following written consent for analysis of their urine for research most widely used tool for PCa detection,2 it has been historically programs. All men included were consecutive candidates of PB. Our local characterized by lack of specificity3 while none of the novel urine ethics committee was informed of the study; a formal approval was considered unnecessary, because our observational preliminary study did or blood biomarkers—proposed in the past decade—is currently not change the normal clinical practice: the urine samples used for the EN widely used.4 5 evaluation were immediately dismissed and they were not used for any Recently, Matsumura et al., for the first time, established the other clinical or scientific purpose, while EN findings did not change the feasibility of using urinary volatiles to detect lung cancer. In the clinical behavior of the involved medical staff. field of PCa, Cornu et al.6 performed a double-blind study using A complete blood count, a basic metabolic panel (blood urea nitrogen, an adequately trained dog in order to check its ability to detect creatinine, glucose, electrolytes) and a urinanalysis, were required to all PCa by sniffing urine. The results were surprising, with the dog candidates to PB as standard clinical practice. correctly designating the cancer samples in 30 of the 33 cases, Exclusion criteria for our study were a history of urothelial carcinoma or conferring a 91% of both specificity and sensitivity to the test. other malignant disease, any urine infection, fasting for o12 h before PB or ingestion of foods that might alter the urine smell (that is, asparagus, Although issues about the reproducibility (significantly affected by cauliflower, garlic, meat) in the past 24 h. the canine olfactory receptor polymorphisms that influence odor 7 Each patient was given two sterile containers for urine collection, one for detection performance of sniffer dogs ), costs and duration of the the initial part of the voided urine and the other for the midstream. The method were raised, we believe this study provided the first urine sample collection was performed just before the PB, independently demonstration that PCa gives an odor signature to urine. of the fact that it was or not the first voided urine of that morning. Volatile Using the electronic nose (EN), Roine et al.8 recently proved that organic compound (VOC) analysis was performed before the PB and both malignant and nonmalignant prostate cell lines have distinct without knowing the PB results by medical and engineering staff not smell prints. involved in the PB procedure. Furthermore, the staff performing the PB Based on these findings, we herein present, to the best of our was not aware of the VOC analysis results. An adequately designed obturator was created in order to permit the knowledge, the first study on the outcomes of the EN in the extraction of the urine headspace necessary for the EN analysis (Figure 1). identification of PCa in urine samples of prostate biopsy (PB) Both samples were then analyzed by last version of the EN as developed at candidates. Our aim was to evaluate the performance of this the faculty of Engineering of the University of Rome ‘Tor Vergata’ device as a conventional, non-invasive, quick and easily available (Figure 2).9 All measurements were performed within two hours from the instrument that, added to PSA, may improve PCa diagnosis. collection of the urine samples, in order to maintain unaffected the 1UOC of Urology, Department of Surgery, University of Rome Tor Vergata–Policlinico Casilino, Rome, Italy; 2UOC of Urology, Department of Surgery, University of Tor Vergata, Policlinico Tor Vergata, Rome, Italy; 3Center for Integrated Research—CIR, Unit of Electronics for Sensor Systems, Universita` Campus Bio-Medico, Rome, Italy and 4Department of Electronic Engineering, University of Tor Vergata, Rome, Italy. Correspondence: Dr AD Asimakopoulos, UOC of Urology, Department of Surgery, University of Rome Tor Vergata– Policlinico Casilino, Viale Oxford 81, Rome, 00133, Italy. E-mail: [email protected] Received 7 November 2013; revised 15 January 2014; accepted 9 February 2014; published online 1 April 2014 Prostate cancer and electronic nose AD Asimakopoulos et al 207 cases or controls after pathological examination of the specimens. The peripheral zone of both lobes from the lateral to the paramedian area and from the base to the apex was sampled. For prostate volumes450 ml and on the basis of the clinical/ultrasound characteristics, additional cores were eventually obtained. A dedicated uropathologist examined the slides. Finally, the pathological results were compared with the outcomes of the EN. The overall graphic representation of the measurements obtained from the eight sensors requires an eight-dimensional space. A multivariate analysis obtained with supervised PLS-DA (Partial Least Square– Discriminant Analysis) permitted the graphic representation of the sensors’ measurements on a bi- or three-dimensional model. The PLS-DA model was built by using the leave-one-out as cross- validation criterion. All the statistical data relative to the model are reported in Table 1. All the statistical parameters of the sensor array data Figure 1. The adequately designed obturator created in order to set are reported in Figure 3. permit the extraction of the urine headspace, necessary for the The classification model here presented was built using the PB electronic nose analysis. outcomes as reference diagnosis. Statistical methods The kappa statistic (k) was used as a measure of chance-corrected agreement between PB and EN. k-values ofo0.20, 0.21–0.40, 0.41–0.60 E.N. and40.61 represent poor, fair, moderate and good agreement, respec- tively. Sensitivity, specificity, positive predictive value—negative predictive FILTERS value and their confidence interval (CI) were calculated. SOFTWARE RESULTS Fifty consecutive patients referring to our center for PB were evaluated for eventual analysis of their urine headspace through EN. By applying the exclusion criteria, nine candidates to PB were excluded from the evaluation with EN. Consequently, 41 men URINE aged 54–77 years were included in the study. Nine of them were SAMPLE smokers, seven were diabetics and 34 assumed oral medications for various pathologies. One patient was liver-transplanted. There was no statistically significant difference in the distribu- Figure 2. Electronic nose (E.N.). tion of smokers vs non-smokers, alcohol consumers vs non- alcohol consumers and between patients under oral medications or not between the control and cancer group (Table 2). PSA ranged between 1.95 and 14.02 ng ml À 1. A mean of 12.84 cores was obtained from the PBs (range 12–17). At the final histology, 14 olfactory characteristics of these samples. All the principles of the reference patients (34%) were positive for PCa; 12 of them had a Gleason standard (European Standard, EN 13725, Air quality–Determination of score of 6 (3 þ 3) and two a score of 7 (3 þ 4). odour concentration by dynamic olfactometry) that is used to provide a Midstream urine did not correlate significantly neither with a scientifically objective method for assessing odours were respected. The device used is equipped by eight non-selective gas sensors. Each positive nor with a negative PB; the outcomes on these samples sensor is coated with different metallo-porphyrins. Cu-Tetra Phenyl were consequently excluded from the final analysis. On the Porphyrin (TPP), Co-TPP, Zn-TPP, Mn-TPP, Fe-TPP, Sn-TPP, Ru-TPP and contrary, we obtained significantly different results on the initial Cr-TPP are the eight metals selected, showing a large affinity toward part of the urine stream between positive and negative PBs.