To evaluate the efficacy of prostate cancer (PCa) detection by the electronic nose (EN) on human urine samples.
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 <12 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.
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.
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 (PCa) is the most frequent noncutaneous malignancy in men.1 Although PSA blood testing remains the most widely used tool for PCa detection,2 it has been historically characterized by lack of specificity3 while none of the novel urine or blood biomarkers—proposed in the past decade—is currently widely used.4
Recently, Matsumura et al.,5 for the first time, established the feasibility of using urinary volatiles to detect lung cancer. In the field of PCa, Cornu et al.6 performed a double-blind study using an adequately trained dog in order to check its ability to detect PCa by sniffing urine. The results were surprising, with the dog correctly designating the cancer samples in 30 of the 33 cases, conferring a 91% of both specificity and sensitivity to the test. Although issues about the reproducibility (significantly affected by the canine olfactory receptor polymorphisms that influence odor detection performance of sniffer dogs7), costs and duration of the method were raised, we believe this study provided the first demonstration that PCa gives an odor signature to urine.
Using the electronic nose (EN), Roine et al.8 recently proved that both malignant and nonmalignant prostate cell lines have distinct smell prints.
Based on these findings, we herein present, to the best of our knowledge, the first study on the outcomes of the EN in the identification of PCa in urine samples of prostate biopsy (PB) candidates. Our aim was to evaluate the performance of this device as a conventional, non-invasive, quick and easily available instrument that, added to PSA, may improve PCa diagnosis.
Materials and methods
Urine samples were obtained from Caucasian patients recruited in our center following written consent for analysis of their urine for research programs. All men included were consecutive candidates of PB. Our local ethics committee was informed of the study; a formal approval was considered unnecessary, because our observational preliminary study did not change the normal clinical practice: the urine samples used for the EN evaluation were immediately dismissed and they were not used for any other clinical or scientific purpose, while EN findings did not change the clinical behavior of the involved medical staff.
A complete blood count, a basic metabolic panel (blood urea nitrogen, creatinine, glucose, electrolytes) and a urinanalysis, were required to all candidates to PB as standard clinical practice.
Exclusion criteria for our study were a history of urothelial carcinoma or other malignant disease, any urine infection, fasting for <12 h before PB or ingestion of foods that might alter the urine smell (that is, asparagus, cauliflower, garlic, meat) in the past 24 h.
Each patient was given two sterile containers for urine collection, one for the initial part of the voided urine and the other for the midstream. The urine sample collection was performed just before the PB, independently of the fact that it was or not the first voided urine of that morning. Volatile organic compound (VOC) analysis was performed before the PB and without knowing the PB results by medical and engineering staff not involved in the PB procedure. Furthermore, the staff performing the PB was not aware of the VOC analysis results.
An adequately designed obturator was created in order to permit the extraction of the urine headspace necessary for the EN analysis (Figure 1). Both samples were then analyzed by last version of the EN as developed at the faculty of Engineering of the University of Rome ‘Tor Vergata’ (Figure 2).9 All measurements were performed within two hours from the collection of the urine samples, in order to maintain unaffected the olfactory characteristics of these samples. All the principles of the reference standard (European Standard, EN 13725, Air quality–Determination of odour concentration by dynamic olfactometry) that is used to provide a scientifically objective method for assessing odours were respected.
The device used is equipped by eight non-selective gas sensors. Each sensor is coated with different metallo-porphyrins. Cu-Tetra Phenyl Porphyrin (TPP), Co-TPP, Zn-TPP, Mn-TPP, Fe-TPP, Sn-TPP, Ru-TPP and Cr-TPP are the eight metals selected, showing a large affinity toward multiple different VOCs. Each sensor (transducer+metallo-porphyrin) adsorbs on its surface the VOCs and changes its frequency of resonance because of its mass variation as regulated by the proportional law of Sauerbrey.10 The interaction between the VOCs and the sensors is regulated by weak bonds, such as Van der Waals, dipole–dipole and hydrogen. The change on the frequency of each sensor with respect to the baseline constitutes the response of that sensor. The final output consists of a fingerprint of the eight sensor responses registered for each sample. A matrix containing all the measurements performed is then extracted by means of a dedicated software.
Two hundred seconds were required for the analysis of each sample (with a flow rate of 50 Standard cc3 per min) while 600 s of flow with dehumidified air were required between two consecutive samples in order to clean the sensors.
All the components with which the urine samples and the urine headspace get in contact during the analysis (teflon for the obturator and tygon for the tubes bringing the headspace to the sensors) are chemically inert, odorless and they present a low potential of absorption towards the VOCs that remain consequently not affected.
Subsequently, all patients underwent a transperineal, transrectal-ultrasound-guided PB according to a standard procedure (12 cores) under local anaesthesia with an 18-G biopsy needle. They were classified as 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 volumes>50 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 set are reported in Figure 3.
The classification model here presented was built using the PB outcomes as reference diagnosis.
The kappa statistic (κ) was used as a measure of chance-corrected agreement between PB and EN. κ-values of<0.20, 0.21–0.40, 0.41–0.60 and>0.61 represent poor, fair, moderate and good agreement, respectively. Sensitivity, specificity, positive predictive value—negative predictive value and their confidence interval (CI) were calculated.
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 aged 54–77 years were included in the study. Nine of them were 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 distribution 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−1. A mean of 12.84 cores was obtained from the PBs (range 12–17). At the final histology, 14 patients (34%) were positive for PCa; 12 of them had a Gleason score of 6 (3+3) and two a score of 7 (3+4).
Midstream urine did not correlate significantly neither with a positive nor with a negative PB; the outcomes on these samples were consequently excluded from the final analysis. On the contrary, we obtained significantly different results on the initial part of the urine stream between positive and negative PBs.
The agreement between PB and EN was 85%, that is, significantly higher than the expected by chance (P<0.001) with a κ value of 0.66.
In particular, the EN correctly recognized 10 out of the 14 cases (that is, sensitivity 71.4% (CI 42–92%)) of PCa while four were false negatives. Both patients with Gleason 7 disease were correctly recognized by the EN. More importantly, 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 (Table 3).
The two false-positive patients were followed-up with digital rectum exam and PSA evaluation at 6 months after the initial biopsy. A rebiopsy was required to one of them for rising PSA, resulting again negative for PCa.
Globally, the positive predictive value of the device is 83.3% (CI 52–98%), while the negative predictive value is 86.2% (CI 68–96%).
Our results are depicted in Figure 4.
The mean number of positive bioptic cores among the correctly identified PCa patients was 3.2 (range: 1 positive core with 10% involvement of the core–7 positive cores with 10–80% involvement of the cores).
The mean number of positive bioptic cores among the four misclassified patients was 1.75 (range 1 positive core with 5% involvement–3 positive cores with 10–40% involved). No statistically significant difference between the mean number of positive cores between correctly identified PCa and misclassified patients was observed.
Figure 5 represents the receiver operating characteristic curve for the discrimination between positive and negative subjects.
The early diagnosis of cancer constitutes a basic and diachronic objective of oncology. Animal models are increasingly used in this context. Canines are mainly adopted due to their superior olfactive apparatus, characterized by a detection threshold of parts per trillion. The scientific basis of this ability of dogs to detect the odor signature of cancer is linked to the VOCs produced by malignant cells.11 In fact, during tumor growth, protein changes in malignant cells lead to peroxidation of the cell membrane components and produce VOCs that can be detected in the headspace of the cells.12, 13
The utilization of dogs for cancer detection merged after the first case report in 1989 about a melanoma detected by a dog on his owner’s leg.14 There followed another study on melanoma detection by dogs directly on tissue that was correct in 75–86% of the cases.15 Other studies documented an impressive performance of the canine olfatus in detecting lung cancer by sniffing the exhaled breath (sensitivity 71%, specificity 93%, independent of the presence of chronic obstructive pulmonary disorder or tobacco smoke),16 ovarian cancer in both blood and tissue samples (reaching a sensitivity of 100% and specificity of 98%),17 breast cancer in breath samples (sensitivity and specificity of dog detection of 88% and 98%, respectively),18 colorectal cancer (sensitivity of 91% and 97% in breath and fecal samples, respectively, and a specificity of 99% for both sample types)19 and bladder cancer by sniffing urine samples, with a diagnostic success rate of 41%.20
In the specific field of PCa, Cornu et al.6 showed for the first time that a trained dog can distinguish a PCa urine sample among controls with powerful results: the dog correctly designating the cancer samples in 30 of the 33 cases, conferring a 91% of both specificity and sensitivity to the test. The main success of this study was the demonstration that PCa gives an odor signature to the urine.
However, these results were obtained with only one dog, questioning the reproducibility of this method. Furthermore, the type of dog used in the study could have influenced the results as canine olfactory receptor polymorphisms have been shown to influence odor detection performance by sniffer dogs.7 The study was costly and long in duration, making it difficult to conceive of an extended use for this test in clinical practice. Finally, concerns regarding the lack of communication with the dogs, the difficulties of an adequate training, several hygienic and even ethical issues regarding dogs subjected to forced inhalation should be further considered.
Although canine cancer detection is not feasible for wide-spread clinical use, these studies have inspired a race between scientists to develop suitably sensitive analytical instrumentation for quick and easy clinical use that show similar accuracy to the mammalian sense of smell.23 By the term EN it is intended an instrument able to generate digital maps of complex odours or chemical images. The fundamental elements of the EN that we used are the piezoelectric chemical sensors which operating as miniaturized transducers do respond reversely to the chemical volatile compounds generating electrical signals in function of the gas concentration in real time. The operating principle of the piezoelectric chemical sensors (also defined mass sensors) located in the EN narici, is based on the variation of frequency of resonating quartz crystals (Thickness Shear Mode Resonators) caused by their mass variation, that its turn is determined by the adsorption or desorption processes of gas molecules on the chemical interactive materials (metal-tetrafenilporfirine) of the sensors. The odour classification is obtained by a kind of multivariate statistical analysis.
The interaction between VOC with an array of sensors generates a characteristic fingerprint, which can be recognized by comparing it with previously recorded patterns in the recognition system.
The common features of all ENs are the relatively fast response and the satisfactory stability. These devices are used in the food and agricultural sectors, in the ambient monitoring, in space applications and for security issues. In the medical field, they are used as diagnostic instruments for skin disease and internal pathology detection, such as diabetes, cancers, urinary infections, tuberculosis, Helicobacter pylori and so on.21, 24
Focusing on the field of oncology, dynamic olfactometry, obtained with EN, is currently studied as an alternative to the animal model for the early detection of several human tumors. Successful detection of lung,25, 26, 27, 28, 29, 30 gastric,31 ovarian,32 colorectal,33 breast18, 34 and skin cancer35 by EN are well described in the published literature, with results similar, even if somewhat lower, to those of the dogs.
In the field of urological oncology, the EN has already shown promising results concerning the detection of urinary tract tumors in urine samples. EN was able to discriminate urine samples of healthy patients from those of patients with bladder cancer, with a diagnostic accuracy of 100%,36 overcoming the one described for dogs.20
The diagnostic accuracy of EN in PCa was not verified till recently, when Roine et al.8 analyzed through EN the smell prints of collected prostatic nonmalignant (EP-156 T and controls) and malignant (LNCaP) cell lines. The device differentiated the nonmalignant and malignant cell lines from each other with very low misclassification rates (2.9–3.6%), suggesting that malignant and nonmalignant cell lines have distinct smell prints.8
With our study, we demonstrated, we believe for the first time, in the clinical setting (and not in cell lines) a potential role of the EN in identifying, by smelling urine samples, healthy patients (with negative PB) while maintaining an acceptable sensitivity. The overall agreement between biopsy and EN was 85% while the κ value (0.66) indicates a good agreement between the two methods.
The first part of the urine stream (and not the midstream) allowed to distinguish between positive and negative PBs, probably because it contains elements of prostatic secretion.
The influence of eventual confounding factors that might alter the urine smell influencing the EN accuracy has been also evaluated. Alcohol, oral medications and smoke do not seem to influence the accuracy of the device.
Overall, the results obtainable with the EN are chemical images with a rather high probability of cancer recognition. The high specificity of the device, if confirmed, could allow to reduce the number of first PBs or rebiopsies. It would appear that if the test results negative, the PB will be almost always (93%) negative; thus, both extra costs to the health-care system and potential complications to the patients of further diagnostic procedures may be spared.
Two issues regarding the sensors should be further commented: their statistical contribution and their consistency with respect to prostate disease-alleged biomarkers. It is worth mentioning that the used EN consists of an array of non-selective gas sensors, thus each latent variable is a linear combination of all the sensors: they search for characteristic smell prints of particular conditions in the urine headspace without distinguishing the constituent VOCs. Over-fitting condition is avoided by the selection of the correct number of LVs (see Table 1). Moreover, as can be observed in the loading plots in Figure 6, none of the sensors in the array shows negligible load values with respect to the other ones. Concerning sensors’ pertinence with respect to PCa diagnosis, the possible discrimination of cancer is based on fingerprint differentiation, thus the volatile biomarkers of PCa remain to be identified, in order to use selective adsorbing material in sensor arrays.
Some limitations of our study should be mentioned. The number of cases is small to permit definitive conclusions on the diagnostic accuracy of the EN, although the preliminary data appears at least promising. Only two cases of Gleason score 7 disease were included in the study, and none with Gleason score⩾8; consequently, the ability of the device in the diagnosis of high-grade PCa cannot be evaluated. However, it seems promising that both cases of Gleason 7 were correctly classified by the device as positive. The minimum number of positive cores/minimum percentage of core involvement that correlates to an accurate diagnosis were not addressed. However, in our preliminary analysis, there was not a significant difference between mean number of positive cores in correctly identified PCa vs misclassified patients. Finally, any correlation between the EN results and the outcomes of the radical prostatectomy specimen was not explored.
Table 3 suggests crucial areas for further future research on EN.
To the best of our knowledge, this is the first demonstration of an olfactory imprinting of the initial part of the urine stream in patients with PCa that was accurately revealed by an EN. The high specificity of the device, if confirmed in larger studies, may overcome the known specificity gap of the PSA serum test, and it may permit a more accurate selection of the PB candidates.
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The authors declare no conflict of interest.
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Asimakopoulos, A., Del Fabbro, D., Miano, R. et al. Prostate cancer diagnosis through electronic nose in the urine headspace setting: a pilot study. Prostate Cancer Prostatic Dis 17, 206–211 (2014). https://doi.org/10.1038/pcan.2014.11
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