Clara Viberti

Shorter Leukocyte Telomere Length Is Independently Associated with Poor Survival in Patients with Bladder Cancer

Background: Shorter telomere length (TL) has been reported to be associated with increased risk of early death in elder individuals. Telomere shortening has been also related to chromosomal instability, which may possibly contribute to the development of several types of digestive or urogenital system cancers and smoking-related tumors. Therefore, we investigated the impact of TL on bladder cancer survival. Methods: TL was measured in leukocyte DNA from whole peripheral blood using quantitative real-time PCR in 463 patients with bladder cancer from a total 726 cases who were followed for up to 18 years. Results: Patients with muscle-invasive tumor/any grade had shorter telomere than patients with non–muscle-invasive tumor/high-grade and with non–muscle-invasive tumor/non–high-grade (TL reference 0.7 ± 0.2; vs. respectively, 0.8 ± 0.2, P = 3.4 × 10−2 and 0.8 ± 0.2, P = 3.6 × 10−2). Moreover, patients in the lowest quartiles of TL were associated with decreased survival after diagnosis (log-rank test, P = 3.9 × 10−4). A Cox regression adjusted by age, cancer aggressiveness, Bacillus Calmette-Guérin, radical cystectomy, radiotherapy, and chemotherapy showed an independent effect of TL on bladder cancer survival (HR, 3.9; 95% confidence interval, 1.7–9.1; P = 1.2 × 10−3). Conclusions: Our results suggest that leukocyte TL is only partly related to tumor aggressiveness and that shorter telomeres act as independent prognostic predictor of survival in patients with bladder cancer. TL information may allow to better select therapeutic approaches in patients with the same stage and grade. Impact: Blood leukocyte TL levels could provide an additional noninvasive prognostic marker to better predict survival and personalize therapies in patients with bladder cancer. Cancer Epidemiol Biomarkers Prev; 23(11); 2439–46. ©2014 AACR.

Detection of multiple mutations in urinary exfoliated cells from male bladder cancer patients at diagnosis and during follow-up

Most bladder cancer (BC) patients need life-long, invasive and expensive monitoring and treatment, making it a serious burden on the health system. Thus, there is a pressing need for an accurate test to assist diagnosis and surveillance of BC as an alternative to cystoscopy. Mutations in human TERT, FGFR3, PIK3CA, and RAS genes have been proposed as potential molecular markers in bladder tumor. Their concomitant presence in urine samples has not been fully explored. We investigated a panel of mutations in DNA from exfoliated urinary cells of 255 BC patients at diagnosis. Forty-one mutations in TERT, FGFR3, PIK3CA, and RAS were analyzed by SNaPshot assay in relation to clinical outcome. In 81 of these patients under surveillance, the same set of mutations was screened in additional 324 samples prospectively collected. The most common mutations detected in urine at diagnosis were in the TERT promoter. In non-invasive BC, these mutations were related to high risk and grade (p<0.0001) as well as progression to muscle-invasive disease (p=0.01), whereas FGFR3 mutations were observed in low-grade BC (p=0.02) and patients with recurrences (p=0.05). Stronger associations were observed for combined TERT and FGFR3 mutations and number of recurrences (OR: 4.54 95% CI: 1.23-16.79, p=0.02). Analyses of the area under the curve for combinations of mutations detected at diagnosis and follow-up showed an accuracy of prediction of recurrence of 0.80 (95% CI: 0.71-0.89). Mutations in urine of BC patients may represent reliable biomarkers. In particular, TERT and FGFR3 mutations have a good accuracy of recurrence prediction.

H2AX phosphorylation level in peripheral blood mononuclear cells as an event-free survival predictor for bladder cancer

Bladder cancer (BC) has a typical aetiology characterized by a multistep carcinogenesis due to environmental exposures, genetic susceptibility, and their interaction. Several lines of evidence suggest that DNA repair plays a role in the development and progression of BC. In particular, the study of individual susceptibility to DNA double strand breaks (DSBs) may provide valuable information on BC risk, and help to identify those patients at high‐risk of either recurrence or progression of the disease, possibly personalizing both surveillance and treatment. Among the different DSB markers, the most well characterized is phosphorylation of the histone H2AX (γ‐H2AX). We assessed any potential role of γ‐H2AX as a molecular biomarker in a case‐control study (146 cases and 146 controls) to identify individuals with increased BC risk and at high‐risk of disease recurrence or progression. We investigated γ‐H2AX levels in peripheral blood mononuclear cells before and after their exposure to ionizing radiation (IR). We did not find any significant difference among cases and controls. However, we observed a significant association between γ‐H2AX basal levels and risk of disease recurrence or progression. In particular, both BC patients as a whole and the subgroup of non‐muscle invasive BC (NMIBC) with high basal H2AX phosphorylation levels had a decreased risk of recurrence or progression (for all BC HR 0.70, 95%CI 0.52–0.94, P = 0.02; for NMIBC HR 0.68, 95%CI 0.50–0.92, P = 0.01), suggesting a protective effect of basal DSB signaling. Our data suggest that γ‐H2AX can be considered as a potential molecular biomarker to identify patients with a higher risk of BC recurrence.

Increased micronucleus frequency in peripheral blood lymphocytes predicts the risk of bladder cancer.

Background:Bladder cancer (BC) is among the most common malignancies worldwide. The identification of new biomarkers for early BC detection, recurrence/progression is urgently needed. The cytokinesis-block micronucleus assay (CBMN) evaluates chromosome damage in cultured human lymphocytes and micronuclei (MN) provide a convenient and reliable index of both chromosome breakage and loss.Methods:Chromosomal damage (expressed as frequencies of MN, nucleoplasmic bridges and nuclear buds (NBUD)) was evaluated by CBMN assay in cryopreserved lymphocytes from 158 age/smoking-matched pairs of cases and controls in relation to BC risk, recurrence or progression. Moreover, non-muscle invasive BC (NMIBC) patients were characterised for 783 DNA repair gene polymorphisms for their possible association with the investigated cytogenetic end points.Results:MN and NBUD frequencies were significantly higher in cases than in controls (P=0.001 and P=0.006, respectively), with the associations being stronger in NMIBC. In a logistic regression model, for each increase of one unit in the MN frequency, a 1.12 increased risk of developing NMIBC was observed. In NMIBC cases, 10 polymorphisms were associated with different MN frequencies after genotype stratification.Conclusions:A model including traditional BC risk factors, MN frequency and the selected polymorphisms differentially distributed in cases and controls improved BC patient identification. Understanding the meaning of systemic chromosomal damage in BC patients with respect to the general population may help to adopt specific prevention strategies and therapeutic intervention.

An Ecosystem with HTII Response and Predators’ Genetic Variability

AbstractA new model to investigate environmental effects of genetically distinguishable predators is presented. The Holling type II response function, modelling feeding satiation, leads to persistent systems oscillations, as in classical population models. An almost complete classification of the cases arising in the Routh–Hurwitz stability conditions mathematically characterizes the paper. It is instrumental as a guideline in the numerical experiments leading to the findings on the limit cycles. This result extends what found in an earlier parallel investigation containing a standard bilinear response function.

microRNA profiles in urine by next-generation sequencing can stratify bladder cancer subtypes

Bladder cancer (BC) is the most frequent malignancy of the urinary tract with a high incidence in men and smokers. Currently, there are no non-invasive markers useful for BC diagnosis and subtypes classification that could overcome invasive procedures such as cystoscopy. Dysregulated miRNA profiles have been associated with numerous cancers, including BC. Cell-free miRNAs are abundantly present in a variety of biofluids including urine and make them promising candidates in cancer biomarker discovery. In the present study, the identification of miRNA fingerprints associated with different BC status was performed by next-generation sequencing on urine samples from 66 BC and 48 controls. Three signatures based on dysregulated miRNAs have been identified by regression models, assessing the power to discriminate different BC subtypes. Altered miRNAs according to invasiveness and grade were validated by qPCR on 112 cases and 65 controls (among which 46 cases and 16 controls were an independent group of subjects while the rest were replica samples). The area under the curve (AUC) computed including three miRNAs (miR-30a-5p, let-7c-5p and miR-486-5p) altered in all BC subtypes showed a significantly increased accuracy in the discrimination of cases and controls (AUC model = 0.70; p-value = 0.01). In conclusions, the non-invasive detection in urine of a selected number of miRNAs altered in different BC subtypes could lead to an accurate early diagnosis of cancer and stratification of patients.

Abstract 4615: H2AX phosphorylation assays, gene expression and epigenomic profiles as markers in bladder cancer: an integrated approach

Bladder cancer (BC), which is the fourth most common malignancy among men in the Western world, has a typical aetiology characterized by a multistep carcinogenesis, reflecting that multiple lesions in the DNA are required for tumour development. DNA repair capacity (DRC) is a complex marker comprising the sum of several factors such as gene variants, gene expression, stability of gene products, and effect of inhibitors/stimulators and might account for different susceptibility of developing this cancer. Individuals with low DRC will tend to accumulate more damage than those who have a better ability to repair such damage. The identification of patients with particular DRC and at high-risk of BC and/or recurrence of the disease could help in personalizing both surveillance and treatment. We aimed at studying the relationship between DRC evaluated by H2AX phosphorylation assays and BC, integrating gene expression and epigenetic profile data (methylation levels and microRNA expression) in cryopreserved lymphocytes from 159 BC cases and 159 controls matched by age and smoking habits. We observed a significant association between γ-H2AX basal levels and H2AX dephosphorylation capacity and risk of BC recurrence. Patients with high basal DSB signaling had a better event-free survival (HR 0.35, 95% CI 0. 19-0.63, p = 0.0005). Additionally, there was an association when considering the percentage of dephosphorylation after 3h divided into two categories (above and under the calculated best cut-off). Interestingly, when compared with the lower category (“slow repair”), the other group (“fast repair”) had a better event-free survival (HR 0.41, 95%CI 0.23-0.72, p = 0.002). Our data suggest that γ-H2AX can be considered as a possible molecular biomarker to identify patients with a higher risk of BC recurrence. A subgroup of cases and controls (39 and 44, respectively) was also analyzed for evaluating their gene expression (Illumina HumanHT-12 Expression BeadChip array) and methylation profile data (Illumina HumanMethylation450 BeadChip array). We found a set of CpG islands that were differentially methylated among cases and controls and a set of genes whose expression was deregulated among different subgroups (e.g. cases vs healthy controls, controls vs BC patients with muscle invasive tumor, etc). We performed an integrated analysis on the genotype/phenotype correlation in the analysed BC cases and healthy controls provided with detailed description of the follow-up for response to therapy/recurrence/survival. This integrated approach will help in elucidating the role of DRC (and its determinants) as predictive and prognostic marker and to identify DNA repair phenotypic assays to be performed in blood cells as non-invasive predictive method. Results will be presented at the AACR Annual Meeting. Citation Format: Barbara Pardini, Alessandra Allione, Simonetta Guarrera, Valentina Turinetto, Giovanni Fiorito, Clara Viberti, Alessia Russo, Paolo Vineis, Carlotta Sacerdote, Claudia Giachino, Giuseppe Matullo. H2AX phosphorylation assays, gene expression and epigenomic profiles as markers in bladder cancer: an integrated approach. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4615. doi:10.1158/1538-7445.AM2015-4615

MMP23B expression and protein levels in blood and urine are associated with bladder cancer

Urothelial bladder cancer (UBC) represents a public health problem because of its high incidence/relapse rates. At present, there are no suitable biomarkers for early diagnosis or relapse/progression prognosis. To improve diagnostic accuracy and overcome the disadvantages of current diagnostic strategies, the detection of UBC biomarkers in easily accessible biofluids, such as urine, represents a promising approach compared with painful biopsies. We investigated the levels of MMP23 genes (microarray and qPCR) and protein (western blot and enzyme-linked immunosorbent assay) in a set of samples (blood, plasma and urine) from patients with UBC and controls as biomarkers for this cancer. MMP23B and its pseudogene MMP23A resulted downregulated in blood cells from UBC compared with controls (66 cases, 70 controls; adjusted P-value = 0.02 and 0.03, respectively). In contrast, MMP23B protein levels in plasma (53 UBC, 49 controls) and urine (59 UBC, 57 controls) increased in cases, being statistically significant in urine. MMP23B dosage observed in urine samples was related to both tumor risk classification and grading. As the lack of correlation between mRNA and protein levels could be due to a posttranscriptional regulation mediated by microRNAs (miRNAs), we investigated the expression of urinary miRNAs targeting MMP23B. Five miRNAs resulted differentially expressed between cases and controls. We reported the first evidence of MMP23B secretion in plasma and urine, suggesting a role of this poorly characterized metalloproteinase in UBC as a potential non-invasive biomarker for this cancer. Further analyses are needed to elucidate the mechanism of regulation of MMP23B expression by miRNAs.

Abstract 1061: MicroRNA profiling by a next generation sequencing approach in urine and plasma samples: from genomics to diagnostics and prognostics of bladder cancer

Bladder cancer (BC) is one of the leading causes of cancer-related death worldwide. Most BCs are non-muscle invasive (NMIBC), which generally have a good prognosis but frequently recur or progress to muscle invasion (MIBC) within 5 years. By contrast, MIBC has a poor prognosis and treatment requires a multidisciplinary approach with radical surgery, radiotherapy, and chemotherapy. BC is among the most expensive cancer per patient because it requires frequent surveillance and repeated treatments over many years. Reliable predictors of disease and progression for BC are lacking so developing novel noninvasive diagnostics is imperative to both improve patient outcomes and decrease health costs. MicroRNAs (miRNAs) are aberrantly expressed in many cancers, including BC, and may be isolated from various biological specimens, including plasma and urine. Next generation sequencing (NGS) technology provides novel information about miRNA expression and is likely to become the gold standard method for comprehensive miRNA analysis in cancer genomics. So far, only few studies investigated miRNA signatures in BC by NGS and only on tissues. To investigate miRNA signatures in surrogate tissues may be a useful alternative to reduce invasiveness of biopsies, allowing repetitive samplings during follow-up and reducing health care costs for detection, monitoring of progression and treatment. We aim to identify specific miRNA signatures in urine and plasma samples from 20 NMIBC, 20 MIBC patients and 40 healthy controls using a NGS approach able to accurately distinguish BC patients and predict disease outcome. The measurement of miRNA levels in both urine and plasma samples from the same patients will allow us to compare the levels of promising biomarkers in two surrogate tissues. Urine is the best and closest surrogate tissue for BC, since it is in direct contact with the tissue of tumor origin; while plasma is one of the best surrogate tissue for distant metastasis and advanced cancer detection. In plasma samples, 5 miRNAs were differentially expressed among cases and controls (miR-222, miR-146b, miR-126, miR221 upregulated and miR-5096 downregulated). However, after repetition of the analyses stratifying cases for NMIBC (the most represented group in BC cases), only 2 of the previously described miRNAs (miR-222 and miR-126) resulted dysregulated among NMIBC and controls. In urine, 31 miRNAs were differentially expressed among BC cases and controls (adjusted p-value ranging from 0.0007 to 0.05). On the other hand, 26 and 31 miRNAs resulted differentially expressed among NMIBC and controls and MIBC and controls, respectively (for NMIBC: adjusted p-value from 0.0009 to 0.04; for MIBC adjusted p-value from 7.2×10-9 to 0.04). Interestingly, miRNAs differentially expressed among cases and controls were able to discriminate not only BC cases from controls, but also NMIBC and MIBC. Citation Format: Barbara Pardini, Francesca Cordero, Alessandra Allione, Clara Viberti, Alessio Naccarati, Marco Oderda, Mirko Preto, Marco Allasia, Maddalena Arigoni, Federica Riccardo, Raffaele Calogero, Carlotta Sacerdote, Giuseppe Matullo. MicroRNA profiling by a next generation sequencing approach in urine and plasma samples: from genomics to diagnostics and prognostics of bladder cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1061.