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.

Genomic rearrangements of the CDKN2A locus are infrequent in Italian malignant melanoma families without evidence of CDKN2A/CDK4 point mutations.

Predisposition to familial cutaneous malignant melanoma has been associated with mutations in the CDKN2A and CDK4 genes. However, only a small subgroup of melanoma pedigrees harbour CDKN2A or CDK4 germline mutations. It is possible that other types of CDKN2A rearrangements, not detectable by routine PCR-based approaches, are involved in a fraction of melanoma cases negative for point sequence changes. In order to gain insights on the possible role of CDKN2A large deletions or duplications in melanoma susceptibility in the Italian population, we screened a series of 124 cutaneous malignant melanoma families referred to five national medical/cancer genetics centres. All probands were negative for point mutations in CDKN2A and CDK4. All samples were tested by MLPA (multiplex ligation-dependent probe amplification), and the results were confirmed by real-time quantitative PCR in a subset of 53 cases. No genomic rearrangements were detected in this series, one of the largest so far investigated. These data suggest that large deletions/duplications in the CDKN2A locus are infrequently involved in the development of familial melanoma in the Italian population. Based on these results, routine search for these rearrangements in CDKN2A- and CDK4-mutation negative melanoma families is not warranted, although it would be reasonable to pursue it in selected cases with very strong family history and/or showing linkage to 9p21.

The p.G23S CDKN2A founder mutation in high-risk melanoma families from Central Italy.

We have investigated the frequency and spectrum of CDKN2A/CDK4 mutations in 23 cutaneous melanoma families from Central Italy (Tuscany). Three distinct mutations were identified in five families. One mutation, p.G23S, was present in three families. Several lines of evidence indicate that p.G23S is a pathogenic mutation: it is located in the functionally important first ankyrinic domain of p16, it was not detected in a sample of 100 control individuals, and it was present in all tested affected individuals from the three families. Haplotype analysis showed a common ancestral origin of the p.G23S mutation. Our data show that the p.G23S mutation is an important cause of hereditary melanoma in Tuscany.

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.

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.