SANIST: a rapid mass spectrometric SACI/ESI data acquisition and elaboration platform for verifying potential candidate biomarkers.

RATIONALE: Surface-Activated Chemical Ionization/Electrospray Ionization mass spectrometry (SACI/ESI-MS) is a technique with high sensitivity and low noise that allows accurate biomarker discovery studies. We developed a dedicated SACI/ESI software, named SANIST, for both biomarker fingerprint data acquisition and as a diagnostic tool, using prostate cancer (PCa) as the disease of interest. METHODS: Liquid chromatography (LC)/SACI/ESI-MS technology was employed to detect a potential biomarker panel for PCa disease prediction. Serum from patients with histologically confirmed or negative prostate biopsies for PCa was employed. The biomarker data (m/z or Thompson value, retention time and extraction mass chromatogram peak area) were stored in an ascii database. SANIST software allowed identification of potential biomarkers. A Bayesian scoring algorithm developed in house allowed sample separation based on comparison with samples in the database. RESULTS: Biomarker candidates from the carnitine family were detected at significantly lower levels in patients showing histologically confirmed PCa. Using these biomarkers, the SANIST scoring algorithm allowed separation of patients with PCa from biopsy negative subjects with high accuracy and sensitivity. CONCLUSIONS: SANIST was able to rapidly identify and perform a preliminary evaluation of the potential diagnostic efficiency of potential biomarkers for PCa.

Practical recommendations for performing ultrasound scanning in the urological and andrological fields.

AIM: US scanning has been defined as the urologist's stethoscope. These recommendations have been drawn up with the aim of ensuring minimum standards of excellence for ultrasound imaging in urological and andrological practice. A series of essential recommendations are made, to be followed during ultrasound investigations in kidney, prostate, bladder, scrotal and penile diseases. METHODS: Members of the Imaging Working Group of the Italian Society of Urology (SIU) in collaboration with the Italian Society of Ultrasound in Urology, Andrology and Nephrology (SIEUN) identified expert Urologists, Andrologists, Nephrologists and Radiologists. The recommendations are based on review of the literature, previously published recommendations, books and the opinions of the experts. The final document was reviewed by national experts, including members of the Italian Society of Radiology. RESULTS: Recommendations are listed in 5 chapters, focused on: kidney, bladder, prostate and seminal vesicles, scrotum and testis, penis, including penile echo-doppler. In each chapter clear definitions are made of: indications, technological standards of the devices, the method of performance of the investigation. The findings to be reported are described and discussed, and examples of final reports for each organ are included. In the tables, the ultrasound features of the principal male uro-genital diseases are summarized. Diagnostic accuracy and second level investigations are considered. CONCLUSIONS: Ultrasound is an integral part of the diagnosis and follow-up of diseases of the urinary system and male genitals in patients of all ages, in both the hospital and outpatient setting. These recommendations are dedicated to enhancing communication and evidence-based medicine in an inter- and multi-disciplinary approach. The ability to perform and interpret ultrasound imaging correctly has become an integral part of clinical practice in uro-andrology, but intra and inter-observer variability is a well known limitation. These recommendations will help to improve reliability and reproducibility in uro-andrological ultrasound scanning.

Surface-activated chemical ionization-electrospray ionization source improves biomarker discovery with mass spectrometry.

RATIONALE: Mass spectrometry (MS) is increasingly employed for the discovery of clinical biomarkers. However, due to sensitivity limitations related to in-source ionization yield, many potential biomarkers are not detected by standard mass spectrometers. Therefore, more efficient ion-source technologies are needed to improve MS applications in biomarker discovery. METHODS: Among novel ion-source technologies, Surface-Activated Chemical Ionization (SACI), although endowed with high sensitivity linked to its ability to reduce chemical noise in mass spectra, has seen limited application in biomarker discovery to date, due to its selectivity for highly polar compounds. However, in combination with an Electrospray Ionization (ESI) source, SACI selectivity can be enlarged in the range of less polar compounds. To validate the new SACI-ESI approach in biomarker discovery, we applied it to a translational setting in oncology. We performed MS profiles of 101 human serum samples from a male population, aged 40 or older, coming to the clinic for prostate cancer evaluation based on multiple PSA exams, digital rectal examination and echography. The SACI-ESI MS spectra were analyzed and classified with an innovative bioinformatic approach based on the MS-search freeware developed in house. RESULTS: Here we demonstrate that the SACI-ESI combination can produce MS spectra with greater sensitivity and lower noise than those obtained with the common ESI alone. We found that the SACI-ESI combination increased the number of detectable compounds and produced better quality of profiles in liquid chromatography (LC) coupled with MS (LC/MS) analysis of human serum samples, improving disease prediction potential. CONCLUSIONS: SACI-ESI can facilitate MS-based discovery of potential biomarkers in human serum. Combined with the proposed bioinformatic approach (based on XCMS and NIST data elaboration) for the analysis of the MS spectra obtained, the potential for developing biomarkers with diagnostic capabilities are demonstrated in a prostate cancer diagnosis clinical setting.

Role of imaging and biopsy to assess local recurrence after definitive treatment for prostate carcinoma (surgery, radiotherapy, cryotherapy, HIFU).

PURPOSE: Defining the site of recurrent disease early after definitive treatment for a localized prostate cancer is a critical issue as it may greatly influence the subsequent therapeutic strategy or patient management. METHODS: A systematic review of the literature was performed by searching Medline from January 1995 up to January 2011. Electronic searches were limited to the English language, and the keywords prostate cancer, radiotherapy [RT], high intensity focused ultrasound [HIFU], cryotherapy [CRIO], transrectal ultrasound [TRUS], magnetic resonance [MRI], PET/TC, and prostate biopsy were used. RESULTS: Despite the fact that diagnosis of a local recurrence is based on PSA values and kinetics, imaging by means of different techniques may be a prerequisite for effective disease management. Unfortunately, prostate cancer local recurrences are very difficult to detect by TRUS and conventional imaging that have shown limited accuracy at least at early stages. On the contrary, functional and molecular imaging such as dynamic contrast-enhanced MRI (DCE-MRI), and diffusion-weighted imaging (DWI), offers the possibility of imaging molecular or cellular processes of individual tumors. Recently, PET/CT, using 11C-choline, 18F-fluorocholine or 11C-acetate has been successfully proposed in detecting local recurrences as well as distant metastases. Nevertheless, in controversial cases, it is necessary to perform a biopsy of the prostatic fossa or a biopsy of the prostate to assess the presence of a local recurrence under guidance of MRI or TRUS findings. CONCLUSION: It is likely that imaging will be extensively used in the future to detect and localize prostate cancer local recurrences before salvage treatment.

Serum Steroid Ratio Profiles in Prostate Cancer: A New Diagnostic Tool Toward a Personalized Medicine Approach.

BACKGROUND: Serum steroids are crucial molecules altered in prostate cancer (PCa). Mass spectrometry (MS) is currently the elected technology for the analysis of steroids in diverse biological samples. Steroids have complex biological pathways and stoichiometry and it is important to evaluate their quantitative ratio. MS applications to patient hormone profiling could lead to a diagnostic approach. METHODS: Here, we employed the Surface Activated Chemical Ionization-Electrospray-NIST (SANIST) developed in our laboratories, to obtain quantitative serum steroid ratio relationship profiles with a machine learning Bayesian model to discriminate patients with PCa. The approach is focused on steroid relationship profiles and disease association. RESULTS: A pilot study on patients affected by PCa, benign prostate hypertrophy (BPH), and control subjects [prostate-specific antigen (PSA) lower than 2.5 ng/mL] was done in order to investigate the classification performance of the SANIST platform. The steroid profiles of 71 serum samples (31 controls, 20 patients with PCa and 20 subjects with benign prostate hyperplasia) were evaluated. The levels of 10 steroids were quantitated on the SANIST platform: Aldosterone, Corticosterone, Cortisol, 11-deoxycortisol, Androstenedione, Testosterone, dehydroepiandrosterone, dehydroepiandrosterone sulfate (DHEAS), 17-OH-Progesterone and Progesterone. We performed both traditional and a machine learning analysis. CONCLUSION: We show that the machine learning approach based on the steroid relationships developed here was much more accurate than the PSA, DHEAS, and direct absolute value match method in separating the PCa, BPH and control subjects, increasing the sensitivity to 90% and specificity to 84%. This technology, if applied in the future to a larger number of samples will be able to detect the individual enzymatic disequilibrium associated with the steroid ratio and correlate it with the disease. This learning machine approach could be valid in a personalized medicine setting.

A PSA-guided approach for a better diagnosis of prostatic adenocarcinoma based on MALDI profiling and peptide identification.

BACKGROUND: Prostate cancer (PCa) is the second cause of mortality in men worldwide. The prostate-specific antigen (PSA) test is routinely adopted in diagnosis; nevertheless more reliable biomarkers are continuously under investigation by monitoring the release of molecules into the bloodstream. The serum protein profiles appear to provide cancer-specific fingerprints that help to discriminate patients (especially with low PSA level) from controls, improving the performance of existing clinical tests. METHODS: Samples from healthy controls and PCa patients with low (≤4 ng/mL) and high PSA (>4 ng/mL) levels were analyzed by MALDI profiling, and by a multi fractionation approach coupled to ESI-MS for peaks identification. RESULTS: MALDI profiling achieved to detect 10 and 14 changed peaks (p-value <0.05), respectively, in PCa patients with low and high PSA versus controls. In particular, a peak identified as C3f fragment, resulted overexpressed in low PSA PCa patients. CONCLUSIONS: PSA test, coupled to MALDI profiling, is able to detect changes, specifically related to PCa, in low molecular weight protein range. Furthermore, for the first time in prostate cancer research, the identification and quantification of the small peptide C3f appears to be relevant for the detection of false negatives, providing an additive diagnostic power to PSA (p<0.01) and suggesting its use in clinical tests.

[Role of ultrasonography-guided prostatic biopsy of hypoechoic areas associated with systematic biopsies in patients with normal and high PSA levels]. / Il ruolo delle biopsie prostatiche mirate sulle aree ipoecogene associate alle biopsie a sestanti nei pazienti con PSA elevato e nella norma.

OBJECTIVE: The aim of the study is to evaluate the need to perform directed biopsies to hypoechoic areas at transrectal ultrasound associated with a prostatic mapping in patients with normal and elevated levels of PSA. MATERIALS AND METHODS: Since January 1987, 517 consecutive patients (mean age: 65.5 +/- 5.2 yrs) underwent selective prostatic biopsies of hypoechoic areas and systematic sextant biopsies with 10 samples in patients with a prostatic volume < 60 g and 12 samples in prostatic volume > 60 g. RESULTS: The median PSA value was 7.2 +/- 4.6 ng/ml (SD). 52% of the patients had a positive digital rectal examination. Cancer was detected in 47% of the patients (245/517), in 18% (14/78) of patients with PSA level < 4.0 ng/ml, in 42% (109/256) with PSA level from 4 to 10 ng/ml, in 66% (122/183) with PSA > 10 ng/ml. The PSA value was statistically higher (PSA = 14.9 +/- 17) in patients with positive prostatic biopsies compared to patients with negative biopsies (PSA = 8.5 +/- 8.3 ng/ml) (p > 0.0001). The PPV (positive predictive value) of the hypoechoic lesions was 36% (187/517). Cancer was detected only in directed biopsies of the hypoechoic areas regardless of PSA value in the 20% of patients (49/245). Sextant biopsies were positive with negative directed biopsies in 24% (58/245) of the patients, while both directed and sextant biopsies were positive in 56% (138/245) of the patients. COMMENTS: The hypoechoic lesion is the prostatic area in which prostatic cancer is most likely to be located in spite of the fact that the PPV of a hypoechoic area is less than 40%. The combination of sextant and lesion-directed biopsies maximizes the detection rate using the lowest possible number of biopsy cores. In the case of a TRUS visible lesion, the optimal number and placement of added systematic biopsies is yet to be defined. Due to the multifocality of prostate cancer, in the future, it is probable that, by adding more biopsies to the sextant standard scheme, the necessity of biopsying single small hypoechoic lesions will no longer be necessary.

[Role of prostate fossa ultrasonography in the diagnosis of local recurrence after radical prostatectomy in case of PSA failure]. / Il ruolo dell'ecografia della loggia prostatica nella diagnosi delle recidive locali dopo prostatectomia radicale in caso di rialzo del PSA.

OBJECTIVES: The aim of this study is to verify the diagnostic accuracy of transrectal ultrasound (TRUS) of vesico-urethral anastomosis in patients with PSA elevation (> or = 0.2 ng/mL) after radical prostatectomy, who received 4-6 random anastomotic biopsies of the prostatic fossa plus additional biopsies directed to TRUS detectable lesions. MATERIAL AND METHODS: Since 1992 up to now, 102 patients (mean age: 68.3 +/- 5.4 years) with PSA elevation after radical prostatectomy underwent TRUS of the vesico-urethral anastomosis and 4-6 TRUS-guided random biopsies plus 1-2 additional biopsies directed to TRUS detectable lesions. Pathologic stage was B (ASS classification) in 60% of cases, C in 36% and D in 4% (patients without hormonal treatment who underwent TRUS-guided biopsy because of TRUS detectable or palpable lesion). RESULTS: The mean PSA at biopsy time was 2.1 +/- 4.6 (SD) ng/mL (range: 0.2-31.6 ng/mL) with median PSA of 0.9 ng/mL. DRE was positive in 37% of cases, while TRUS was positive in 73%. Recurrent adenocarcinoma was detected in 51% of all patients and in 45% (26/57) of patients with PSA < 1.0 ng/mL. TRUS sensitivity was higher (80%) than DRE (50%), but specificity was lower (37% vs 81%). The positive predictive value of TRUS detectable lesion was 60%. TRUS sensitivity and specificity increase with PSA elevation and sonographic aspects of prostatic fossa are statistically correlated with histology when PSA > 1.2 ng/mL. CONCLUSIONS: TRUS of the vesico-urethral anastomosis seems to be more sensitive but less specific than DRE for prostatic cancer local recurrence. More than half of TRUS detectable lesions is positive at biopsy. TRUS and TRUS-guided biopsy accuracy are directly correlated with PSA elevation.