Evaluation of Matrix Metalloproteases by Artificial Intelligence Techniques in Negative Biopsies as New Diagnostic Strategy in Prostate Cancer.

Usually, after an abnormal level of serum prostate-specific antigen (PSA) or digital rectal exam, men undergo a prostate needle biopsy. However, the traditional sextant technique misses 15-46% of cancers. At present, there are problems regarding disease diagnosis/prognosis, especially in patients' classification, because the information to be handled is complex and challenging to process. Matrix metalloproteases (MMPs) have high expression by prostate cancer (PCa) compared with benign prostate tissues. To assess the possible contribution to the diagnosis of PCa, we evaluated the expression of several MMPs in prostate tissues before and after PCa diagnosis using machine learning, classifiers, and supervised algorithms. A retrospective study was conducted on 29 patients diagnosed with PCa with previous benign needle biopsies, 45 patients with benign prostatic hyperplasia (BHP), and 18 patients with high-grade prostatic intraepithelial neoplasia (HGPIN). An immunohistochemical study was performed on tissue samples from tumor and non-tumor areas using specific antibodies against MMP -2, 9, 11, and 13, and the tissue inhibitor of MMPs -3 (TIMP-3), and the protein expression by different cell types was analyzed to which several automatic learning techniques have been applied. Compared with BHP or HGPIN specimens, epithelial cells (ECs) and fibroblasts from benign prostate biopsies before the diagnosis of PCa showed a significantly higher expression of MMPs and TIMP-3. Machine learning techniques provide a differentiable classification between these patients, with greater than 95% accuracy, considering ECs, being slightly lower when considering fibroblasts. In addition, evolutionary changes were found in paired tissues from benign biopsy to prostatectomy specimens in the same patient. Thus, ECs from the tumor zone from prostatectomy showed higher expressions of MMPs and TIMP-3 compared to ECs of the corresponding zone from the benign biopsy. Similar differences were found for expressions of MMP-9 and TIMP-3, between fibroblasts from these zones. The classifiers have determined that patients with benign prostate biopsies before the diagnosis of PCa showed a high MMPs/TIMP-3 expression by ECs, so in the zone without future cancer development as in the zone with future tumor, compared with biopsy samples from patients with BPH or HGPIN. Expression of MMP -2, 9, 11, and 13, and TIMP-3 phenotypically define ECs associated with future tumor development. Also, the results suggest that MMPs/TIMPs expression in biopsy tissues may reflect evolutionary changes from prostate benign tissues to PCa. Thus, these findings in combination with other parameters might contribute to improving the suspicion of PCa diagnosis.

The role of morphometric and respiratory factors in predicting the severity and evolution of urinary symptoms in patients with obstructive sleep apnea syndrome.

OBJECTIVES: To assess the severity, evolution, and behaviour of several urinary symptoms in patients with obstructive sleep apnea syndrome (OSAS) before and after the treatment with continuous positive airway pressure (CPAP). METHODS: A prospective study was performed on patients with a recent diagnosis of sleep apnea confirmed by nocturnal sleep polygraphy and absence of medical urological past history. The symptom incidence was analysed seeking predictive factors for initial nocturia, nocturnal polyuria (NP), and unfavourable International Prostate Symptoms Score (IPSS) before and after a 1-year period of treatment using a CPAP device. Morphometric variables (body mass index, BMI; neck and abdominal diameter) and functional respiratory variables (FEV1, FVC, and FEV1/FVC) were analysed. A multivariate analysis was performed with a calculation of Pearson's correlation coefficient to establish a linear relation between the variables. RESULTS: A total of 43 patients completed the two-step study (IPSS and bladder diary before and after the CPAP treatment). IPSS decreased by 3.58 points. Nocturia decreased to once per night. Neck diameter, FEV1, and FEV1/FVC significantly predicted the initial severity of some lower urinary tract symptoms (LUTS), (p=0.015, p=0.029, p=0.008, respectively). Neck diameter, abdominal perimeter, and FEV1/FVC significantly predicted the LUTS evolution throughout the study (p=0.023, p=0.007, p=0.05, respectively). CONCLUSION: Some pre-treatment morphometry and spirometry parameters such as abdominal or neck diameter, FEV1, and FEV1/FVC were predictive of the severity and evolution of LUTS in patients with OSAS.

Prostate Cancer Tumor Stroma: Responsibility in Tumor Biology, Diagnosis and Treatment.

Prostate cancer (PCa) is a common cancer among males globally, and its occurrence is growing worldwide. Clinical decisions about the combination of therapies are becoming highly relevant. However, this is a heterogeneous disease, ranging widely in prognosis. Therefore, new approaches are needed based on tumor biology, from which further prognostic assessments can be established and complementary strategies can be identified. The knowledge of both the morphological structure and functional biology of the PCa stroma compartment can provide new diagnostic, prognostic or therapeutic possibilities. In the present review, we analyzed the aspects related to the tumor stromal component (both acellular and cellular) in PCa, their influence on tumor behavior and the therapeutic response and their consideration as a new therapeutic target.

Gene Expression Profile of Stromal Factors in Cancer-Associated Fibroblasts from Prostate Cancer.

Recent investigations point at the stromal microenvironment to assess additional diagnostic information and provide new therapeutic targets in cancer. The aim of the study was to contribute to the characterization of the phenotype of cancer-associated fibroblasts (CAFs) in prostate cancer (PCa) compared with normal prostate-associated fibroblasts (NAFs) and fibroblasts from benign prostatic hyperplasia (BPH). Three patient populations were prospectively recruited: 23 patients with new localized PCa, 14 patients with advanced PCa treated with androgenic deprivation therapy (ADT), and 7 patients with BPH. Gene expression of 20 stroma-derived factors, including the androgen receptor (AR), chaperones (HSPA1A and HSF1), growth factors (FGF2, FGF7, FGF10, HGF, PDGFB, and TGFß), proteins implicated in invasion (MMP2, MMP9, and MMP11), inflammation (IL6, IL17RB, NFκB, and STAT3), and in-stroma/epithelium interaction (CDH11, CXCL12, CXCL14, and FAP), was evaluated. Localized PCa CAFs showed a significant higher expression of FGF7, IL6, MMP2, and MMP11 compared with NAFs or IL17RB compared with BPH fibroblasts, but significantly lower expression of FGF10 and IL17RB compared with NAFs or CXCL14 compared with BPH fibroblasts. In addition, CAFs from ADT-resistant PCa showed significantly higher MMP11 and NFκB but significant lower TGFß expression compared with CAFs from ADT-sensitive tumors. Our results contribute to defining the CAFs phenotypes associated to PCa progression, which may contribute to the diagnosis and design of alternative therapies in PCa.

Relationship between metalloprotease expression in tumour and stromal cells and aggressive behaviour in prostate carcinoma: Simultaneous high-throughput study of multiple metalloproteases and their inhibitors using tissue array analysis of radical prostatectomy samples.

OBJECTIVE: The aim of this study was to detect a potential association between clinicopathological factors of prostate cancer aggressiveness and the expression of matrix metalloproteases and their inhibitors in tumour and stromal cells. MATERIAL AND METHODS: A tissue array technique and immunochemistry with specific antibodies against matrix metalloproteinases (MMPs)-1, 2, 7, 9, 11, 13, 14, and their tissue inhibitors (TIMPs)-1, 2 and 3 were used to analyse the surgical specimens of 133 patients treated by radical prostatectomy. For each antibody preparation, the cellular location of immunoreactivity was determined. RESULTS: The expression of MMP-2 was negatively associated with high tumour grade. With regard to stromal fibroblasts, TIMP-3 expression was positively associated with histological grade. MMP-7 expression was negatively associated with pretreatment serum levels of PSA, whereas MMP-13 was positively associated with higher levels of the antigen. TIMP-2 expression by mononuclear inflammatory cells correlated significantly and negatively with tumour grade. CONCLUSIONS: The expression of TIMP-3 by fibroblasts was associated with a higher Gleason score. An increased expression of MMP-13 by fibroblasts was associated with a greater preoperative level of PSA. In contrast, MMP-2 expression by tumour as well as TIMP-2 expression by peritumoral inflammatory cells was associated with less aggressive prostate carcinoma characteristics.

Myopodin methylation is associated with clinical outcome in patients with T1G3 bladder cancer.

PURPOSE: Bacillus Calmette-Guerin is standard treatment to decrease tumor recurrence and delay progression of high risk, nonmuscle invasive bladder tumors. However, it is not yet clear which T1G3 cases are more prone to more aggressive clinical behavior or susceptible to respond to bacillus Calmette-Guerin. We evaluated the role of myopodin methylation as a clinical outcome prognosticator and predictive biomarker for the bacillus Calmette-Guerin response in patients with T1G3 bladder tumors. MATERIALS AND METHODS: We analyzed the methylation status of myopodin in tumor specimens from 170 patients with T1G3 bladder cancer, including a subset of 108 who underwent bacillus Calmette-Guerin treatment. Myopodin methylation was assessed by methylation specific polymerase chain reactions. Recurrence, progression to muscle invasive tumors and disease specific overall survival were analyzed using competing risks regression analysis. RESULTS: Of the 170 cases analyzed 72 recurred (42.4%) and 36 progressed (21.2%). A total of 24 patients (14.1%) died of the disease. Univariate and multivariate survival analysis revealed that myopodin methylation was significantly associated with an increased recurrence rate (p = 0.004), progression (p = 0.002) and shorter disease specific overall survival (p = 0.020). In a subset treated with bacillus Calmette-Guerin myopodin methylation was also related to an increased recurrence rate (p = 0.011), progression (p = 0.030) and shorter disease specific overall survival (p = 0.028). CONCLUSIONS: Epigenetic analysis revealed that myopodin methylation was associated with tumor aggressiveness and clinical outcome in patients with T1G3 disease. Myopodin methylation distinguished patients responding to bacillus Calmette-Guerin from those who may require a more aggressive therapeutic approach.

Sympathetic skin response in patients with erectile dysfunction.

OBJECTIVES: To evaluate the role of the sympathetic skin response (SSR) in men with erectile dysfunction (ED), focusing on detecting SSR in the penis. PATIENTS AND METHODS: We assessed the SSR in 82 patients with ED, as an indicator of abnormalities both in amyelinic C-fibres and in autonomic pathways in these patients. The SSR was carried out according to the to the Technical Standards of the International Federation of Clinical Neurophysiology. Electrical stimulation was applied through superficial electrodes over the contralateral median nerve. Values were recorded with superficial electrodes on the skin in the contralateral hand and foot, as well as in the penis. The percentage of SSR (SSR%) was classified into three groups, i.e. 0-20%, 21-89% and 90-100%. Results of latency were also classified into three groups of normal or abnormal (increased) latency, and response blocking (no response), the last two being considered pathological conditions. RESULTS: In the penis, the mean (sd) SSR% was 52.8 (43.19)% and significantly lower than responses in hands and feet. There was a significant correlation of the SSR% between the palm of the hand and the sole of the foot (P = 0.01) and between the sole of foot and penis (P = 0.05). Diabetics showed a significant decrease (P = 0.001) in the mean SSR% in the palm of the hand and sole of the foot. Although not statistically different, the mean SSR% in the penis was lower in diabetics than in patients with other risk factors for ED. Likewise, the mean SSR% in hand, foot and penis increased with an increase in the International Index of Erectile Function. In the penis, latency was normal (<1.5 ms) in 14 and abnormal in 37 patients. There was a significant association between pathological chronic re-innervation in the bulbocavernosus muscle and SSR latencies in the foot (P = 0.002) and penis (P = 0.03). Bulbocavernosus muscle electromyography showed a higher frequency of chronic bilateral axonomnesis in patients with abnormal latencies (28%) than in patients with normal SSR latencies in the penis. CONCLUSION: These results establish an indication of the SSR in patients with ED, registering responses not only in classic locations like the palm of the hand or sole of the foot, but also in the penis. The SSR% was useful as an indicator of the effect on efferent C fibres. Despite SSR being a polysynaptic potential of long latency and regulated by the cerebral cortex, the present results show that it is advisable to record the latencies of SSR in the three areas registered, and especially in the penis, where it seems be more useful as a marker of lumbosacral and/or pudendal alterations.

Stromal factors involved in human prostate cancer development, progression and castration resistance.

PURPOSE: To detect new predictive markers from the prostate cancer tissue, to study the expression by cultured cancer-associated fibroblasts (CAFs) of stromal factors implicated in prostate carcinogenesis, and to compare their expressions in localized, metastatic, castration-sensitive (CSCP), castration-resistant prostate tumors (CRCP) as well as in fibroblasts from benign prostatic hyperplasia (BPH). MATERIALS AND METHODS: The genomic expression of 20 stroma-derived factors, including the androgen receptor (AR), growth factors (FGF2, FGF7, FGF10, HGF, TGFß, PDGFB), protein implicated in invasion (MMP-2, MMP-9 and MMP-11), inflammation (IL-6, IL-17, STAT-3 and NFκB), stroma/epithelium interaction (CDH11, FAP, CXCL12 and CXCL14) and chaperones (HPA1A and HSF1), was evaluated in cultured fibroblasts both from BHP and prostate carcinomas (PCa). After isolation and culture of fibroblasts by biopsy specimens, RNA was isolated and genomic studies performed. RESULTS: Finally, 5 BPH and 37 PCa specimens were selected: clinically localized (19), metastatic (5), CSCP (7) and CRPC (6). Interleukin-17 receptor (IL-17RB) was highly expressed in CAFs compared with fibroblasts from BPH. However, metalloproteinase-2 and chemokine ligand 14 (CXCL14) were expressed at higher levels by fibroblasts from BPH. The fibroblastic growth factor-7 was highly expressed by CAFs from localized tumors, but metalloproteinase-11 in metastatic tumors. MMP-11, androgen receptor (AR) and heat-shock-70kda-protein-1A (HSPA1A) expressions were significantly higher in CAFs from CRPC. CONCLUSIONS: These results demonstrate a CAFs heterogeneity among prostate carcinomas with regard to some molecular profile expressions that may be relevant in tumor development (IL-17RB), progression (MMP-11) and castration resistance (AR, MMP-11 and HSPA1A).

Molecular classification of non-muscle-invasive bladder cancer (pTa low-grade, pT1 low-grade, and pT1 high-grade subgroups) using methylation of tumor-suppressor genes.

The role of epigenetics in distinguishing pathological and clinical subgroups in bladder cancer is not fully characterized. We evaluated whether methylation of tumor-suppressor genes (TSGs) would classify non-muscle-invasive (NMI) bladder cancer subgroups and predict outcome. A retrospective design included the following paraffin-embedded primary NMI tumor types (n = 251): pTa low grade (LG) (n = 79), pT1LG (n = 81), and pT1 high grade (HG) (n = 91). Methylation of 25 TSGs was measured using methylation-specific, multiplex, ligation-dependent probe amplification. The TSGs most frequently methylated in the overall series were STK11 (96.8%), MGMT2 (64.5%), RARB (63.0%), and GATA5 (63.0%). TSG methylation correlated to clinicopathological variables in each subgroup and in the overall NMI series. Methylation of RARB, CD44, PAX5A, GSTP1, IGSF4 (CADM1), PYCARD, CDH13, TP53, and GATA5 classified pTa versus pT1 tumors whereas RARB, CD44, GSTP1, IGSF4, CHFR, PYCARD, TP53, STK11, and GATA5 distinguished LG versus HG tumors. Multivariate analyses indicated that PAX5A, WT1, and BRCA1 methylation independently predicted recurrence in pTaLG, PAX6, ATM, CHFR, and RB1 in pT1LG disease; PYCARD, in pT1HG disease; and PAX5A and RB1, in the overall series. Methylation of TSGs provided a molecular classification of NMI disease according to clinicopathological factors. Furthermore, TSG methylation predicted recurrence in NMI subgroups.

Analysis of the expression of interleukins, interferon ß, and nuclear factor-κ B in prostate cancer and their relationship with biochemical recurrence.

There are accumulating epidemiological, experimental, and genetic data supporting that prostate inflammation may contribute to prostate carcinogenesis, and several inflammatory-related molecules have been linked to tumorigenesis and prognosis in several tumors. The aim of this study was to evaluate tumor expression of inflammatory-related factors in prostate carcinomas and their possible relationship with biochemical recurrence (elevation of prostate-specific antigen serum levels). An immunohistochemical study was conducted using tissue microarrays and specific antibodies against interleukin-1ß (IL-1ß), IL-6, IL-10, IL-17, interferon ß (IFNß), and nuclear factor-κ B (NF-κB). Determinations in cancer specimens from 118 patients with primary prostate cancer (78 without and 40 with recurrence during the follow-up period) were performed. Immunostaining for all the studied proteins was localized both in tumor cells and in stromal cells in the majority of tumors. High-score values for IL-1ß or low-score values for IFNß were significantly associated with biochemical recurrence. The analysis defined a score value of 160 for IL-1ß and of 170 for IFNß as the optimal cutoff points that identified 32.7% and 73.2% of patients, respectively, having high probability of biochemical recurrence. Multivariate analysis according to a Cox model indicated that the cutoff point 170 for IFNß (P=0.035) was an independent factor associated with biochemical recurrence in patients with prostate cancer. Both IL-1ß and IFNß may be new biomarkers to distinguish high-risk/low-risk patients with prostate cancer, and to select appropriate therapeutic approaches.

Polyamine-modulated factor-1 methylation predicts Bacillus Calmette-Guérin response in patients with high-grade non-muscle-invasive bladder carcinoma.

BACKGROUND: Bacillus Calmette-Guérin (BCG) is a standard treatment to reduce tumor recurrence and delay progression of high-risk non-muscle-invasive (NMI) bladder tumors. However, it is not clear yet which patients are more likely to respond to BCG. OBJECTIVE: The aim was to evaluate the role of polyamine-modulated factor-1 (PMF-1) methylation in predicting clinical outcome of T1 high-grade (T1HG) bladder tumors treated with BCG. DESIGN, SETTING, AND PARTICIPANTS: In a retrospective design, PMF-1 methylation was analyzed on tumor specimens belonging to 108 patients with T1HG NMI bladder cancer undergoing BCG treatment. Median follow-up was 77 mo (range: 5-235 mo). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: PMF-1 methylation was assessed by methylation-specific polymerase chain reactions. Recurrence, progression into muscle-invasive tumors, and disease-specific survival rates were analyzed using competing risks regression analysis. RESULTS AND LIMITATIONS: Among the 108 patients analyzed, 35 had recurring disease (32.4%), 21 progressed (19.4%), and 16 died of disease (14.8%); 71.3% of tumors had PMF-1 methylation. Univariate analyses using cumulative incidence curves revealed that an unmethylated PMF-1 was significantly associated with increased recurrence (p=0.026), progression (p=0.01), and shorter disease-specific survival (log-rank, p=0.03). Multivariate analyses indicated that among sex, age, focality, tumor size, and concomitant carcinoma in situ, only PMF-1 methylation provided significant hazard ratios (HRs) for recurrence of (HR: 2.032; p=0.042), and progression (HR: 2.910; p=0.020). Limitations of the study include its retrospective design, lymphovascular invasion status not available, short maintenance BCG, and that a second transurethral resection was not performed. CONCLUSIONS: Epigenetic analyses revealed that the methylation status of PMF-1 was associated with the clinical outcome of patients with T1HG tumors undergoing BCG treatment. An unmethylated PMF-1 correlated to recurrence and progression in T1HG disease using univariate and multivariate analyses. Thus, assessing the methylation status of PMF-1 may serve to distinguish patients responding to BCG from those who may require more aggressive therapeutic approaches.