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Digital archive of theses discussed at the University of Pisa


Thesis etd-03222013-131158

Thesis type
Tesi di dottorato di ricerca
Thesis title
Academic discipline
Course of study
relatore Prof. Naccarato, Antonio Giuseppe
tutor Prof. Bevilacqua, Generoso
relatore Dott.ssa Zavaglia, Katia
  • biomarkers bladder cancer
Graduation session start date
Bladder cancer (BC) is the seventh most common cancer worldwide and its incidence has increased in the last 10 years. It is predominantly a disease of older males, with a median age at presentation of 60-65 years. There are geographical variations in incidence, in Europe the highest incidence rates for men are in northern Italy, Spain and Geneva, Switzerland.
The etiology is well defined, with the most common association being cigarette smoking and other factors including exposure to dyes and some industrial solvents, exhaust fumes.
Nearly 80% of new cases originate from diffuse flat hyperplasia also referred to as low-grade intraurothelial neoplasia. They are typically of low histologic grade, growing as superficial papillary protrusions, and have high propensity for recurrence but they practically never invade the bladder wall or metastasize. The remaining 20% of tumors are non-papillary and develop from severe displasia or carcinoma in situ (CIS), also termed high-grade intraurothelial neoplasia. About 10-15% of low grade superficial papillary tumors progress to invade the bladder wall and may metastasize [1].
Both types of cancer are significant problem for public health: papillary tumors are not usually life threatening but because of their high recurrence rate, they contribute to bladder cancer’s rank as most expensive in terms of clinical management. Although about half of muscle-invasive tumors do initially respond cisplatin-based chemotherapy, the development of drug resistance is the major problem, and the disease progression in resistant tumors is rapid and uniformly fatal [2].
Many factors, such as chromosomal anomalies, genetic polymorphisms, genetic and epigenetic alterations, contribute to tumorigenesis and progression of bladder cancer.
Several authors have reported that the detection of single genetic abnormalities can improve diagnosis and surveillance of the disease
Different genes are well known to be involved independently in the development of BC, leading to loss of cell proliferation control (genes in FGFR3-p53 pathway), apoptosis resistance (i.e. Survivn) and promoting Epithelial Mesenchimal Transition (EMT) (i.e.: CK19, CK20, E cadherin and CD44).
MicroRNAs (miRNAs) are part of a class of small ribonucleic acid (RNAs). They are important regulatory molecules, involved in several cell processes, such as developmental timing, stem cell division and apoptosis. Dysregulated miRNAs have been identified in several human malignancies, including bladder cancer tissue samples, and may confer a “tumour signature” that can be exploited for diagnostic purposes.
To determine whether there is a correlation between the genetic profile and the histo-pathological feature of BC in an Italian population, we genetically characterized 66 Italian patients affected by BC, analyzing both TP53 and FGFR3 mutational status, and CK19-20, E cadherin, Survivin and CD44 gene expression levels.
We report also a prospective pilot study investigating the diagnostic ability of a two miRNAs in voided urine samples collected from patients with bladder cancer just prior to bladder tumour resection
Survivin was significantly over-expressed (p=0.019) in invasive tumors related to CK20 down-regulation (p=0.025). Using multivariate analysis we observed a significant positive relationship between CK19 and CK20 (p=0.0006), a significant association between E cadherin and CK20 in both High Grade (HG) and Low Grade (LG) tumors (p=0.0064 and p=0.0000, respectively) and a strong positive association between Survivin and CD44 (p= 0.0000) only in HG tumors. TP53 and FGFR3 mutations showed a heterogenic distribution.
Gene expression data obtained by RT-PCR on CD44, E-cadherin and Survivin in urine sediments don’t show any significant value in discriminating BC patients from healthy donor group. On the contrary, the two miRNAs of interest were significantly up regulated in urine of BC patients than in healthy controls. Moreover, different statistical methods, like Discriminant Analysis (StatGraphics software) and Artificial Neural Network (ANN), showed a really strong predictive value (around 89% and 83% respectively) using miRNA expression value together in discriminating BC patents from healthy group.
Taken together, our findings on TP53 and FGFR3 mutational status support the well-known genetic heterogeneity of BCs. However our results put in evidence the role of CD44, CK20, CK19 and Survivin on invasiveness ability and apoptosis resistance in HG tumors.
The results on miRNA analysis provide rationale for further studies on validation of candidate miRNAs in voided urine and may potentially lead to the development of a non-invasive and sensitive test for bladder cancer diagnosis and recurrence surveillance.