Development of castration resistance in prostate cancer patients treated with luteinizing hormone-releasing hormone analogues (LHRHa): results of the ANARESISTANCE study.

PURPOSE: Evaluate the percentage of patients with prostate cancer treated with luteinizing hormone-releasing hormone analogues (LHRHa) that develop castration resistance after a follow-up period of 3 years. The secondary objective is to evaluate the variables potentially related to the progression to castration resistant prostate cancer (CRPC). METHODS: A post-authorization, nation-wide, multicenter, prospective, observational, and longitudinal study that included 416 patients treated with LHRHa between 2012 and 2017 is presented. Patients were followed for 3 years or until development of CRPC, thus completing a per-protocol population of 350 patients. A Cox regression analysis was carried out to evaluate factors involved in progression to CRPC. RESULTS: After 3 years of treatment with LHRHa 18.2% of patients developed CRPC. In contrast, in the subgroup analysis, 39.6% of the metastatic patients developed CRPC, compared with 8.8% of the non-metastatic patients. The patients with the highest risk of developing CRPC were those with a nadir prostate-specific antigen (PSA) > 2 ng/ml (HR 21.6; 95% CI 11.7-39.8; p < 0.001) and those receiving concomitant medication, most commonly bicalutamide (HR 1.8; 95% CI 1-3.1, p = 0.0431). CONCLUSIONS: The proportion of metastatic patients developing CRPC after 3 years of treatment with LHRHa is consistent with what has been previously described in the literature. In addition, this study provides new findings on CRPC in non-metastatic patients. Concomitant medication and nadir PSA are statistically significant predictive factors for the time to diagnosis of CRPC, the nadir PSA being the strongest predictor.

Impedance-based Real-time Monitoring of Neural Stem Cell Differentiation.

We present here the first impedance-based characterization of the differentiation process of two human mesencephalic fetal neural stem lines. The two dopaminergic neural stem cell lines used in this study, Lund human mesencephalic (LUHMES) and human ventral mesencephalic (hVM1 Bcl-XL), have been developed for the study of Parkinsonian pathogenesis and its treatment using cell replacement therapy. We show that if only relying on impedance magnitude analysis, which is by far the most usual approach in, e.g., cytotoxicity evaluation and drug screening applications, one may not be able to distinguish whether the neural stem cells in a population are proliferating or differentiating. However, the presented results highlight that equivalent circuit analysis can provide detailed information on cellular behavior, e.g. simultaneous changes in cell morphology, cell-cell contacts, and cell adhesion during formation of neural projections, which are the fundamental behavioral differences between proliferating and differentiating neural stem cells. Moreover, our work also demonstrates the sensitivity of impedance-based monitoring with capability to provide information on changes in cellular behavior in relation to proliferation and differentiation. For both of the studied cell lines, in already two days (one day after induction of differentiation) equivalent circuit analysis was able to show distinction between proliferation and differentiation conditions, which is significantly earlier than by microscopic imaging. This study demonstrates the potential of impedance-based monitoring as a technique of choice in the study of stem cell behavior, laying the foundation for screening assays to characterize stem cell lines and testing the efficacy epigenetic control.

Note: Differential configurations for the mitigation of slow fluctuations limiting the resolution of digital lock-in amplifiers.

The resolution of digital lock-in amplifiers working with a narrow bandwidth (<100 Hz) is limited by slow fluctuations, which can be two orders of magnitude larger (µV range) than the noise of the input amplifier (tens of nV). In order to tackle this issue, affecting state-of-the-art laboratory instrumentation and here systematically quantified, three differential sensing configurations are presented. They adapt to different setup conditions and are based on manual and automatic tuning of dummy references, allowing a 25-fold resolution improvement for enhanced long-term tracking of impedance sensors.

Fully inkjet-printed organic photodetectors with high quantum yield.

Bulk-heterojunction based organic photodetectors are fabricated by means of drop-on-demand inkjet printing with vertical topology, inverted structure, and small footprint (about 100 µm x 100 µm). Due to optimization of the deposition technique, an external quantum efficiency in excess of 80% at 525 nm and a -3dB bandwidth of a few tens of kHz is achieved.

Pathway analysis using genome-wide association study data for coronary restenosis--a potential role for the PARVB gene.

BACKGROUND: Coronary restenosis after percutaneous coronary intervention (PCI) still remains a significant limitation of the procedure. The causative mechanisms of restenosis have not yet been fully identified. The goal of the current study was to perform gene-set analysis of biological pathways related to inflammation, proliferation, vascular function and transcriptional regulation on coronary restenosis to identify novel genes and pathways related to this condition. METHODS: The GENetic DEterminants of Restenosis (GENDER) databank contains genotypic data of 556,099SNPs of 295 cases with restenosis and 571 matched controls. Fifty-four pathways, related to known restenosis-related processes, were selected. Gene-set analysis was performed using PLINK, GRASS and ALIGATOR software. Pathways with a p<0.01 were fine-mapped and significantly associated SNPs were analyzed in an independent replication cohort. RESULTS: Six pathways (cell-extracellular matrix (ECM) interactions pathway, IL2 signaling pathway, IL6 signaling pathway, platelet derived growth factor pathway, vitamin D receptor pathway and the mitochondria pathway) were significantly associated in one or two of the software packages. Two SNPs in the cell-ECM interactions pathway were replicated in an independent restenosis cohort. No replication was obtained for the other pathways. CONCLUSION: With these results we demonstrate a potential role of the cell-ECM interactions pathway in the development of coronary restenosis. These findings contribute to the increasing knowledge of the genetic etiology of restenosis formation and could serve as a hypothesis-generating effort for further functional studies.

Systematic testing of literature reported genetic variation associated with coronary restenosis: results of the GENDER Study.

BACKGROUND: Coronary restenosis after percutaneous coronary intervention still remains a significant problem, despite all medical advances. Unraveling the mechanisms leading to restenosis development remains challenging. Many studies have identified genetic markers associated with restenosis, but consistent replication of the reported markers is scarce. The aim of the current study was to analyze the joined effect of previously in literature reported candidate genes for restenosis in the GENetic DEterminants of Restenosis (GENDER) databank. METHODOLOGY/PRINCIPAL FINDINGS: Candidate genes were selected using a MEDLINE search including the terms 'genetic polymorphism' and 'coronary restenosis'. The final set included 36 genes. Subsequently, all single nucleotide polymorphisms (SNPs) in the genomic region of these genes were analyzed in GENDER using set-based analysis in PLINK. The GENDER databank contains genotypic data of 2,571,586 SNPs of 295 cases with restenosis and 571 matched controls. The set, including all 36 literature reported genes, was, indeed, significantly associated with restenosis, p = 0.024 in the GENDER study. Subsequent analyses of the individual genes demonstrated that the observed association of the complete set was determined by 6 of the 36 genes. CONCLUSION: Despite overt inconsistencies in literature, with regard to individual candidate gene studies, this is the first study demonstrating that the joint effect of all these genes together, indeed, is associated with restenosis.

Annexin A5 prevents post-interventional accelerated atherosclerosis development in a dose-dependent fashion in mice.

BACKGROUND: Activated cells in atherosclerotic lesions expose phosphatidylserine (PS) on their surface. Annexin A5 (AnxA5) binds to PS and is used for imaging atherosclerotic lesions. Recently, AnxA5 was shown to inhibit vascular inflammatory processes after vein grafting. Here, we report a therapeutic role for AnxA5 in post-interventional vascular remodeling in a mouse model mimicking percutaneous coronary intervention (PCI). METHODS AND RESULTS: Associations between the rs4833229 (OR = 1.29 (CI 95%), p(allelic) = 0.011) and rs6830321 (OR = 1.35 (CI 95%), p(allelic) = 0.003) SNPs in the AnxA5 gene and increased restenosis-risk in patients undergoing PCI were found in the GENDER study. To evaluate AnxA5 effects on post-interventional vascular remodeling and accelerated atherosclerosis development in vivo, hypercholesterolemic ApoE(-/-) mice underwent femoral arterial cuff placement to induce intimal thickening. Dose-dependent effects were investigated after 3 days (effects on inflammation and leukocyte recruitment) or 14 days (effects on remodeling) after cuff placement. Systemically administered AnxA5 in doses of 0.1, 0.3 and 1.0mg/kg compared to vehicle reduced early leukocyte and macrophage adherence up to 48.3% (p = 0.001) and diminished atherosclerosis development by 71.2% (p = 0.012) with a reduction in macrophage/foam cell presence. Moreover, it reduced the expression of the endoplasmic reticulum stress marker GRP78/BiP, indicating lower inflammatory activity of the cells present. CONCLUSIONS: AnxA5 SNPs could serve as markers for restenosis after PCI and AnxA5 therapeutically prevents vascular remodeling in a dose-dependent fashion, together indicating clinical potential for AnxA5 against post-interventional remodeling.

A genome-wide association study identifies a region at chromosome 12 as a potential susceptibility locus for restenosis after percutaneous coronary intervention.

Percutaneous coronary intervention (PCI) has become an effective therapy to treat obstructive coronary artery diseases (CAD). However, one of the major drawbacks of PCI is the occurrence of restenosis in 5-25% of all initially treated patients. Restenosis is defined as the re-narrowing of the lumen of the blood vessel, resulting in renewed symptoms and the need for repeated intervention. To identify genetic variants that are associated with restenosis, a genome-wide association study (GWAS) was conducted in 295 patients who developed restenosis (cases) and 571 who did not (controls) from the GENetic Determinants of Restenosis (GENDER) study. Analysis of ~550 000 single nucleotide polymorphisms (SNPs) in GENDER was followed by a replication phase in three independent case-control populations (533 cases and 3067 controls). A potential susceptibility locus for restenosis at chromosome 12, including rs10861032 (P(combined) = 1.11 × 10(-7)) and rs9804922 (P(combined) = 1.45 × 10(-6)), was identified in the GWAS and replication phase. In addition, both SNPs were also associated with coronary events (rs10861032, P(additive) = 0.005; rs9804922, P(additive) = 0.023) in a trial based cohort set of elderly patients with (enhanced risk of) CAD (PROSPER) and all-cause mortality in PROSPER (rs10861032, P(additive) = 0.007; rs9804922, P(additive) = 0.013) and GENDER (rs10861032, P(additive) = 0.005; rs9804922, P(additive) = 0.023). Further analysis suggests that this locus could be involved in regulatory functions.

Matrix metalloproteinases 2 and 3 gene polymorphisms and the risk of target vessel revascularization after percutaneous coronary intervention: Is there still room for determining genetic variation of MMPs for assessment of an increased risk of restenosis?

OBJECTIVE: Mixed results have been reported of matrix metalloproteinases (MMP) and their association with restenosis after percutaneous coronary intervention (PCI). The current study examines whether multiple single nucleotide polymorphisms (SNPs), covering the full genomic region of MMP2 and MMP3, were associated with restenosis in the GENDER study population. METHODS AND RESULTS: The GENetic DEterminants of Restenosis (GENDER) study enrolled 3104 consecutive patients after successful PCI. The primary endpoint was clinical restenosis, defined as target vessel revascularization (TVR), occurring in 9.8% of the patients. From the Hapmap database, 19 polymorphisms of MMP2 and 11 of MMP3 were selected. Furthermore, in a subpopulation, a genome-wide association analysis (GWA) was performed. No significant association was found with any of the investigated SNPs, including the previously reported 5A/6A polymorphism (rs3025058), with regard to TVR using single SNP analysis or haplotype analysis. CONCLUSION: We found no significant association of MMP2 or MMP3 with TVR with this SNP-broad gene approach. Although we did not test all the known polymorphisms of these genes, using tagging analyses we examined those SNPs covering all known haplotypes of MMP2 and MMP3 to conclude that these genes do not correlate with a genetic risk of coronary restenosis after successful PCI.

A genome wide association analysis in the GENDER study.

Percutaneous coronary intervention (PCI) has become an effective therapy to treat coronary artery diseases. However, one of the major drawbacks of PCI is the occurrence of restenosis in 8 to 40% of all treated patients. The GENetic Determinants of Restenosis (GENDER) project was designed to study the association between genetic polymorphisims and clinical restenosis. The discovery of genetic variants associated to the occurrence of restenosis after PCI may provide a more tailored therapy and may serve as rationale for new antirestenotic therapies. So far, several candidate gene approaches had already been performed in the GENDER samples but a Genome Wide Association Scan (GWAS) was still lacking. Here, we present preliminary results from the GWAS we are currently carrying out in the GENDER population. (Neth Heart J 2009;17:262-4.).

p27kip1-838C>A single nucleotide polymorphism is associated with restenosis risk after coronary stenting and modulates p27kip1 promoter activity.

BACKGROUND: The cyclin-dependent kinase inhibitor p27(kip1) is a key regulator of smooth muscle cell and leukocyte proliferation in vascular disease, including in-stent restenosis. We therefore hypothesized that common genetic variations or single nucleotide polymorphisms in p27(kip1) may serve as a useful tool in risk stratification for in-stent restenosis. METHODS AND RESULTS: Three single nucleotide polymorphisms concerning the p27(kip1) gene (-838C>A, rs36228499; -79C>T, rs34330; +326G>T, rs2066827) were determined in a cohort of 715 patients undergoing coronary angioplasty and stent placement. We discovered that the p27(kip1)-838C>A single nucleotide polymorphism is associated with clinical in-stent restenosis; the -838AA genotype decreases the risk of target vessel revascularization (hazard ratio, 0.28; 95% confidence interval, 0.10 to 0.77). This finding was replicated in another cohort study of 2309 patients (hazard ratio, 0.61; 95% confidence interval, 0.40 to 0.93). No association was detected between this end point and the p27(kip1)-79C>T and +326G>T single nucleotide polymorphisms. We subsequently studied the functional importance of the -838C>A single nucleotide polymorphism and detected a 20-fold increased basal p27(kip1) transcriptional activity of the -838A allele containing promoter. CONCLUSIONS: Patients with the p27(kip1)-838AA genotype have a decreased risk of in-stent restenosis corresponding with enhanced promoter activity of the -838A allele of this cell-cycle inhibitor, which may explain decreased smooth muscle cell proliferation.

Palaeogenetic evidence supports a dual model of Neolithic spreading into Europe.

The peopling of Europe is a complex process. One of the most dramatic demographic events, the Neolithic agricultural revolution, took place in the Near East roughly 10000 years ago and then spread through the European continent. Nevertheless, the nature of this process (either cultural or demographic) is still a matter of debate among scientists. We have retrieved HVRI mitochondrial DNA sequences from 11 Neolithic remains from Granollers (Catalonia, northeast Spain) dated to 5500 years BP. We followed the proposed authenticity criteria, and we were also able, for the first time, to track down the pre-laboratory-derived contaminant sequences and consequently eliminate them from the generated cloning dataset. Phylogeographic analysis shows that the haplogroup composition of the Neolithic population is very similar to that found in modern populations from the Iberian Peninsula, suggesting a long-time genetic continuity, at least since Neolithic times. This result contrasts with that recently found in a Neolithic population from Central Europe and, therefore, raises new questions on the heterogeneity of the Neolithic dispersals into Europe. We propose here a dual model of Neolithic spread: acculturation in Central Europe and demic diffusion in southern Europe.

Nanoscale capacitance imaging with attofarad resolution using ac current sensing atomic force microscopy.

Nanoscale capacitance imaging with attofarad resolution (∼1 aF) of a nano-structured oxide thin film, using ac current sensing atomic force microscopy, is reported. Capacitance images are shown to follow the topographic profile of the oxide closely, with nanometre vertical resolution. A comparison between experimental data and theoretical models shows that the capacitance variations observed in the measurements can be mainly associated with the capacitance probed by the tip apex and not with positional changes of stray capacitance contributions. Capacitance versus distance measurements further support this conclusion. The application of this technique to the characterization of samples with non-voltage-dependent capacitance, such as very thin dielectric films, self-assembled monolayers and biological membranes, can provide new insight into the dielectric properties at the nanoscale.

The genetics of the pre-Roman Iberian Peninsula: a mtDNA study of ancient Iberians.

The Iberians developed a surprisingly sophisticated culture in the Mediterranean coast of the Iberian Peninsula from the 6th century BC until their conquest by the Romans in the 2nd century BC. They spoke and wrote a non-Indo-European language that still cannot be understood; their origins and relationships with other non-Indo-European peoples, like the Etruscans, are unclear, since their funerary practices were based on the cremation of bodies, and therefore anthropology has been unable to approach the study of this people. We have retrieved mitochondrial DNA (mtDNA) from a few of the scarce skeletal remains that have been preserved, some of them belonging to ritualistically executed individuals. The most stringent authentication criteria proposed for ancient DNA, such as independent replication, amino-acid analysis, quantitation of template molecules, multiple extractions and cloning of PCR products, have been followed to obtain reliable sequences from the mtDNA hypervariable region 1 (HVR1), as well as some haplogroup diagnostic SNPs. Phylogeographic analyses show that the haplogroup composition of the ancient Iberians was very similar to that found in modern Iberian Peninsula populations, suggesting a long-term genetic continuity since pre-Roman times. Nonetheless, there is less genetic diversity in the ancient Iberians than is found among modern populations, a fact that could reflect the small population size at the origin of the population sampled, and the heterogenic tribal structure of the Iberian society. Moreover, the Iberians were not especially closely related to the Etruscans, which points to considerable genetic heterogeneity in Pre-Roman Western Europe.

Shot noise in linear macroscopic resistors.

We report on direct experimental evidence of shot noise in a linear macroscopic resistor. The origin of the shot noise comes from the fluctuation of the total number of charge carriers inside the resistor associated with their diffusive motion under the condition that the dielectric relaxation time becomes longer than the dynamic transit time. The present results show that neither potential barriers nor the absence of inelastic scattering are necessary to observe shot noise in electronic devices.

Unravelling migrations in the steppe: mitochondrial DNA sequences from ancient central Asians.

This study helps to clarify the debate on the Western and Eastern genetic influences in Central Asia. Thirty-six skeletal remains from Kazakhstan (Central Asia), excavated from different sites dating between the fifteenth century BC to the fifth century AD, have been analysed for the hypervariable control region (HVR-I) and haplogroup diagnostic single nucleotide polymorphisms (SNPs) of the mitochondrial DNA genome. Standard authentication criteria for ancient DNA studies, including multiple extractions, cloning of PCR products and independent replication, have been followed. The distribution of east and west Eurasian lineages through time in the region is concordant with the available archaeological information: prior to the thirteenth-seventh century BC, all Kazakh samples belong to European lineages; while later an arrival of east Eurasian sequences that coexisted with the previous west Eurasian genetic substratum can be detected. The presence of an ancient genetic substratum of European origin in West Asia may be related to the discovery of ancient mummies with European features in Xinjiang and to the existence of an extinct Indo-European language, Tocharian. This study demonstrates the usefulness of the ancient DNA in unravelling complex patterns of past human migrations so as to help decipher the origin of present-day admixed populations.

Liver cancer risk is increased in patients with porphyria cutanea tarda in comparison to matched control patients with chronic liver disease.

BACKGROUND/AIMS: Patients with porphyria and chronic liver disease could be at high risk of developing hepatocellular carcinoma. To define the incidence of primary liver cancer and identify variables associated with the risk of cancer in patients with porphyria cutanea tarda in comparison to control patients. METHODS: Fifty-three patients with porphyria cutanea tarda were enrolled in a prospective study (median follow-up 72 +/- 54.1 months; range 12-216) and matched individually to a control case according to age (+/-5 years), sex, duration of follow up (+/- 5 years), severity of liver disease, and hepatitis C virus infection. RESULTS: During follow-up hepatocellular carcinoma developed in 18 patients with porphyria and in four control patients. Incidence of primary liver cancer was 4.8 and 1.3 x 100 patients/year in the overall series of patients and of controls, respectively. The cumulative probability of being tumor free was significantly lower in porphyria cutanea tarda than in matched controls (75 vs 95%). Variables independently associated with the risk of liver cancer were the presence of porphyria and cirrhosis at enrollment (Odds ratios: 5.3, 95% CI 1.4-19.3 and 3.0, 95% CI 1.2-7.6, respectively). CONCLUSIONS: Patients with porphyria are at higher risk of developing liver cancer than matched control patients.

Autoantibodies to human cytosol: a marker of sporadic porphyria cutanea tarda.

The enzymes potentially involved in the pathogenesis of sporadic porphyria cutanea tarda (PCT) reside in liver cytosoles and microsomes. PCT is frequently associated with hepatitis C virus (HCV) infection, which is in turn associated with autoimmune manifestations. To investigate whether autoimmune reactions, possibly triggered by HCV, are involved in the pathogenesis of PCT, we measured by immunoblot autoantibodies to human cytosolic and microsomal liver fractions in 82 patients with PCT (77% with HCV infection), 105 with other liver disorders and 40 healthy subjects. Anti-liver cytosolic antibodies were more frequent in PCT patients (38/82, 46%) than in pathological controls (P < 0.05-P < 0.001) or in healthy subjects (3/40, 8%, P < 0.001). Among PCT patients, anticytosolic antibodies were more frequent in HCV positive (36/63, 57%) than in HCV negative (2/19, 11%, P < 0.05) cases. Reactivity to a 40-kDa cytosolic polypeptide was present in 20 PCT patients (19 HCV positive), being more frequent than in all pathological controls (P < 0.01-P < 0.0001). Histological activity index (P = 0.04) and antibodies to HCV (P = 0.027) - but not HCV RNA - were associated independently with anticytosolic antibodies as assessed by multivariate analysis. In contrast, frequency of antiliver microsomal antibodies was similar in PCT patients (24/82, 29%) and pathological controls (8-26%), being higher in the autoimmune hepatitis control group (23/23, 100%, P < 0.0001). In conclusion, anticytosolic antibodies, particularly to a 40-kDa polypeptide, are frequent in PCT and associated with HCV infection and severity of liver damage.

Hyperferritinemia, iron overload, and multiple metabolic alterations identify patients at risk for nonalcoholic steatohepatitis.

OBJECTIVE: The aim of this study was to define in patients with hyperferritinemia and normal transferrin saturation the relationships among hyperferritinemia, iron overload, HFE gene mutations, the presence of metabolic alterations, and nonalcoholic steatohepatitis (NASH). METHODS: Forty patients with increased serum ferritin, resistant to dietary restriction and normal transferrin saturation, 90 with ultrasonographic evidence of hepatic steatosis, and 60 obligate heterozygotes for hemochromatosis, all negative for alcohol abuse, hepatitis virus infections, and inflammation were studied. Transferrin saturation, serum ferritin, uric acid, lipids, glucose tolerance, insulin resistance, HFE gene mutations, liver histology, and hepatic iron concentration were analyzed. RESULTS: Of the 40 patients with hyperferritinemia, 29 (72%) had biochemical metabolic abnormalities, 18 of the 26 examined (69%) had insulin resistance, 26 (65%) had the presence of one of the two HFE gene mutations (normal controls, 33 of 128 [26%], p < 0.0001), and all had increased liver iron concentration. Thirty-one patients (77%) had histology compatible with NASH. At univariate analysis, NASH was significantly associated with the presence of metabolic alterations, the C282Y mutation, and severity of fibrosis. At multivariate analysis, NASH was associated with the coexistence of multiple metabolic alterations (odds ratio = 5.2, 95% CI = 0.95-28.7). The risk of having NASH augmented in the presence of higher values of ferritin and liver iron concentration. Among the 90 patients with ultrasonographic evidence of hepatic steatosis, 24 (27%) had increased serum ferritin with normal transferrin saturation, but only six remained hyperferritinemic after dietary restriction. CONCLUSION: Increased ferritin with normal transferrin saturation is frequently found in patients with hepatic steatosis, but it reflects iron overload only in those patients in whom it persists despite an appropriate diet. The simultaneous disorder of iron and glucose and/or lipid metabolism, in most of the cases associated with insulin resistance, is responsible for persistent hyperferritinemia and identifies patients at risk for NASH.

Mutations in the HFE gene and their interaction with exogenous risk factors in hepatocellular carcinoma.

The possible role of iron in facilitating the development of liver cancer is still debated. The aims of this study were to define the prevalence of the mutations 845G --> A and 187C --> G (C282Y and H63D) in the HFE gene associated with hereditary hemochromatosis in Italian patients with hepatocellular carcinoma occurring in cirrhosis and to analyze the interaction between these mutations and other established risk factors for hepatocellular carcinoma. The HFE gene mutations, performed by polymerase chain reaction, were analyzed in 81 patients (63 males, 18 females) with hepatocellular carcinoma. None of the patients had a phenotype compatible with homozygous hereditary hemochromatosis. Interaction between HFE mutations and exogenous risk factors was analyzed by collecting information on alcohol consumption, hepatitis B and C virus infections, and iron status at the time of diagnosis of chronic liver disease. This analysis was performed only in males to rule out gender influence on patients' iron status by using the case-only approach specifically designed to estimate departure from multiplicative risk ratios under the assumption of independence between genotype and environmental exposure. The prevalence of the C282Y mutation was significantly higher in patients with hepatocellular carcinoma than in normal controls (8.6% vs 1.6%, P < 0.03). At univariate analysis, iron overload was significantly associated with both HFE mutations (P < 0.0001), whereas ongoing hepatitis B virus infection was associated with the C282Y mutation (P < 0.05). By multivariate analysis, a trend for an increased risk of being positive for hepatitis virus markers (OR 2.9, CI 95% 0.9-9.5) and of having been alcohol abusers (OR 3, CI 95% 0.7-14) was observed in patients heterozygous for the HFE mutations. These data indicate that the prevalence of the main mutation associated with hereditary hemochromatosis is significantly higher in cirrhotic Italian patients with hepatocellular carcinoma compared to a normal population and suggest that heterozygotes for HFE mutations exposed to hepatitis virus infections or who had been alcohol abusers could have an increased risk of developing cirrhosis and later liver cancer than people without the mutations exposed to the same risk factors.