Loss of heterozygosis on chromosome 18q21-23 and muscle-invasive bladder cancer natural history.

Loss of heterozygosis (LOH) on chromosome (Chr) 18q21-23 was reported to be one of the most common genetic alterations identified in bladder cancer. The current study aimed to determine the prognostic role of LOH on Chr 18q21-23 in patients diagnosed with muscle-invasive urothelial bladder carcinoma (MIBC). A total of 34 consecutive patients were enrolled in the present prospective study. LOH on Chr 18 was assessed by performing multiplex polymerase chain reaction on paired blood and tumour tissue samples from each patient. The following primers were used in the present study: D18S51, MBP LW and MBP H. These data were then compared with follow-up information. The main outcome measure was patient status at the end of the follow-up. Cox regression was used to evaluate the impact of each parameter on cancer-specific survival and the Kaplan Meier test for disease-free survival was plotted in order to estimate survival. Out of 34 patients, 18 (52.9%) exhibited ≥1 alteration in one of the loci analysed on chromosome 18, while 16 (47.1%) revealed no alterations. No correlation was identified with stage (P=0.18) or grade (P=0.06); however, LOH on Chr 18q21-23 was significantly associated with a lower recurrence-free probability (P<0.0001). Kaplan-Meier curves demonstrated a significant association between patient status at follow-up and LOH on Chr 18 (P<0.001). In addition, multivariate analysis identified LOH on Chr 18 (P<0.001) and stage (P=0.01) as independent survival predictors. Furthermore, artificial neural network analysis was consistent with the results of the multivariate analysis. In conclusion, the present study highlighted the role of LOH on Chr 18q21-23 in predicting the clinical outcome of patients with MIBC.

High resolution melting analysis of deletion/insertion polymorphisms: A new method for the detection and quantification of mixed chimerism in allogeneic stem cell transplantation.

Increasing mixed chimerism after allogeneic stem cell transplantation has been associated with a high risk of relapse and probable graft failure in patient with hematological malignancies as well as non-malignant conditions. We evaluated a new method for chimerism detection, based on the quantitative High Resolution Melting Analysis (HRMA) of deletion/insertion polymorphisms (DIPs). The study consisted in the selection of a panel of DIPs, all generating genotype-specific melting curves, and in the use of samples containing opposite molecular species (homozygous INS/INS and DEL/DEL) mixed in different percentages to create a standard curve for each polymorphism. The detection of mixed chimerism with the HRMA attained a sensitivity of <1%, as well as good accuracy and precision with Percent Errors and Coefficients of Variation not exceeding 30% in reconstruction experiments with DNA mixtures. The present approach provides accurate and precise estimates of mixed chimerism and makes the method open to evaluation for its use in clinical practice.

Loss of heterozygosis on IFN-alpha locus is a prognostic indicator of bacillus Calmette-Guerin response for nonmuscle invasive bladder cancer.

PURPOSE: We evaluated the role of loss of heterozygosity on the interferon-alpha locus to predict the response to bacillus Calmette-Guerin therapy in patients with nonmuscle invasive bladder cancer. MATERIALS AND METHODS: A total of 117 consecutive patients were selected, including 77 with nonmuscle invasive bladder cancer and 40 controls. Loss of heterozygosity on the interferon-alpha locus (chromosome 9p21) was assessed in blood and urine samples before transurethral resection. All patients underwent transurethral resection and then 6 weekly bacillus Calmette-Guerin instillations. Those with nonmuscle invasive bladder cancer were assigned to groups 1 and 2 with and without loss of heterozygosity on the interferon-alpha locus, respectively. RESULTS: Of the 77 patients with nonmuscle invasive bladder cancer 39 (50.6%) had loss of heterozygosity on the interferon-alpha locus (group 1) and 38 (49.4%) had no alteration (group 2). Only 1 of 40 controls showed loss of heterozygosity on the interferon-alpha locus. At the end of followup 13 patients in group 1 and 27 in group 2 were alive without recurrence. We noted a significant difference between loss of heterozygosity on interferon-alpha and followup status (dF 01, LR 11.252, p = 0.003). Kaplan-Meier analysis revealed a significant difference in recurrence probability (response to bacillus Calmette-Guerin) and loss of heterozygosity on interferon-alpha (p <0.0001). On multivariate analysis loss of heterozygosity (HR 4.09, 95% CI 2.59-6.28, p = 0.002), grade (grade 3 HR 3.31, 95% CI 1.38-3.35, p = 0.03) and the number of lesions (3 or greater HR 2.31, 95% CI 1.38-3.25, p = 0.03) were independent predictors of the bacillus Calmette-Guerin response. CONCLUSIONS: This study highlights the predictive value of loss of heterozygosity analysis on interferon-alpha in patients with nonmuscle invasive bladder cancer treated with bacillus Calmette-Guerin.

Prognostic role of loss of heterozygosity on chromosome 18 in patients with low-risk nonmuscle-invasive bladder cancer: results from a prospective study.

BACKGROUND: Somatic alterations on chromosome (Chr) 18q21-23, such as loss of heterozygosity (LOH), have been indicated as a critical step in bladder carcinogenesis. The aim of this study was to evaluate the prognostic role of LOH on Chr 18q21-23 in patients affected by low-grade, nonmuscle-invasive bladder cancer (NMIBC). MATERIALS AND METHODS: A group of 108 consecutive subjects (65 affected by low-risk NMIBC and 43 controls) were selected for this prospective study. LOH on Chr 18 was assessed in the blood and urine samples. The primers used were D18S51, MBP LW, and MBP H. The data obtained were compared with follow-up information. Results were also analyzed by using artificial neural networks (ANN). RESULTS: Out of the 65 patients with NMIBC, 38 (58.4%) showed at least one alteration on Chr 18, while 27 (41.6%) showed no alteration. In the control group, only 2 out of 43 subjects showed LOH on Chr 18. At the end of follow-up, 29 patients were alive without recurrence, while 36 had at least one recurrence. A significant correlation between LOH on Chr 18 and status at follow-up was found (P = 0.022). Kaplan-Meier curves demonstrated a significant correlation between recurrence-free status and LOH on Chr 18 (P = 0.0003). At multivariate analysis, LOH on Chr 18 (P = 0.002) and the number of lesions (P = 0.03) were identified as independent predictors of recurrence-free probability. ANN analysis confirmed the results from multivariate analysis. CONCLUSIONS: This study highlights the role of LOH analysis on Chr 18 in improving recurrence prediction in patients affected by low-grade NMIBC.

Loss of P16 expression and chromosome 9p21 LOH in predicting outcome of patients affected by superficial bladder cancer.

OBJECTIVES: To evaluate the prognostic role of p16 expression and loss of heterozygosity (LOH) on chromosome 9p21 in patients affected by low-grade (G1-G2) urothelial bladder cancer. METHODS: Fifty-six consecutive patients with diagnosis of urothelial bladder cancer were enrolled in this prospective study. LOH analysis was performed on a blood/tumor pair sample of each patient, by using polymerase chain reaction analysis. The D9S171 (9p21) locus on chromosome 9 was investigated. All tumors were stained immunohistochemically for p16. Data from p16 and LOH analyses were compared with follow-up data to evaluate the prognostic role of these molecular markers. RESULTS: Loss of p16 expression was found in 33 patients (58.9%) and was significantly associated with the reduced recurrence-free probability (P < 0.0001). No correlations were reported with stage (P = 0.162) or grade (P = 0.051). Forty-three patients (76.7%) showed LOH on chromosome 9p21 (D9S171). A significant association was observed between loss of p16 expression and LOH on chromosome 9p21 (P = 0.005). The Kaplan-Meier curves showed a significant correlation between recurrence-free status and p16 expression (P = 0.0001). By multivariate analysis, p16 expression (P = 0.002) and number of lesions (P = 0.002) were identified as independent tumor recurrence factors. CONCLUSIONS: Our study highlights the prognostic role of p16 in predicting the recurrence-free probability in patients affected by low-grade urothelial bladder and highlights the fact that this method could be used in everyday urologic clinical practice to better characterize the natural history of urothelial bladder carcinomas.

E-cadherin mRNA expression analysis in evaluating the natural history of urothelial bladder cell carcinoma: results from a long-term follow-up study.

Many studies have indicated that the E-cadherin (E-CAD) expression loss is associated with the loss of cellular differentiation and increased cellular invasiveness and can be correlated with poor prognosis in urothelial carcinoma (UC) of the urinary bladder. The aim of this study was to define the role of E-CAD mRNA expression on recurrence, progression and survival in UC of the urinary bladder over a long follow-up period. From 30 patients with bladder UC, enrolled in our previous study, 27 were selected for this study. All patients were re-analyzed in terms of clinical and tumor characteristics, tumor pathological analysis and tumor E-CAD mRNA expression. The data were correlated to 12-year follow-up results. Significant correlations between stage (p=0.002), grade (p=0.008) and E-CAD mRNA expression were reported. E-CAD did not show any correlation in predicting recurrence or progression in bladder UC. The survival analysis demonstrated a significant relationship (p=0.019) between patients with expressed E-CAD mRNA levels and cancer-specific survival. Multivariate analysis demonstrated that expression of E-CAD mRNA levels is an independent prognostic factor in terms of cancer-specific survival in UC of the urinary bladder (p=0.002). Our study is the first to demonstrate that mRNA extraction and Northen blot analysis is to be considered a reliable method to evaluate E-CAD mRNA levels for predicting survival rate in patients affected by urothelial bladder cancers. We stress that a long follow-up period is needed to evaluate the role of molecular factors in predicting prognosis in patients affected by bladder UC.

Prognostic role of the tumor-associated tissue inflammatory reaction in transitional bladder cell carcinoma.

Many authors have indicated that the presence of an inflammatory response within the tumor may predict not only recurrence and progression but also survival in several tumors, including transitional cell carcinoma (TCC) of the urinary bladder. Several studies have been performed with a mean follow-up period that is often too limited for predicting patient outcome. The aim of the present study was to define the influence of inflammatory cell infiltrate on recurrence, progression and survival in TCC of the bladder over a long follow-up period. Between January and December 1995, 410 consecutive patients, who had undergone transurethral or open surgery for bladder tumors at the same urologic center, were selected for the study. All cases were reviewed to assess histotype, stage and grade of the tumor and presence or absence of tumor-associated inflammatory reaction. To better evaluate the prognostic role of each single factor in TCC, a follow-up of 10 years after surgery was performed. Pathologic evaluation showed superficial TCC in 312 patients, while 98 had an invasive bladder tumor. Three among 410 bladder tumors were squamous cell carcinomas. Out of 407 TCCs, 119 (29.23%) presented inflammation within the tumor or the lamina propria. At 10 years follow-up, a statistically significant association was shown between the presence of inflammation within the tumor or lamina propria and the number of recurrences (p<0.0001). Moreover, the absence of inflammatory infiltrate in the tumor established the relative risk of suffering more than one recurrence at 2.287 (95% CI 1.180-3.346). The Mann-Whitney test confirmed a statistically significant difference between superficial bladder tumors with inflammation and those without (26.3 vs 11.5 months, p<0.001). In terms of survival rate, a statistically significant difference was reported between carcinomas with and without inflammation (p=0.0261). On multivariate analysis, the presence of inflammation within the tumor was found to be an independent predictor of survival in patients with TCC of the bladder (p=0.027). Survival analysis by means of the Kaplan-Meier curves showed a statistically significant difference between patients with tumor-associated inflammatory reaction and those without (p=0.0098). These results confirm that the presence of inflammatory reaction has a good prognostic value in transitional bladder cell carcinoma. However, to better define its prognostic significance, the characterization of inflammatory cells in tumor-associated tissue reaction must be accomplished.

Pre and postoperative quantitative detection of fragments of cytokeratins 8 and 18 (UBC IRMA) as markers of early recurrence of superficial bladder tumor.

OBJECTIVES: The Urinary Bladder Cancer (UBC) test is a marker that detects urinary fragments of cytokeratin 8 and 18. The aim of this study is to evaluate the usefulness of the pre and post operative UBC test to detect early recurrences of a bladder tumor in the first year after the transurethral resection of a bladder tumor. MATERIALS AND METHODS: A multicentric perspective study on 36 patients with superficial bladder cancer (pTa-pT1) treated with transurethral resection (TUR) was performed. Each patient underwent 4 specific urine collections: 1) preoperatively, 2) 3 days after TUR, 3) 7 days after TUR, 4) 30 days after TUR. UBC was analysed on urine with the IRMA method and the cut off value of 12 mg/L was used. Cystoscopy was performed after 3, 6, 9, and 12 months after TUR, with the aim of identifying all cancer recurrences in the first year postoperatively. Statistical analyses to identify differences between patients with or without early recurrence were performed in accordance with Fisher's exact test and Chi-square analysis. RESULTS: Of the 36 patients included in the study 15 showed early recurrence and 21 were recurrence free 1 year after surgery. UBC levels measured in recurrence free patients 30 days after TUR showed normal values, values decreasing as compared with preoperative levels or both circumstances, even if a statistically significant difference was not found between the two groups. CONCLUSIONS: In this study we reported an insignificant correlation between the postoperative modifications of UBC levels and the risk of tumor recurrence during the first year of follow-up. A larger study group with longer follow-ups will probably allow better evaluation of the real power of UBC tests in clinical practice.

Multiplex polymerase chain reaction for microsatellite analysis of urine sediment cells: a rapid and inexpensive method for diagnosing and monitoring superficial transitional bladder cell carcinoma.

PURPOSE: Several urinary markers have been recently introduced in clinical practice for improving the noninvasive diagnosis of transitional cell carcinoma. Although microsatellite analysis must be considered the best method in terms of results, its cost and method time are unacceptable for daily use. We validated a more rapid and inexpensive method of determination using rapid DNA extraction and automatic multiplex polymerase chain reaction amplification. MATERIALS AND METHODS: A total of 120 patients who presented consecutively to a urological office, including 73 with transitional cell carcinoma and 43 who served as controls, were selected for study. Microsatellite analysis was performed in the blood/urine pair using 3 multiplex polymerase chain reactions per patient. Urine sediment inflammatory cells were assessed by urine dipstick test. Ten microsatellite loci were investigated. Numerical data collected during electrophoresis of the amplified segment in an ABI Prism 310 Genetic Analyzer were used to calculate the cutoff for allelic imbalance. Method sensitivity, specificity, and positive and negative predictive values were calculated. RESULTS: A total of 66 patients had microsatellite analysis alterations in urine sediment, of whom 59 had transitional cell carcinoma, while 7 had other urological diseases. Test sensitivity and specificity were 80.8% and 85.1%, respectively. Statistical analysis did not indicate any significant influence of inflammatory status on microsatellite analysis diagnostic performance. In the control group the allelic imbalance on chromosome 9 was significantly lower than on other chromosomes (p = 0.0143). This could confirm that chromosome 9 has a specific role in transitional cell carcinoma. The multiplex microsatellite analysis method was low cost and not time-consuming. CONCLUSIONS: Multiplex microsatellite analysis is a noninvasive, rapid, inexpensive and reproducible method for screening for and monitoring superficial transitional cell carcinoma. It should be considered an alternative method to urinary cytology and it should also be considered in the presence of urine sediment inflammatory cells.

Early diagnosis and monitoring of superficial transitional cell carcinoma by microsatellite analysis on urine sediment.

It has recently been shown that allelic abnormalities, detected by microsatellite analysis of the DNA extracted from urine sediment, can be successfully used for the detection of transitional cell carcinoma (TCC) of the bladder. The diagnostic accuracy of urinary cytology, urinary bladder cancer (UBC) marker, bladder tumor antigen (BTA) and microsatellite sequence alterations was compared in 42 patients who were recruited for the study. Of them, 30 had been diagnosed with TCC at cystoscopy plus biopsy (group A). Seven patients without any apparent lesions after trans-urethral resection (TUR) and 6 subsequent weeks of endovesical administration of bacillus Calmette-Guerin (BCG), had irritative symptoms. None of them had positive cytology or TCC bladder mucosa biopsies (group B). In the control group were 5 other subjects who were affected by benign prostatic hypertrophy and candidates for prostatectomy (group C). Urine and blood samples were obtained from all of the patients before surgery. Tumor tissue and normal mucosa samples were taken from groups A and C during surgery. Different urinary sediment analyses were performed by using both nuclear medicine and molecular tests. UBC and BTA-t analyses were carried out using monoclonal antibody tests while microsatellite analyses were performed using extracted DNA and electrophoresis of polymerase chain reaction (PCR) products on 13 different primers. Urinary cytological examinations were carried out using the Autocyte Preparation System(R). Urinary cytology confirmed the presence of TCC in 13.3% of patients. The BTA-t marker allowed the identification of 73.3% of cancers with 50% specificity; the UBC marker identified 63.3% of the cases with 41.6% specificity. Microsatellite analysis permitted the identification of 83.3% of the tumors with 100% specificity. DNA analysis demonstrated high sensitivity in patients affected by superficial (81.4%) or G1 (80%) tumors, even when cytological studies demonstrated little or no sensitivity. Microsatellite analysis is a highly-sensitive and specific marker for TCC diagnosis and its monitoring, especially in patients with low-stage and low-grade tumors. Other testing procedures failed to increase urinary cytological diagnostic significance.

Molecular urinary sediment analysis in patients with transitional cell bladder carcinoma.

BACKGROUND: Nowadays urinary cytology methods in early diagnosis of superficial bladder transitional carcinoma (TCC) allow the identification of about 35-50% of tumors. Cytoscopy and biopsy are reliable but invasive. It has been recently shown that allelic abnormalities detected by microsatellite analysis of DNA extracted from urine sediment can be successfully used in TCC. We performed a comparative study between urinary cytology and microsatellite sequence alterations in patients affected by TCC. MATERIALS AND METHODS: Fifty-eight patients were recruited for the study. Of these, 45 had cystoscopic diagnosis of TCC, while 7 were without apparent lesions after TUR but presented urinary irritative symptoms after BCG endocavitary administration, and 6 who underwent open surgery for benign prostatic hypertrophy represented the control groups. DNA extraction and PCR analysis were performed by using 13 different primers, while urinary cytology was performed by using an Autocyte Preparation System. RESULTS: Urinary cytology confirmed the presence of TCC in 22% of patients while in 15.5% of them a displastic/inflammatory status was found. Microsatellite analysis allowed the identification of 82% of tumors with a 100% specificity. A high sensitivity was obtained in patients affected by superficial (79%) or G1(80%) tumors. CONCLUSION: Microsatellite analysis represents a highly sensitive and specific marker in TCC diagnosis and monitoring.