MicroRNA and hepatocellular carcinoma: biology and prognostic significance.

Hepatocellular carcinoma (HCC) is the most common primary liver cancer and the third most common cause of cancer-related death worldwide. In 90% of cases, HCC arises on a background of cirrhosis which, in turns, results from hepatitis (HBV and HCV) infections, alcohol abuse, metabolic disorders including NASH, and genetic metabolic diseases, autoimmune hepatitis, primary biliary cirrhosis and exposure to environmental carcinogens. The molecular mechanisms underlying HCC development are still only partially known. Despite a high molecular variability, the deregulation of definite oncogenic pathways has been confirmed as a common finding in HCC. Among these, the molecular ways controlling proliferation, apoptosis and migration play a major role. In recent years, a new class of regulatory RNAs, the microRNAs, has been discovered and their deregulated expression has been linked to the molecular pathogenesis of many cancers because of their ability to strongly impact on the expression of crucial messenger RNAs. This review focuses on some of the most relevant evidence concerning the contribution of microRNA aberrant expression to HCC development.

Aberrant fragile histidine triad gene transcripts in primary hepatocellular carcinoma and liver cirrhosis.

To determine whether transcriptional alterations of the fragile histidine triad (FHIT) gene play a role in the development and progression of human hepatocellular carcinoma (HCC) we used reverse transcription-PCR to examine mRNA FHIT expression in 28 paired samples of HCC (24 in cirrhotic and 4 in noncirrhotic livers) and matched noncancerous tissue and in 10 normal livers. We also assessed loss of heterozygosity of the polymorphic D3S1300 microsatellite marker in the intron between exons 5 and 6 of the FHIT gene. Abnormal FHIT transcripts were detected in 13 cases (46.4%): 10 in the cancerous tissue only, 1 with the same pattern in both cancerous and matched noncancerous tissue, and 2 in the noncancerous tissue only. The four HCCs that arose in noncirrhotic liver all showed abnormal FHIT transcripts. No alterations were found in normal livers. Sequence analysis of abnormally sized transcripts revealed that they were generated by the fusion of exons 3 or 4 with exons 8 or 9. Among the cancerous specimens, one case showed only an abnormal sized transcript derived from the fusion of exons 4 and 9 in the absence of any normal-sized transcript, and another case showed deletion of a sequence comprised between nucleotides -35 and 399 resulting in an exon 4-9 fusion not respecting the exons' bounds. Loss of heterozygosity was found in two cases with abnormal FHIT transcripts and in only one case with normal transcript. Patients with aberrant FHIT transcripts showed a significantly higher relapse rate and shorter recurrence time (P = 0.001). This could be related to a primary genomic instability affecting particularly susceptible regions like FRA3B and could be associated with an increasing risk of recurrence without involving a causative role.

MicroRNAs in liver cancer: a model for investigating pathogenesis and novel therapeutic approaches.

MicroRNAs (miRNAs) constitute a large class of short RNAs (e.g., 20-24 nucleotides in length), whose main function is to posttranscriptionally regulate the expression of protein-coding genes. Their importance in tumorigenesis has been demonstrated over the past decade, and correspondingly, they have emerged as potential therapeutic molecules and targets. Liver cancer is one of the most common neoplastic diseases worldwide, and it currently has a poor prognosis owing to largely ineffective therapeutic options. Liver cancer is also an excellent model for testing miRNA-based therapy approaches as it can be easily targeted with the systemic delivery of oligonucleotides. In recent years, the role of miRNAs in hepatocellular carcinoma (HCC) has been established with molecular studies and the development of animal models. These studies have also provided the basis for evaluating the therapeutic potential of miRNAs, or anti-miRNAs. In general, the safety of miRNAs has been proven and antitumor activity has been observed. Moreover, because of the absence or presence of mild side effects, the prophylactic use of miRNA-based approaches may be foreseen.

Duplex-Doppler evaluation of the effects of propranolol and isosorbide-5-mononitrate on portal flow and splanchnic arterial circulation in cirrhosis.

BACKGROUND: A decrease in portal flow is an important pharmacological effect of drugs used for the prophylaxis of variceal bleeding. AIM: To assess the acute and chronic effects of propranolol, and the effect of the acute addition of isosorbide-5-mononitrate, on splanchnic circulation. METHODS: Measurements of portal blood flow volume (PBFV) and of Doppler ultrasound pulsatility index of the superior mesenteric, femoral and interlobar renal arteries were performed in 10 cirrhotic patients with varices at baseline, 90 min after propranolol or placebo, after 30 days of chronic propranolol treatment and 45 min after the addition of isosorbide-5-mononitrate. RESULTS: The mean PBFV was significantly lower at all times than at baseline, with the greatest mean percentage decrease achieved after the addition of isosorbide-5-mononitrate (> or = 20% in all patients). Acute changes, however, did not predict the chronic effects in many patients. Isosorbide-5-mononitrate significantly increased the mesenteric and femoral pulsatility indices, whereas no significant change was observed in the kidney. CONCLUSIONS: Propranolol significantly decreases PBFV, but chronic effects cannot be reliably predicted by the acute change. All patients achieved a decrease in PBFV of > or = 20% after the acute addition of isosorbide-5-mononitrate to chronic propranolol treatment.

Determination of xanthine oxidase in human serum by a competitive enzyme-linked immunosorbent assay (ELISA).

Xanthine oxidase was purified from human milk and used to immunise rabbits. A competitive immunoenzymatic assay with purified enzyme and rabbit antiserum was optimised to measure xanthine oxidase in human serum, the lowest detectable amount being 0.03 pmol of enzymatic protein. Thus, the test (i) is sensitive enough to determine xanthine oxidase in human serum, being more sensitive than the spectrophotometric method, (ii) it is more convenient for clinical laboratories than other sensitive tests and (iii) it has the advantage over the enzyme activity-based assays of also detecting inactive enzyme molecules. A competitive enzyme-linked immunosorbent assay (ELISA) was used to measure the serum xanthine oxidase level in healthy donors and in patients with liver diseases, and it was found that any concentration below 1 mg/L is in the normal range.

Relationship between splanchnic, peripheral and cardiac haemodynamics in liver cirrhosis of different degrees of severity.

OBJECTIVE: To investigate the relationships between changes in splanchnic and systemic haemodynamics in liver cirrhosis. DESIGN AND METHODS: Abdominal and peripheral duplex-Doppler sonography and Doppler echocardiography were performed in 42 cirrhotic patients with (group A, ascitic) or without ascites (group NA, non-ascitic) and in a control group of 36 healthy volunteers. RESULTS: There were significant differences (P < 0.05 at ANOVA) between the three groups in portal vein flow velocity (controls, groups NA and A, respectively, 29.2, 21.4 and 20.0 cm/s), portal diameter (9.3, 12.2 and 12.0 mm), superior mesenteric artery (SMA) resistance index (RI) (0.889, 0.854 and 0.816), femoral artery RI (0.988, 0.974 and 0.945), mean arterial pressure (MAP) (101.4, 102.0 and 87.3 mmHg), peripheral vascular resistance (1579, 1404 and 1094 dyn/cm5/s) and cardiac index (CI) (2.91, 3.46 and 3.77 l/min/m2). Multiple regression analysis identified renal interlobular- and SMA RI (respectively, r = -0.58 and r = 0.51) in group A as the two regional vascular beds correlated to MAP. CONCLUSION: The deterioration of the cirrhotic hyperdynamic circulation in the presence of ascites and the correlation between MAP and mesenteric and renal resistances are consistent with the peripheral arterial vasodilation hypothesis. The positive correlation between MAP and SMA RI in ascitic patients shows a link between this region and the general circulation. This seems to suggest that splanchnic hyperafflux plays a part in the formation of ascites.

Different haemodynamic effects of a single dose of long-acting isosorbide-5-mononitrate in healthy subjects and patients with cirrhotic portal hypertension.

BACKGROUND: The action pathways of nitrates are hypothesised to be deranged in cirrhosis. AIM: In order to confirm it, the acute haemodynamic effects of isosorbide-5-mononitrate in cirrhotic patients and controls was investigated. PATIENT: Nine cirrhotics and nine healthy controls. METHODS: Evaluation in the fasting state, 90 min after isosorbide-5-mononitrate or placebo (double-blind on two different days) and then 30 and 120 min after eating a standard meal. Various systemic and splanchnic haemodynamic parameters, including arterial impedance, assessed as Doppler pulsatility index, were measured. RESULTS: isosorbide-5-mononitrate reduced arterial pressure and increased heart rate and mesenteric pulsatility index both in controls and in cirrhotics, whereas the following parameters behaved differently in the two groups (P < 0.05): hepatic pulsatility index decreased (-9%) and the portal velocity increased (+13%) in controls, whereas hepatic pulsatility increased (+18%) and portal velocity decreased (-18%) in cirrhotics. The two groups presented a similar pattern of changes in most variables under placebo after a meal. In controls, the administration of isosorbide-5-mononitrate blunted the postprandial mesenteric vasodilation and related changes in splanchnic and systemic circulation, expected at 30 min, in comparison to those observed under placebo. In cirrhotics, instead, the postprandial pattern was similar under placebo and isosorbide-5-mononitrate. CONCLUSIONS: The acute administration of isosorbide-5-mononitrate produces different haemodynamic effects in healthy and diseased livers, both in the fasting state and after a meal, consistent with the hypothesis of a deranged response of the intrahepatic microcirculation to nitrates in cirrhosis.

Left ventricular volumes in liver cirrhosis.

BACKGROUND: Patients with alcoholic cirrhosis have left ventricular dimensions similar to controls. Few data have been reported in patients with cirrhosis of viral origin. AIM: To assess left ventricular dimensions in patients with pure viral cirrhosis. PATIENTS AND METHODS: Thirty patients with virus-related cirrhosis, 23 patients with alcoholic cirrhosis and 12 healthy controls were submitted to measurement of left ventricular volumes, cardiac output, mean arterial pressure and total peripheral resistance. RESULTS: Patients with cirrhosis showed a similar increase in cardiac index and heart rate and reduction of mean arterial pressure and peripheral vascular resistance in comparison to controls, irrespective of the aetiology. Left ventricular end systolic volume index was lower (p<0.01) and ejection fraction higher (p<0.01) in virus-related cirrhotic patients [mean +/- SD, respectively 12.4+/-4.1 ml/sqm and 77.9%) in comparison both to controls (21.5+/-6.3 ml/sqm and 66.8%) and alcoholics (20.6+/-7.0 ml/sqm and 68.8%). End diastolic volume index was not significantly different between the three groups. CONCLUSIONS: Our findings indicate smaller left ventricular volumes and higher ejection fraction in pure virus-related cirrhosis than in alcoholic cirrhosis and controls. Since peripheral haemodynamics proved similar in virus- and alcohol-related cirrhosis, a subclinical alcohol cardiomyopathy may be hypothesised to account for the absence of such left ventricular pattern in alcoholic patients.

Intra- and extrahepatic arterial resistances in chronic hepatitis and liver cirrhosis.

Thirty patients with chronic hepatitis (CH), 84 with liver cirrhosis (LC) and 42 controls, underwent noninvasive measurement of hepatic artery resistance index (RI) by means of Doppler ultrasound (US), at the porta hepatis and in the intrahepatic branches, in order to investigate possible changes related to: (a) the liver disease; (b) the site of measurement; and (c) ageing. The intrahepatic RI differed among LC, CH and controls (0.731, 0.690 and 0.643, p < 0.05), whereas the RI at the porta hepatis did not (0.754, 0.748 and 0.729, respectively). Intrahepatic RI correlated with age in LC (r = 0.51, p < 0.0001) and in controls (r = 0.49, p < 0.001). In LC, it correlated also with the presence and size of esophageal varices (r = 0.32, p < 0.05). In conclusion, an increase of hepatic artery RI in chronic liver diseases can be demonstrated when assessed in the intraparenchymal branches. The increase of hepatic artery RI with ageing should be considered in future studies.

Value of splanchnic Doppler ultrasound in the diagnosis of portal hypertension.

The accuracy of various Doppler parameters of portal circulation in the diagnosis of relevant portal hypertension (presence of gastroesophageal varices) was prospectively validated. The following parameters were compared in 51 patients with chronic liver disease (40 with cirrhosis and 11 with chronic hepatitis): portal vein flow velocity and congestion index, hepatic and splenic arteries resistance indexes (RI), modified liver vascular index (portal flow velocity/hepatic artery RI) and portal hypertension index, a new index calculated as: [(hepatic artery RI x 0.69) x (splenic artery RI x 0.87)]/portal vein flow velocity. Highest accuracy was achieved by the splenic artery RI and the portal hypertension index (both around 75%) at cut-offs, respectively, of 0.60 and 12 cm/s(-1), which appeared to be, therefore, the most favorable parameters for the clinical practice. Their use may limit the need for endoscopy to search for varices.

MiR-221 controls CDKN1C/p57 and CDKN1B/p27 expression in human hepatocellular carcinoma.

The identification of target mRNAs is a key step for assessing the role of aberrantly expressed microRNAs in human cancer. MiR-221 is upregulated in human hepatocellular carcinoma (HCC) as well as in other malignancies. One proven target of miR-221 is CDKN1B/p27, whose downregulation affects HCC prognosis. Here, we proved that the cyclin-dependent kinase inhibitor (CDKI) CDKN1C/p57 is also a direct target of miR-221. Indeed, downregulation of both CDKN1B/p27 and CDKN1C/p57 occurs in response to miR-221 transfection into HCC-derived cells and a significant upregulation of both CDKN1B/p27 and CDKN1C/p57 occurs in response to antimiR-221 transfection. A direct interaction of miR-221 with a target site on the 3' UTR of CDKN1C/p57 mRNA was also demonstrated. By controlling these two CDKIs, upregulation of miR-221 can promote growth of HCC cells by increasing the number of cells in S-phase. To assess the relevance of these studies in primary tumors, matched HCC and cirrhosis samples were assayed for miR-221, for CDKN1B/p27 and CDKN1C/p57 expression. MiR-221 was upregulated in 71% of HCCs, whereas CDKN1B/p27 and CDKN1C/p57 proteins were downregulated in 77% of cases. A significant inverse correlation between miR-221 and both CDKN1B/p27 and CDKN1C/p57 was found in HCCs. In conclusion, we suggest that miR-221 has an oncogenic function in hepatocarcinogenesis by targeting CDKN1B/p27 and CDKN1C/p57, hence promoting proliferation by controlling cell-cycle inhibitors. These findings establish a basis toward the development of therapeutic strategies aimed at blocking miR-221 in HCC.

Alteration of DNA ploidy and cell nuclearity in human hepatocellular carcinoma associated with HBV infection.

BACKGROUND/AIMS: Hepatocellular carcinoma usually arises in cirrhotic livers as a complication of chronic liver disease, and may show a variable trend towards increasing ploidy. The aim of this study was to investigate possible associations between different etiological factors, particularly hepatitis B virus and hepatitis C virus infection, and alteration of DNA-ploidy and nuclearity of neoplastic hepatocytes. METHODS: DNA-ploidy, the percentage of binucleated cells in the total cell population and the fraction of mononucleated hepatocytes in the polyploid compartment were assessed by image cytometry on cellular suspensions obtained by fine-needle biopsy from 60 hepatocellular carcinomas in patients whose viral status had previously been assessed. RESULTS: Significantly higher DNA-ploidy values (p = 0.005), with a reduction in the percentage of binucleated hepatocytes (p = 0.003) and an increase in the fraction of mononucleated hepatocytes in the polyploid compartment (p < 0.0001), were found in hepatocellular carcinoma with actual or previous hepatitis B virus infection (including also HCV+ve patients) in comparison to those not associated with hepatitis B virus infection, but not when HCV+ve hepatocellular carcinomas were compared to HCV-ve ones. Statistically significant differences for ploidy values (p < 0.05), percentage of binucleated hepatocytes (p < 0.05) and fraction of mononucleated hepatocytes in the polyploid compartment (p = 0.003) were also found between hepatocellular carcinoma associated only to hepatitis B virus infection ("pure" hepatitis B virus cases) and those associated only to hepatitis C virus infection ("pure" hepatitis C virus cases). CONCLUSIONS: Hepatocellular carcinoma associated with a previous or actual hepatitis B virus infection shows a peculiar phenotypical appearance, characterized by a trend towards increasing ploidy and reduction of binuclearity.

Diagnostic and prognostic value of DNA ploidy and cell nuclearity in ultrasound-guided liver biopsies.

BACKGROUND: Focal nodule lesions in patients with cirrhotic livers may be visualized by using imaging techniques; however, the diagnostic and prognostic judgment of biopsies from borderline lesions may be difficult using conventional histologic criteria. METHODS: The diagnostic and prognostic value of DNA ploidy analysis determined by image cytometry of Feulgen-stained isolated hepatocytes was investigated in ultrasound-guided biopsies from 50 nodular lesions found in patients with cirrhotic livers (39 hepatocellular carcinomas [HCCs] and 11 macroregenerative nodules) and from 10 patients with livers affected by viral chronic hepatitis. Of the 11 macroregenerative nodules, 7 presented a subsequent neoplastic behavior. Specimens from the morphologically normal livers of five patients who underwent liver surgery served as control tissues. Image cytometry was performed on Feulgen-stained cytologic preparations, obtained by enzymatic digestion of formalin fixed biopsies. The DNA ploidy of the main stem line and the distribution of mononucleated and binucleated hepatocytes (nuclearity) were compared using histologic diagnosis, Edmondson's grade, tumor size, and patient follow-up. RESULTS: The main stem line was peridiploid in all benign specimens and in 31 clinically confirmed HCCs, peritetraploid in 11 HCCs, perioctaploid in 1 HCC, and aneuploid in 3 HCCs. The fraction of mononucleated polyploid hepatocytes was found to be the best diagnostic parameter in euploid HCCs and was significantly correlated with the Edmondson grade and the nodular size. Survival information was available for 43 patients, with a median observation period of 350 days. A DNA ploidy value of the main stem line greater than 3c was an important determinant of survival as a single parameter and in association with histologic grade and greatest dimension of tumor. CONCLUSIONS: This study suggests that the ploidy distribution analysis of mononucleated and binucleated hepatocytes can provide valuable information for making correct diagnoses and for predicting survival outcome for patients with HCCs.

Pilot study of triple antiviral therapy for chronic hepatitis C in interferon alpha non-responders.

BACKGROUND: No effective therapy exists for interferon non-responding chronic hepatitis C patients. AIMS: Pilot study evaluating the potential efficacy and safety of triple antiviral therapy in interferon-alpha non-responders. PATIENTS AND METHODS: Twenty consecutive adult patients with chronic hepatitis C who had failed to respond to a 6-month course of interferon alpha were randomly assigned to receive a combination of interferon alpha + oral ribavirin (double therapy), or the same combination + oral amantadine (triple therapy), for 6 months. RESULTS: By the end of therapy, normal alanine transaminase (biochemical response) was obtained in 2 out of 10 patients on double therapy but in 7 out of 10 on triple therapy (p < 0.05), and negative serum hepatitis C virus (HCV) RNA (virological response) occurred in 1 out of 10 patients on double therapy but in 7 out of 10 patients on triple therapy (p < 0.01). Six months after therapy, biochemical response was sustained in 1 (double therapy) and 4 patients (triple therapy), respectively, and the virological response was sustained in no patient on double therapy but in 3 patients on triple therapy. CONCLUSIONS: Triple antiviral therapy seems to be able to induce biochemical and virological responses in interferon alpha non-responders with chronic hepatitis C.

Superior mesenteric artery impedance in chronic liver diseases: relationship with disease severity and portal circulation.

OBJECTIVE: The increase of splanchnic blood flow volume in liver cirrhosis is attributed to decreased arterial resistance. The aim of this study was to noninvasively investigate superior mesenteric artery impedance in patients with chronic liver diseases and to assess its relationship with portal hemodynamics and with clinical parameters. METHODS: Superior mesenteric artery (SMA) pulsatility (SMA-PI) and resistance (SMA-RI) indices and portal vein flow parameters (velocity, volume, and congestion index) were measured by duplex-Doppler ultrasound in 14 patients with chronic hepatitis, in 73 cirrhotics, in 30 liver transplant recipients, and in 31 control subjects. RESULTS: SMA-PI significantly differed among the five groups (p < 0.0001), being lower in cirrhotics (2.55+/-0.70) and transplanted patients (2.77+/-0.69) than in chronic hepatitis (3.28+/-0.57) and control subjects (3.42+/-0.92). SMA-PI was lower in ascitic cirrhosis (2.40+/-0.71) than in compensated cirrhosis (2.71+/-0.70) (p < 0.01) and in cirrhotics with large varices (2.30+/-0.67) than in those without varices (2.75+/-0.65) (p < 0.05). Moreover SMA-PI correlated with numeric Child-Pugh score (r=-0.28) and portal vein congestion index (r=-0.36). CONCLUSION: Hyperdynamic splanchnic circulation, noninvasively assessed by a decrease of mesenteric artery impedance, occurs in cirrhosis since the early stage of the disease and tends to worsen in relation to liver failure and the severity of portal hypertension. Furthermore, the persistent SMA-PI decrease in transplant recipients suggests a consistent contribution to this circulatory alteration from a patent portosystemic collateral circulation.

In hepatocellular carcinoma AgNOR protein expression correlates with tumour mass doubling time.

AIMS/METHODS: The relationship between AgNOR protein expression and doubling time was evaluated in 20 untreated nodules of hepatocellular carcinoma arising in cirrhotic liver. AgNOR protein quantity within the lesion was defined by image cytometry on histological sections from frozen biopsies obtained under ultrasound-guidance, selectively stained for AgNOR proteins. Tumour doubling time was calculated 6 months after diagnosis by measuring the volume variations of the nodules over a fixed period by "real time" ultrasonography. RESULTS: The doubling time of nodules characterized by high AgNOR protein area values (> 5.50 microns2, corresponding to the median AgNOR protein value) was shorter than that of nodules with low AgNOR protein area values (< 5.50 microns2). A highly significant difference in the mean doubling time values between the two groups (6.31 +/- 2.68 (E.S.) versus 15.92 +/- 3.03 (E.S.) months, respectively; p = 0.009) was found. Moreover, when the relationship between AgNOR protein and doubling time values was tested by linear regression analysis, a significant inverse correlation was observed (r = -0.68; p < 0.005). CONCLUSIONS: Our results indicate that AgNOR protein quantity represents a reliable parameter for predicting the tumour growth rate of untreated hepatocellular carcinoma nodules. Among the procedures commonly employed for the assessment of cell proliferation, the evaluation of the AgNOR parameter seems to be particularly suitable for kinetic analysis of ultrasound-guided fine-needle liver biopsies.

Cytometric measurement of cell proliferation in echo-guided biopsies from focal lesions of the liver.

Increased proliferative activity determined in surgical specimens of hepatocellular carcinoma (HCC) has been associated with tumor grade and patient survival. The measurement of cell proliferation in echo-guided biopsies of small focal liver lesions might provide useful information for the early recognition of malignancy and for predicting the aggressiveness of small HCCs. We assessed the diagnostic and prognostic value of cell proliferation in 91 echo-guided needle biopsies of focal liver lesions using the monoclonal antibody Ki-67, which detects a human nuclear antigen that is present in proliferating cells. Measurements were performed by image cytometry as the percentage of Ki-67 positive hepatocytes nuclei over total hepatocyte nuclei in the biopsy. At the histological examination, 27 lesions were diagnosed as chronic hepatitis, 10 as cirrhosis, 11 as macroregenerative nodule, and 43 as HCC in cirrhotic liver. Although the highest Ki-67 values (> 20%) were found in less-differentiated HCCs, most well-differentiated HCCs and nine borderline nodules were completely devoid of Ki-67-positive hepatocytes. A sustained Ki-67 labeling (up to 16%) was found in hepatitis and cirrhosis, similar to that found in several malignant tumors. In the HCC subset, Ki-67 labeling was strongly correlated to the Edmondson-Steiner histological grade. However, survival analysis did not indicate a better outcome for those patients with low-proliferating tumors.

Duplex Doppler findings in splenic arteriovenous fistula.

Splenic arteriovenous fistula (AVF) is a rare cause of portal hypertension. An early diagnosis is crucial because lethal complications may occur if the disease is not treated. We report a case of AVF in a noncirrhotic patient in whom the diagnosis was made by duplex sonography. This technique can differentiate between AVF and other, more common vascular abnormalities in the left upper quadrant in patients with portal hypertension. Duplex sonography can therefore be recommended as the first imaging approach in this population.

Allelic imbalance on 16q in small, unifocal hepatocellular carcinoma: correlation with HBV and HCV infections and cellular proliferation rate.

In advanced hepatocellular carcinoma (HCC), allelic loss on chromosome 16q may occur. To better define the frequency of this alteration in small HCC and to more closely identify the affected region for further positional cloning of the putative tumor suppressor gene contained in this region, microsatellite polymorphism analysis was conducted on small, unifocal HCC, without signs of intrahepatic or systemic spread. We also tried to assess its possible correlation with hepatitis virus infections (HBV and HCV) and cellular proliferation rate. DNA from 35 small (<4 cm), unifocal HCC and from the corresponding nontumorous surrounding tissue was analyzed by 10 sets of microsatellite polymorphic markers. Serologic markers for hepatitis virus B and C infections were investigated in all cases. AgNOR protein quantity was assessed by image analysis on cryostatic sections stained with silver. The percentage of tumours with allelic imbalance ranged from 11.1 to 37%. The minimal involved region was assessed at 16q24.3, corresponding to the D16S413 marker, which was also the most commonly affected locus (10 of 27 informative cases, 37%). Allelic imbalance on chromosome 16q was significantly associated with HBV infection: 8 of 10 cases showed an actual or previous HBV infection in the group showing allelic imbalance, versus 6 with a previous HBV infection out of 25 in the control group (P < 0.01). No difference was found as far as HCV infection is concerned. The mean (+/-SE) AgNOR protein value in six cases showing allelic imbalance was 8.36 +/- 1.2 microm2, compared to 6.45 +/- 0.68 microm2 in 13 cases retaining both the alleles at 16q but the difference proved not statistically significant. In conclusion, in this series of small, unifocal HCC the minimal region of allelic imbalance on 16q was restricted to 16q24.3. It was found to be associated with HBV infection but not with increased cellular proliferation rate.

Assessment of vascular patterns of small liver mass lesions: value and limitation of the different Doppler ultrasound modalities.

OBJECTIVES: This study aimed to investigate the value and limitation of the different Doppler ultrasound modalities (spectral analysis, color, and power Doppler imaging) in the differential diagnosis of small liver tumors to identify the optimal diagnostic approach with the presently available Doppler technology. METHODS: Presence and distribution of color and power Doppler signals, Doppler peak frequency, resistive index, and systolic acceleration time were examined in 133 liver nodules (< or = 4 cm). RESULTS: Color and power Doppler did not identify specific diagnostic vascular patterns. By discriminant analysis, peak frequency (cut-off 1320 Hz) differentiates small hematocellular carcinoma (< or = 2 cm) from macroregenerative nodules and hemangiomas (accuracy 92.6%); resistive index (cut-off 0.65) differentiates malignancies from benign lesions (accuracy 83.8%); and systolic acceleration time (cut-off 105 ms) differentiates hepatocellular carcinoma from metastases (accuracy 80.9%). CONCLUSIONS: Power Doppler imaging is able to assess vascularity in the majority of small liver nodules, but the pattern distribution of tumoral vascular signals does not provide reliable differential diagnostic criteria. Using conventional Doppler technology, power Doppler should be used to detect vascular signals and spectral analysis, and subsequently to measure quantitative parameters such as high peak frequency and resistive index (which identify malignancy) and prolonged systolic acceleration time (which identifies primary from metastatic liver tumors).