Cell Differentiation and Aging Lead To Up-Regulation of FTO, While the ALKBH5 Protein Level Was Stable During Aging but Up-Regulated During in vitro-Induced Cardiomyogenesis.

FTO and ALKBH5 proteins are essential erasers of N6-adenosine methylation in RNA. We studied how levels of FTO and ALKBH5 proteins changed during mouse embryonic development, aging, cardiomyogenesis, and neuroectodermal differentiation. We observed that aging in male and female mice was associated with FTO up-regulation in mouse hearts, brains, lungs, and kidneys, while the ALKBH5 level remained stable. FTO and ALKBH5 proteins were up-regulated during experimentally induced cardiomyogenesis, but the level of ALKBH5 protein was not changed when neuroectodermal differentiation was induced. HDAC1 depletion in mouse ES cells caused FTO down-regulation. In these cells, mRNA, carrying information from genes that regulate histone signature, RNA processing, and cell differentiation, was characterized by a reduced level of N6-adenosine methylation in specific gene loci, primarily regulating cell differentiation into neuroectoderm. Together, when we compared both RNA demethylating proteins, the FTO protein level undergoes the most significant changes during cell differentiation and aging. Thus, we conclude that during aging and neuronal differentiation, m6A RNA demethylation is likely regulated by the FTO protein but not via the function of ALKBH5.

The significance of calprotectin, CD147, APOA4 and DJ-1 in non-invasive detection of urinary bladder carcinoma.

Aim of the study is to define the diagnostic accuracy of selected urinary protein biomarkers in the non-invasive detection of primary and recurrent urothelial carcinoma of the urinary bladder. The urinary levels of calprotectin, CD147, APOA4 and protein deglycase DJ-1 were examined in 255 individuals, including 60 controls with non-malignant urological disease, 61 patients with a history of urinary bladder cancer with negative cytology and negative cystoscopy and 134 patients with urinary bladder cancer. Urinary concentrations of biomarkers were determined by Enzyme-Linked Immunosorbent Assay (ELISA). During the follow-up of patients with non-muscle invasive bladder cancer (NMIBC), a group of 44 patients with cancer recurrence was compared to the group of 61 patients with a history of NMIBC but with no evidence of disease. Urinary concentrations of the evaluated markers did not reveal any significant difference between these groups. During the primary diagnosis, a group of 90 patients with primary bladder cancer and 60 subjects with benign disease were compared. Urinary levels of CD147 were not significantly higher in patients with tumors. The greatest diagnostic accuracy was observed in APOA4 (sensitivity 55.6, specificity 83.3, AUC 0.75), and lesser in calprotectin (sensitivity 39.4, specificity 87.7, AUC 0.66) and in DJ-1 (sensitivity 61.1, specificity 66.7, AUC 0.64), respectively. Apolipoprotein A4 may be used potentially as a supplemental urinary marker in the diagnosis of primary bladder cancer.

Wilms tumor gene 1 (WT1), TP53, RAS/BRAF and KIT aberrations in testicular germ cell tumors.

PURPOSE: Wilms tumor gene 1 (WT1), a zinc-finger transcription factor essential for testis development and function, along with other genes, was investigated for their role in the pathogenesis of testicular germ cell tumors (TGCT). METHODS: In total, 284 TGCT and 100 control samples were investigated, including qPCR for WT1 expression and BRAF mutation, p53 immunohistochemistry detection, and massively parallel amplicon sequencing. RESULTS: WT1 was significantly (p < 0.0001) under-expressed in TGCT, with an increased ratio of exon 5-lacking isoforms, reaching low levels in chemo-naïve relapsed TGCT patients vs. high levels in chemotherapy-pretreated relapsed patients. BRAF V600E mutation was identified in 1% of patients only. p53 protein was lowly expressed in TGCT metastases compared to the matched primary tumors. Of 9 selected TGCT-linked genes, RAS/BRAF and WT1 mutations were frequent while significant TP53 and KIT variants were not detected (p = 0.0003). CONCLUSIONS: WT1 has been identified as a novel factor involved in TGCT pathogenesis, with a potential prognostic impact. Distinct biologic nature of the two types of relapses occurring in TGCT has been demonstrated. Differential mutation rate of the key TGCT-related genes has been documented.

The prognostic value of T1 bladder cancer substaging: a single institution retrospective study.

OBJECTIVE: To evaluate the prognostic value of the depth of lamina propria invasion in patients with T1 bladder cancer. SUBJECTS AND METHODS: 200 patients were treated between the years 2002 and 2009. Tumours with depth of invasion above the muscularis mucosae level were categorised as pT1a and those with depth of invasion up to or beyond the muscularis mucosae as pT1b. RESULTS: Categorisation for pT1a and pT1b was performed in 176 of 200 patients (88%). In 10 patients a muscle-invasive tumour was found in re-transurethral resection samples. 131 (79%) of 166 analysed patients had pT1a tumour and 35 (21%) had pT1b tumour. During the follow-up, in 101 (61%) patients the tumour had recurred and in 27 (16.3%) the tumour had progressed. Of all the investigated parameters, T1 substaging (p < 0.0001), grade (p = 0.0003) and the number of bacillus Calmette-Guérin instillations (p = 0.0490) were significant in predicting progression. The only significant factor for disease-specific survival was T1 substaging in univariable (p = 0.0008) and multivariable (hazard ratio 4.407) analysis. T1 substaging (p = 0.0149) and tumour multiplicity (p = 0.0448) have a statistically significant prognostic value with respect to overall survival. CONCLUSIONS: Deep invasion of the lamina propria is a significant adverse prognostic factor for tumour progression, disease-specific survival and overall survival.

[Laparoscopic pyeloplasty in adults--mid-term results in 32 laparoscopic pyeloplasties during 2003-2008]. / Laparoskopická pyeloplastika u dospelých--strednedobé výsledky 32 laparoskopických pyeloplastik 2003-2008.

Pyeloureteric iunction obstruction is the most common inborn error of the upper urinary pathways. In the time of routine prenatal ultrasound examination, this disease is discovered in the child's age, when it is treated most frequently. Publication summarizes the results of modern laparoscopic management in the adults, who had not been diagnosed previously and symptoms occurred many years after their birthdate. The surgical treatment has been changed in the approach, but not the goal of desobtruction. The results in the adults are influenced not only by the operation technique, but also by the length of time the obstruction lasted and by the number and severity of the inflammatory changes.

Does the expression of fascin-1 and tumor subclassification help to assess the risk of recurrence and progression in t1 urothelial urinary bladder carcinoma?

INTRODUCTION: To evaluate the prognostic value of T1 subclassification and fascin-1 expression in T1 human urothelial cell carcinoma of the bladder. MATERIALS AND METHODS: In a prospective study with 105 consecutive patients, T1 tumors were subclassified into 2 groups according to the depth of tumor invasion. The tunica muscularis mucosae was used as a landmark. The expression of fascin-1 was examined by using an anti-fascin-1 mouse monoclonal antibody and was evaluated semiquantitatively for both intensity and distribution. The patients were followed up for 27.3 +/- 13.7 months. RESULTS: The T1 tumor subclassification was feasible in 99 patients (94%). T1a tumor was detected in 77 patients (73%), T1b tumor in 22 patients (21%). An invasive tumor was found in 5 patients (4.8%) during the restaging transurethral resection of the bladder. The risk of understaging in patients with T1b tumor was 18%. There was not a significant difference in time to the recurrence in the T1a and the T1b group. The progression-free survival rates were significantly different between both groups (p = 0.0034). No correlation was found between fascin-1 positivity and the depth of tumor invasion. Fascin-1 positivity did not correlate with recurrence or the progression-free intervals. In the multivariate analysis, only the extent of lamina propria invasion was an independent predictor of the tumor progression. The fascin positivity was not an independent prognostic factor relating to the risk of recurrence or progression. CONCLUSION: The finding of T1b tumor was connected with a significantly higher risk of progression and understaging. The fascin-1 expression did not correlate with the depth of tumor invasion or with the tumor recurrence or progression.

Urinary cytology and quantitative BTA and UBC tests in surveillance of patients with pTapT1 bladder urothelial carcinoma.

OBJECTIVES: To compare results of urinary cytology, quantitative detection of human complement factor H-related protein (BTA TRAK), and urinary fragments of cytokeratins 8 and 18 (UBC IRMA) with the recurrence status in patients with pTapT1 bladder cancer and to define the possible role of these methods in a surveillance protocol. METHODS: We collected urine from 88 consecutive patients with primary pTapT1 tumors before the first transurethral resection (TURB) and before each follow-up cystoscopy. In all samples urinary cytology and quantitative BTA and UBC tests were performed. We compared results with recurrence status and with tumor characteristics in the case of recurrence. We constructed receiver operator characteristic (ROC) curves for quantitative methods. In addition, we evaluated individual cutoffs based on pretreatment levels. RESULTS: During the mean follow-up of 16.96 months, we performed 313 cystoscopies, 93 of which were positive in 51 patients. The sensitivity and specificity of cytology, BTA, and UBC were 19.8% and 99%, 53.8% and 83.9%, and 12.1% and 97.2%, respectively. The sensitivity of pTis detection was 66.6%, 0%, and 100%, respectively. With cutoffs set to a sensitivity of 90%, the specificity of BTA and UBC dropped to 24.8% and 20.4%, respectively. Individually calculated cutoffs did not provide a significant benefit. CONCLUSIONS: Because of high specificity and sensitivity in pTis detection, urinary cytology fulfills requirements for an adjunctive method to cystoscopy. Quantitative BTA and UBC tests have a low sensitivity in the detection of bladder cancer recurrence and cannot be used routinely to reduce the number of cystoscopies during follow-up.

[Outcomes of transperitoneal laparoscopic nephrectomy for renal adenocarcinoma]. / Výsledky transperitoneání laparoskopické nefrektomie pro renální adenokarcinom.

Laparoscopic radical nephrectomy (LRN) is method of choice in malignant renal tumors (RCC) stage T1-3. Procedure has proved to be technically safe with low post-operative morbidity and standard oncological results, provided the patients are secondaries free in the time of surgery, and there was no positive margin in resected tissue. There was no recurrence even after 6 years of follow-up. The most influential variables regarding the survival of patients postoperatively were: presence of metastases in the time of surgery p <0.0001, pathological grade p < 0.001, stage p < 0.018 and p < 0.046, respectively, if the tumors were 4 cm and bigger. It could be concluded, that laparoscopic way of removing the tumor with the kidney has proved the same oncological results even in the mid-term follow-up, if compared with classical open surgery done lege artis.

[Cystectomy in the treatment of bladder cancer]. / Cystektomie v léce nádoru mocového mechýre.

Radical cystectomy with pelvic lymphadenectomy and urinary diversion is an important component in the treatment of bladder cancer. It is considered the most important method in the therapy for muscle invasive and selected high-risk non-muscle invasive tumours with excellent local control and high complete remission rate. It consists of complete removal of tumour tissue in the bladder, small pelvis and regional lymph nodes. In males, urinary bladder and prostate are routinely removed; in females, bladder, uterus and anterior vaginal wall are removed. Urethrectomy is indicated only in selected situations. An integral part of the operation is the bilateral pelvic lymphadenectomy. Extravesical disease extension and lymph node positivity are unfavourable prognostic factors. Better prognosis is expected in patients with less than 5 positive nodes. Important prognostic factor is also the number of removed lymph nodes, which is a strong argument for meticulous bilateral pelvic lymphadenectomy. Lymph node density (number of positive nodes/ number of removed nodes) is considered as very important prognostic factor. Better prognosis can be expected in patients with less positive and more removed nodes.

[Detection of prostate cancer by ultrasonographically-guided transrectal prostate biopsies at the Department of Urology of the First Faculty of Medicine, Charles University and General Teaching Hospital in Prague, analysis of 1464 cases]. / Detekce karcinomu prostaty transrektální ultrasonograficky cílenou biopsií na urologické klinice 1. LF UK a VFN v Praze: analýza 1464 pozorování.

BACKGROUND: Early diagnostics of prostate cancer is still the most important factor in tumor-specific survival of patients harbouring this malignant disease. Without better understanding of the etiology and without relevant markers of the disease progression, only the early diagnostics of organ-confined disease can save the patient's life. METHODS AND RESULTS: Throughout 7 consecutive years, 1464 transrectal prostate biopsies in 1302 patients were performed. In all cases, the age, DRE (positive or negative), prostatic volume (ccm), total PSA in peripheral blood (ng/ml) were assigned, as well as free/total PSA ratio (%) in most of them. Apart from previously used linear regression, we applied logistic regression, since only age grows linear and DRE is determined only as positive or negative. The surrogate endpoint was ROC, which determines the area applying to the relations of sensitivity and specificity of any marker. Multifactorial logistic regression then reached best results at values over 0.8 in all tested age categories with maximal deviation of 8%, which had not been achieved before. CONCLUSIONS: Despite hundreds of papers published on this topic, the question of when and how the patient is indicated to the biopsy of the prostate has not been solved. A computer driven model based on 1464 documented examinations on the relatively homogeneous population is presented. On the basis of the combination of biological and statistical methods, the model can give correct predictions in 9 out of 10 cases.

[Progress of laparoscopic surgery in adults at the Department of Urology of the First Faculty of Medicine and General Teaching Hospital]. / Rozvoj laparoskopické operativy dospelých na Urologické klinice 1. LF UK a VFN v Praze.

Laparoscopy is the most modern operative technique, the main advantage of which is the least invasivity. In urology, the laparoscopy has won its yield only in nineties of 20th century, but it was in urology, where the robots were launched as first, and nowadays using them it is possible to perform ablative/reconstructive surgery in a tiny intracorporeal space. The price of laparoscopic operations is high and has been the only limit up to now, but technical approaches and indications have already been fixed.

Molecular cytogenetic characterization and diagnostics of bladder cancer.

Bladder cancer is a heterogenous malignancy with wide scale of clinical manifestation. Different chromosomal aberrations have been already identified in bladder tumors. These aberrations can be detected by multicolor interphase fluorescence in situ hybridization (I-FISH) or comparative genomic hybridization (CGH). The aim of this study was to determine the diagnostic benefits of non-invasive I-FISH method and to comprehensively characterise genetic alterations using CGH in selected patients with bladder tumors. We examined 128 urine samples and correlated our results with histological findings. I-FISH using UroVysion kit showed positivity in 63,6 % of G1 tumors, 64,3 % of G2 tumors and 91,7 % in G3 tumors. We examined also 12 bladder tissue samples by means of CGH and various genetic alterations were ascertained independent on tumor grade. The most frequent gains and losses of DNA material were detected on chromosomes 1, 8, 9, 10, 11, 13, and 14. The contribution of I-FISH is in an early and non-invasive detection of bladder cancer recurrences during follow up of patients after the surgery. CGH provides information about further genetic alterations and some of them could be ascertained as recurrent changes with prognostic significance.

[The p53 positivity in non-tumor mucosa in patients with superficial urinary bladder cancer]. / P53 pozitivita v nenádorove sliznici u pacientu s povrchovým uroteliiálním karcinomem mocoveho mechyre.

BACKGROUND: The aim of the study is to assess the prognostic value of p53 positivity in the non-tumor mucosa of urinary bladder in patients with superficial urinary bladder carcinoma. METHODS AND RESULTS: In 45 patients cold cup non-tumor mucosa samples were taken at the same time with the TUR of superficial urinary bladder carcinoma prospectively. Monoclonal antibody BP53-12-1 was used for the detection of p53 protein. When identifying positive colouring only the nuclear immunoreactivity was being evaluated. 200 nuclei at minimum were examined in several representative fields. The McCarthy method in Bacus modification was used to analyse the findings. It is a semiquantitative method which detects not only the percentage of p53 positive cells but also the intensity of positivity classified into four degrees (0 - negative, 1 - slightly positive, 2 - distinctly positive, 3 - strongly positive). The intensity of p53 positivity was quantified as HSCORE, where HSCORE = 7Pi (i + 1), in which i is one of the four see above degrees and Pi fluctuates from 0 % to 100 %. The result is a numerical figure from 100-400. A negative finding is of HSCORE 100, HSCORE of 400 is the highest possible. The samples were analysed in the analytical system LUCIA. The borderline value was quantified to HSCORE 200. All patients were carefully followed up and treated using usual schemes. The results were evaluated by the use of SAS system (Cary, USA). Thirty patients recurred during the follow-up and 7 of them progressed. The average HSCORE in those who did not recur was 130.2, in patients with the recurrence of tumor it was 162.5 and in patients with progression it was 169.2. We have found a correlation between the HSCORE and the risk of recurrence, which was statistically significant. CONCLUSIONS: The p53 positivity in non-tumor mucosa of urinary bladder in patients with superficial bladder cancer may bring additional information when predicting the risk of recurrence. More extensive studies need to be carried out.

Association of PAX5 expression with clinical outcome in patients with TaT1 transitional cell carcinoma of the bladder.

OBJECTIVES: To assess the frequency and intensity of PAX5 gene messenger ribonucleic acid (mRNA) expression in TaT1 bladder cancer tissue, as well as its correlation with clinicopathologic variables and patient outcome. METHODS: The RNA expression of PAX5 was evaluated with reverse transcriptase polymerase chain reaction in the tumor tissue of 75 patients with stage TaT1 bladder cancer treated with transurethral resection. Patients were observed with cystoscopy and urinary cytologic evaluation. The association between PAX5 expression and clinicopathologic variables and patient outcome was evaluated. Benign urothelium from 8 patients with benign prostatic hyperplasia was obtained. These patients were used as a control group. RESULTS: PAX5 expression was found in 62 patients with bladder cancer (82.7%) but in no patient from the control group. High PAX5 expression (greater than 0.2) was confirmed in 19 patients (25.3%). No significant relationship was observed between quantity of PAX5 expression and clinicopathologic variables. The 3-year recurrence-free and progression-free survival rates in highly positive patients were 13.2% and 71.6%, compared with 40.6% and 92.8%, respectively, in patients with weak or negative expression (log-rank test, P = 0.0075, P = 0.022). Multivariate Cox proportional hazard model analysis identified PAX5 expression as an independent predictor of tumor recurrence. CONCLUSIONS: PAX5 gene expression is a frequent finding in superficial transitional cell carcinoma of the bladder. High levels of PAX5 are associated with poorer recurrence-free and progression-free survival rates. Moreover, PAX5 expression was found to be an independent prognostic factor for recurrence-free survival by a multivariate analysis.

[A case of a patient with solitary prostate metastasis of the colorectal carcinoma three years after abdominoperineal amputation of the rectum]. / Prípad pacienta se solitární metastázou kolorektálního karcinomu v prostate tri roky po abdominoperineální amputaci rekta.

AIM: The aim of this study is to present a case-review of a patient with an extremely rare finding of a solitary metastasis of the colorectal carcinoma in his prostate. METHODOLOGY: A Case-Review. RESULTS: The study describes a case of a 42-year-old patient who underwent abdominoperineal amputation for the rectal carcinoma pT3 N1 M0 with complementary actinotherapy and chemotherapy. Three years after his rectum amputation, a CT scan disclosed a hypodense focus in his prostate of unknown origin. Elevation of the CEA was found upon laboratory examination, other tumor markers including the PSA were not abnormal. The positron emission tomography (PET) did not disclose other distant foci. The patient was administered 4 courses of chemotherapy with, basically, no signs of regression in the prostate foci. Biopsy from the suspicious focus was conducted transperitoneally, followed by its histological examination which confirmed cribriform adenocarcinoma. Our team decided on salvage cystoprostatectomy, securing urine derivation by the ilium conduit. 6 months later, the CT examination showed a massive local relapse and two metastatic foci in the liver, further oncological therapy was not indicated. The patient died 7 moths after the radical cystoprostatectomy. CONCLUSION: This case-review points out a possibility of metastatic prostate affections connected to the colorectal carcinoma disorder, as well as its treatment procedures.

[Bone surgery for unstable hips in patients with cerebral palsy]. / Lécba instabilních kyclí u pacientu s DMO kostními operacemi.

PURPOSE OF THE STUDY: The aim of this retrospective study was to compare the efficacy of femoral osteotomy alone with that of osteotomy combined with an acetabular procedure in patients with unstable hips due to spastic cerebral palsy. MATERIAL: Sixty-one hip joints in 50 patients who had shown distinct subluxation or dislocation of the joint were operated on. Eleven patients underwent bilateral surgery. Before bone surgery, soft-tissue release involving both the flexors and adductors was performed on 19 hips. Femoral osteotomy alone was performed on 29 hip joints and combined femoral and pelvic osteotomy was carried out on 32 joints.Twelve resections of the proximal femur in seven patients were evaluated as a separate group. All treated hip joints were assessed by clinical and radiographic examination at a follow-up of more than 5 years. METHODS: The skiagraphs taken in a strictly neutral position of the lower limbs before surgery and at the final examination were evaluated on the basis of Reimers's migration index and Wiberg's centre-edge angle. The locomotor abilities of each child were categorized according to the Vojta scoring system for locomotor development. The range of motion in the treated hip joint was assessed using the standard S. F. T. R. method. The results obtained were statistically analyzed by the Kruskal- Wallis, one-way ANOVA test. RESULTS: A comparison of the results of femoral osteotomy alone with those of combined femoral and pelvic osteotomy showed that the post-operative values of the migration index and centre-edge angle, as compared with the pre-operative ones, were statistically higher (p<0.05) in the latter. In a long-term perspective, the surgery had no adverse effects on a natural development of locomotor abilities of the child. The children had higher scores by the modified Vojta rating system. There was no change in the range of motion in the treated hip joints after the operation. DISCUSSION: In 28, out of the 32 joints treated by combined femoral and pelvic osteotomy, Salter osteotomy was performed and it showed a high efficacy in providing hip joint stability. The main emphasis during surgery was placed on the maximum acetabular rotation laterally. Femoral osteotomy alone was less effective in providing hip joint stability; in addition, in order to achieve this, tilting of the proximal fragment to a varus position was generally higher than in combined operations. In three patients this treatment resulted in fixed adduction of the hip joint with negative consequences for movement. CONCLUSIONS: In the treatment of unstable hip joints a combination of varus derotation femoral osteotomy and pelvic osteotomy provides better containment of the joint than femoral osteotomy alone.