Rossana Critelli

Considerations on the use of urine markers in the management of patients with low-/intermediate-risk non–muscle invasive bladder cancer

OBJECTIVES Many molecular assays for bladder cancer diagnosis and surveillance have been developed over the past several decades. However, none of these markers have been routinely implemented into clinical decision making. Beyond their potential for screening high-risk populations, urine markers likely have the greatest potential in the follow-up of patients with non-muscle invasive bladder cancer (NMIBC). METHODS Here, we discuss the current options and limitations of the use of urine markers for patient surveillance, focusing on patients with low-/intermediate-risk NMIBC. RESULTS As these patients have a very low risk of tumor progression, the primary goal of surveillance is detection of recurrent disease. Although urine cytology seems to be limited to detection of few patients who would develop high-grade tumors, we conclude that the use of markers with high sensitivity for low-grade disease for patient follow-up has the potential to decrease the frequency of urethrocystoscopy without compromising patient prognosis. Because a single marker may not have sufficient sensitivity for detection of low-grade tumors, different scenarios, e.g., multitesting and reflex or sequential approaches, are discussed. CONCLUSIONS There is consensus that currently available markers have the potential to support clinical decision making in follow-up of patients with low-/intermediate-risk NMIBC. In light of our analysis, further additional randomized controlled studies to effectively assess the clinical usefulness of modern urine markers are required.

Genome-wide association study yields variants at 20p12.2 that associate with urinary bladder cancer.

Genome-wide association studies (GWAS) of urinary bladder cancer (UBC) have yielded common variants at 12 loci that associate with risk of the disease. We report here the results of a GWAS of UBC including 1670 UBC cases and 90 180 controls, followed by replication analysis in additional 5266 UBC cases and 10 456 controls. We tested a dataset containing 34.2 million variants, generated by imputation based on whole-genome sequencing of 2230 Icelanders. Several correlated variants at 20p12, represented by rs62185668, show genome-wide significant association with UBC after combining discovery and replication results (OR = 1.19, P = 1.5 × 10(-11) for rs62185668-A, minor allele frequency = 23.6%). The variants are located in a non-coding region approximately 300 kb upstream from the JAG1 gene, an important component of the Notch signaling pathways that may be oncogenic or tumor suppressive in several forms of cancer. Our results add to the growing number of UBC risk variants discovered through GWAS.

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.

Polymorphisms in the XRCC1 gene modify survival of bladder cancer patients treated with chemotherapy.

Survival of bladder cancer patients depends on several factors including disease stage and grade at diagnosis, age, health status of the patient and the applied treatment. Several studies investigated the role of DNA repair genetic variants in cancer susceptibility, but only few studies investigated their role in survival and response to chemotherapy for bladder cancer. We genotyped 28 single nucleotide polymorphisms (SNP) in DNA repair genes in 456 bladder cancer patients, reconstructed haplotypes and calculated a score for combinations of the SNPs. We estimated Hazard Ratios (adjHR) for time to death. Among patients treated with chemotherapy, variant alleles of five SNPs in the XRCC1 gene conferred better survival (rs915927 adjHR 0.55 (95%CI 0.32–0.94); rs76507 adjHR 0.48 (95%CI 0.27–0.84); rs2854501 adjHR 0.25 (95%CI 0.12–0.52); rs2854509 adjHR 0.21 (95%CI 0.09–0.46); rs3213255 adjHR 0.46 (95%CI 0.26–0.80). In this group of patients, an increasing number of variant alleles in a XRCC1 gene score were associated with a better survival (26% decrease of risk of death for each additional variant allele in XRCC1). By functional analyses we demonstrated that the previous XRCC1 SNPs confer lower DNA repair capacity. This may support the hypothesis that survival in these patients may be modulated by the different DNA repair capacity determined by genetic variants. Chemotherapy treated cancer patients bearing an increasing number of “risky” alleles in XRCC1 gene had a better survival, suggesting that a proficient DNA repair may result in resistance to therapy and shorter survival. This finding may have clinical implications for the choice of therapy.

Environmental and personal determinants of the uptake of disinfection by-products during swimming

BACKGROUND Trihalomethanes (THMs) in exhaled breath and trichloroacetic acid (TCAA) in urine are internal dose biomarkers of exposure to disinfection by-products (DBPs) in swimming pools. OBJECTIVE We assessed how these biomarkers reflect the levels of a battery of DBPs in pool water and trichloramine in air, and evaluated personal determinants. METHODS A total of 116 adults swam during 40min in a chlorinated indoor pool. We measured chloroform, bromodichloromethane, dibromochloromethane and bromoform in exhaled breath and TCAA in urine before and after swimming, trichloramine in air and several DBPs in water. Personal determinants included sex, age, body mass index (BMI), distance swum, energy expenditure, heart rate and 12 polymorphisms in GSTT1, GSTZ1 and CYP2E1 genes. RESULTS Median level of exhaled total THMs and creatinine adjusted urine TCAA increased from 0.5 to 14.4µg/m(3) and from 2.5 to 5.8µmol/mol after swimming, respectively. The increase in exhaled brominated THMs was correlated with brominated THMs, haloacetic acids, haloacetonitriles, haloketones, chloramines, total organic carbon and total organic halogen in water and trichloramine in air. Such correlations were not detected for exhaled chloroform, total THMs or urine TCAA. Exhaled THM increased more in men, urine TCAA increased more in women, and both were affected by exercise intensity. Genetic variants were associated with differential increases in exposure biomarkers. CONCLUSION Our findings suggest that, although affected by sex, physical activity and polymorphisms in key metabolizing enzymes, brominated THMs in exhaled breath could be used as a non-invasive DBP exposure biomarker in swimming pools with bromide-containing source waters. This warrants confirmation with new studies.

Association Between Total Number of Deaths, Diabetes Mellitus, Incident Cancers, and Haplotypes in Chromosomal Region 8q24 in a Prospective Study

The 8q24 region is a gene desert, although chromosomal aberrations and somatic amplification involving this region, including translocations involving the protooncogene c-MYC, have been frequently reported in people with cancer. To investigate the role of variants in 8q24 region, the authors analyzed data from a prospective study (n = 10,372 participants who were followed for 11 years) in which a large number of health events (>1,500) occurred (1993-1998). They genotyped all subjects for 5 candidate single nucleotide polymorphisms (rs672888, rs1447295, rs9642880, rs16901979, and rs6983267) that were identified in previous genome-wide scans. Although significant associations with individual single nucleotide polymorphisms were small in magnitude, the authors observed higher increases in the risks of different types of cancer with specific haplotypes, particularly when subjects were homozygous for the haplotype: for breast cancer and homozygotes for haplotype CAGCT, hazard ratio = 3.40, 95% confidence interval: 1.24, 9.21; for prostate cancer and grouped rare haplotypes, hazard ratio = 7.43, 95% confidence interval: 3.00, 18.37; and for brain cancer and homozygotes for haplotype CGGCT, hazard ratio = 13.48, 95% confidence interval: 3.00, 59.53. Significant associations were also observed between haplotypes and deaths from cardiovascular diseases and cerebrovascular diseases; the most stable association was between homozygotes for haplotypes CGTCG and CAGCT and total deaths in men (hazard ratio = 3.5, 95% confidence interval: 1.8, 6.9, and hazard ratio = 2.8, 95% confidence interval: 1.3, 6.4, respectively). In conclusion, the authors have observed a strong pleiotropic effect of the 8q24 region in a large prospective study. This observation can shed light on the mechanisms underlying reported associations between 8q24 variants and disparate chronic diseases.

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.

Investigation of extended Y chromosome STR haplotypes in Sardinia

Y-chromosomal variation of selected single nucleotide polymorphisms (SNPs) and 32 short tandem repeat (STR) loci was evaluated in Sardinia in three open population groups (Northern Sardinia, n=40; Central Sardinia, n=56; Southern Sardinia, n=91) and three isolates (Desulo, n=34; Benetutti, n=45, Carloforte, n=42). The tested Y-STRs consisted of Yfiler® Plus markers and the seven rapidly mutating (RM) loci not included in the YFiler® Plus kit (DYF399S1, DYF403S1ab, DYF404S1, DYS526ab, DYS547, DYS612, and DYS626). As expected, inclusion of additional Y-STR loci increased haplotype diversity (h), though complete differentiation of male lineages was impossible even by means of RM Y-STRs (h=0.99997). Analysis of molecular variance indicated that the three open populations were fairly homogeneous, whereas signs of genetic heterogeneity could be detected when the three isolates were also included in the analysis. Multidimensional scaling analysis showed that, even for extended haplotypes including RM Y-STR markers, Sardinians were clearly differentiated from populations of the Italian peninsula and Sicily. The only exception was represented by the Carloforte sample that, in accordance with its peculiar population history, clustered with Northern/Central Italian populations. The introduction of extended forensic Y-STR panels, including highly variable RM Y-STR markers, is expected to reduce the impact of population structure on haplotype frequency estimations. However, our results show that the availability of geographically detailed reference databases is still important for the assessment of the evidential value of a Y-haplotype match.

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.

Telomere Length Variation in Juvenile Acute Myocardial Infarction

Leukocyte telomere length (LTL) provides a potential marker of biological age, closely related to the endothelial dysfunction and consequently to the atherosclerotic process. To investigate the relationship between the LTL and the risk of premature acute myocardial infarction and to evaluate the predictive value of LTL on the onset of major cardiovascular events, 199 patients from 18 to 48 years old with first diagnosis of acute myocardial infarction were enrolled and were matched with 190 controls for sex and age (±1 year). Clinical data and coronary artery disease were evaluated at enrollment and at follow up. LTL was measured at enrollment using a quantitative PCR-based method. No significant differences were observed in LTL between cases and controls (p = 0.20) and with the presence of coronary artery disease in patients (p = 0.47). Hypercholesterolemic cases presented LTL significantly longer than cases without hypercholesterolemia (t/s: 0.82±0.16 p = 0.79 and t/s norm: 0.79±0.19 p = 0.01), as confirmed in multivariate regression analysis (p = 0.005, β = 0.09). Furthermore, multivariate regression analysis showed LTL significantly shorter in hypertensive cases than in normotensive cases (p = 0.04, β = −0.07). One hundred seventy-one cases (86%) ended the average follow up of 9±5 years, 92 (54%) presented a major cardiovascular event. At multivariate regression analysis the LTL detected at enrollment did not represent a predictive factor of major cardiovascular events nor it significantly impacted with cumulative events. Based on present cohort of young Italian patients, the LTL did not represent a marker of acute myocardial infarction nor had a predictive role at medium term follow up.