Stefano Maria Giulini

Early and Late Recurrence After Liver Resection for Hepatocellular Carcinoma: Prognostic and Therapeutic Implications

Objective:To evaluate the predictive factors, the therapy, and the prognosis of intrahepatic recurrence (IR) after surgery for hepatocellular carcinoma (HCC). Summary Background Data:The predictive factors of IR are debated. To class the recurrence according to the modality of presentation may help to find a correlation and to select the right therapy for the recurrence. Methods:A total of 213 patients were evaluated. Risk factors for recurrence were related to time (<2 years and >2 years) and type of presentation (marginal, nodular, and diffuse). Prognosis and therapy for the recurrence were studied in each group of patients. Results:IR was observed in 143 patients; 109 were early (group 1) and 34 late recurrences (group 2). Cirrhosis, chronic active hepatitis (CAH) and HCV positivity were independently related to the risk of recurrence with a cumulative effect (92.5% of recurrences in patients with 3 prognostic factors). For group 1, the neoplastic vascular infiltration together with cirrhosis, HCV positivity, CAH, and transaminases were significant; all the 11 patients with 5 negative prognostic factors showed an early recurrence. On the contrary, only cirrhosis was related to a late recurrence. Survival rate was significantly better in late than in early recurrence (61.9%, 27.1% and 25.7%, 4.5% at 3–5 years); a curative procedure was performed in 67.6% in group 1 and 29.3% in group 2. After a radical treatment of IR, the survival was comparable with the group of patients without recurrence. Conclusions:Early and late recurrences are linked to different predictive factors. The modality of presentation of the recurrence together with the feasibility of a radical treatment are the best determinants for the prognosis.

Vascular Hypertrophy and Remodeling in Secondary Hypertension

It has been proposed that several neurohumoral factors may be involved in the genesis of vascular structural changes (remodeling or hypertrophy) frequently observed in essential hypertension. Therefore, in this study we investigated vascular structural alterations of subcutaneous small resistance arteries in patients with secondary forms of hypertension. The study included 70 participants: 11 with pheochromocytoma, 13 with primary aldosteronism, and 17 with renovascular hypertension; 13 normotensive subjects and 16 patients with essential hypertension served as controls. All subjects were submitted to a biopsy of subcutaneous fat. Small resistance arteries were dissected and mounted on a micromyograph, and media-lumen ratio, media thickness, remodeling index, and growth index were evaluated. Endothelial function was evaluated according to the dose-response curve to acetylcholine. In patients with either primary aldosteronism or renovascular hypertension, a marked increase in media-lumen ratio was observed, whereas in patients with pheochromocytoma, the extent of vascular structural alterations was similar to that observed in patients with essential hypertension. The increase in media-lumen ratio in patients with essential hypertension and with pheochromocytoma was mainly due to vascular remodeling (remodeling index, 93% to 94%), whereas in patients with renovascular hypertension, there was vascular growth (remodeling index, 70%; growth index, 53%). Patients with primary aldosteronism had an intermediate pattern compared with the other two forms of secondary hypertension. An evident impairment of endothelial function was observed in all four hypertensive groups. In conclusion, the renin-angiotensin-aldosterone system seems to be more powerful than the adrenergic system in inducing vascular growth.

Vascular Remodeling and Duration of Hypertension Predict Outcome of Adrenalectomy in Primary Aldosteronism Patients

Remodeling of the resistance arteries is a hallmark of arterial hypertension and predicts cardiovascular events, but it was unknown whether it could also predict the blood pressure response to adrenalectomy of patients with an aldosterone-producing adenoma. Therefore, we investigated the outcome of adrenalectomy as a function of vascular remodeling in the context of the preoperative features of aldosterone-producing adenoma patients. At 2 referral centers for hypertension, we prospectively measured the media:lumen ratio of small arteries from fat tissue of 50 consecutive aldosterone-producing adenoma patients treated with adrenalectomy. The blood pressure response to adrenalectomy was assessed by considering the blood pressure values and the number and dosages of antihypertensive medications. Adrenalectomy significantly (P<0.001) lowered plasma aldosterone (from 27.3±4.9 ng/dL to 8.3±11.2 ng/dL), the aldosterone:renin ratio (from 117±35 to 11±2), and blood pressure (from 163±22/98±2 mm Hg to 133±2/84±1 mm Hg), even despite a reduction (from 141±14 to 100±15; P=0.02) of the score of antihypertensive treatment. It provided cure of hypertension in 30% of the aldosterone-producing adenoma patients, normotension with less antihypertensive therapy in 52%, and improved blood pressure control in the rest. The media:lumen ratio and the known duration of hypertension significantly predicted the blood pressure response to adrenalectomy at univariate and multivariate analyses. Because a long duration of hypertension and/or the presence of vascular remodeling imply lower chances of blood pressure normalization at long-term follow-up postadrenalectomy, these findings emphasize the importance of an early diagnosis of aldosterone-producing adenoma.

MicroRNA-23b mediates urokinase and c-met downmodulation and a decreased migration of human hepatocellular carcinoma cells

Urokinase‐type plasminogen activator (uPA) and c‐met play a major role in cancer invasion and metastasis. Evidence has suggested that uPA and c‐met overexpression may be coordinated in human hepatocellular carcinoma (HCC). In the present study, to understand whether the expression of these genes might be coregulated by specific microRNAs (miRs) in human cells, we predicted that Homo sapiens microRNA‐23b could recognize two sites in the 3′‐UTR of uPA and four sites in the c‐met 3′‐UTR by the algorithm pictar. The miR‐23b expression analysis in human tumor and normal cells revealed an inverse trend with uPA and c‐met expression, indicating that uPA and c‐met negative regulation might depend on miR‐23b expression. Transfection of miR‐23b molecules in HCC cells (SKHep1C3) led to inhibition of protein expression of the target genes and caused a decrease in cell migration and proliferation capabilities. Furthermore, anti‐miR‐23b transfection in human normal AB2 dermal fibroblasts upregulated the expression of endogenous uPA and c‐met. Cotransfection experiments in HCC cells of the miR‐23b with pGL4.71 Renilla luciferase reporter gene constructs, containing the putative uPA and c‐met 3′‐UTR target sites, and with the pGL3 firefly luciferase‐expressing vector showed a decrease in the relative luciferase activity. This would indicate that miR‐23b can recognize target sites in the 3′‐UTR of uPA and of c‐met mRNAs and translationally repress the expression of uPA and c‐met in HCC cells. The evidence obtained shows that overexpression of miR‐23b leads to uPA and c‐met downregulation and to decreased migration and proliferation abilities of HCC cells.

Media: lumen ratio in human small resistance arteries is related to forearm minimal vascular resistance.

Background For the evaluation in humans of structural alterations in resistance arteries, most studies have used an indirect index, the measurement of minimal vascular resistance (mean blood pressure divided by maximal postischaemic blood flow) in suitable vascular beds. A sensitive and specific micromyographic technique was recently made available for the study of human small resistance arteries. Whether a correlation really exists between results obtained with the two techniques has not yet been investigated. Objective To evaluate both forearm minimal vascular resistance and media: lumen ratio of omental or subcutaneous small arteries in normotensive subjects and hypertensive patients. Design and methods Thirty-four individuals were included in the study (age range 35–74 years; 24 hypertensive, 10 normotensive). Twenty-five had elective abdominal surgery and nine hypertensive patients had a gluteal biopsy. Omental and subcutaneous small arteries were dissected and mounted on a wire micromyograph (Mulvanys technique), and media: lumen ratio and media thickness were measured. The dose-response curve to noradrenaline was constructed at cumulative concentrations from 3 ± 10–9 to 3 ± 10–5 mol/l. Venous occlusion plethysmography was used to measure blood flow in the forearm, and minimal vascular resistance was calculated from mean blood pressure and postischaemic maximal blood flow (13 min ischaemia plus exercise). Results A statistically significant correlation was found between media: lumen ratio and minimal vascular resistance (r = 0.74, P < 0.001) as well as between media: lumen ratio and systolic (r = 0.44, P < 0.01) and diastolic (r = 0.38, P < 0.05) blood pressures. Similar correlations were observed between media thickness and systolic and diastolic blood pressures. Small arteries from hypertensive patients had a significantly increased reactivity to noradrenaline (by analysis of variance) compared with those from normotensive subjects, in terms of wall tension but not of active media stress. Conclusions The present study demonstrated that the media: lumen ratio of small resistance vessels is significantly related to forearm minimal vascular resistance, suggesting that direct and indirect evaluations of vascular morphology will give similar results.

Long term effectiveness of resection and radiofrequency ablation for single hepatocellular carcinoma <= 3 cm. Results of a multicenter Italian survey.

BACKGROUND & AIMS The aim of this study was to compare liver resection and radiofrequency ablation in patients with single hepatocellular carcinoma ≤3 cm and compensated cirrhosis. METHODS The study involved 544 Child-Pugh A cirrhotic patients (246 in the resection group and 298 in the radiofrequency group) observed in 15 Italian centers. Overall survival and tumor recurrence rates were analyzed using the Kaplan Meier method before and after propensity score matching. Cox regression models were used to identify factors associated with overall survival and tumor recurrence. RESULTS Two cases of perioperative mortality were observed in the resection group and the rate of major complications was 4.5% in the resection group and 2.0% in the radiofrequency group (p=0.101). Four-year overall survival rates were 74.4% in the resection group and 66.2% in the radiofrequency group (p=0.353). Four-year cumulative HCC recurrence rates were 56% in the resection group and 57.1% in the radiofrequency group (p=0.765). Local tumor progression was detected in 20.5% of ablated patients and in one resected patient (p<0.001). After propensity score matching, both survival and tumor recurrence were still not significantly different although a trend towards lower recurrence was observed in resected patients. Older age and higher alpha-fetoprotein levels were independent predictors of poor overall survival while older age and higher alanine-aminotransferase levels resulted to be independent factors associated with higher recurrence rate. CONCLUSIONS In spite of a higher rate of local tumor progression, radiofrequency ablation can provide results comparable to liver resection in the treatment of single hepatocellular carcinoma ≤3 cm occurring in compensated cirrhosis.

A multicentric Western analysis of prognostic factors in advanced, node-negative gastric cancer patients

Background:The presence of lymph node metastasis is one of the most important prognostic factors in patients with gastric carcinoma. Node-negative patients have a better outcome, nevertheless a subgroup of them experience disease recurrence. Aim:To analyze the clinicopathological characteristics of lymph node-negative advanced gastric carcinoma patients submitted to gastrectomy and D2 lymphadenectomy with a retrieved number of nodes greater than 15, after an actual follow-up of almost 5 years, and to evaluate outcome indicators. Study Design:The records of 301 patients who underwent curative gastrectomy for gastric carcinoma and were adequately staged as N0 between 1992 and 2002 were retrospectively analyzed from the prospectively collected database of 7 centers participating to the Italian Research Group for Gastric Cancer. Results:Disease-specific and disease-free survival after 3, 5, and 10 years were 90.4%, 86.1%, 75.9%, and 72.1%, 57.3%, 57.3%, respectively. Mortality was 1.7%. The factors associated with a better disease-free survival at univariate analysis were age <60, T2 tumors, distal location, intestinal histotype, and number of retrieved nodes >25; depth of infiltration and histotype were the only 2 independent predictors of 5-year recurrence-free survival at multivariate analysis. Conclusion:These parameters must be considered to stratify node-negative gastric cancer patients for an adjuvant treatment and follow-up scheduling. Survival was similar to that previously reported by Eastern Centers. Lymphadenectomy is suggested to be effective, and retrieval of more than 25 nodes may be warranted.

u-PA and c-MET mRNA expression is co-ordinately enhanced while hepatocyte growth factor mRNA is down-regulated in human hepatocellular carcinoma

Hepatocyte growth factor/scatter factor (HGF/SF) is one of the most important humoral mediators of liver regeneration. It is potentially related to molecular mechanisms of hepatocarcinogenesis via a paracrine system involving its cellular receptor, c‐met. In this study, the expression patterns of HGF and c‐met were evidenced by multiplex RT‐PCR in different specimens of human hepatic tissues (n = 71). A significant increase of c‐met mRNA expression was detected in hepatitis (P = 0.001), cirrhosis (P = 0.006), and hepatocellular carcinoma (HCC) tissue (P = 0.003) compared with normal parenchyma and steatosis. HGF mRNA expression was significantly higher only in hepatitis (P = 0.01). Over‐expression of c‐met mRNA and under‐expression of HGF mRNA were detected in the HCCs compared with the corresponding peri‐tumoral tissues. Neither HGF nor c‐met expression was related to age, sex, tumor size, grading, presence of pseudocapsula, and proliferative activity of the malignant hepatocytes. A significant inverse correlation was found between c‐met mRNA expression level and survival (in months) of patients (P = 0.007), as previously shown for urokinase‐type plasminogen activator (u‐PA) mRNA (P = 0.027). In addition, c‐met mRNA expression was strictly associated with u‐PA mRNA level in HCC samples (P = 0.001). These data show that a loss of balance concerning HGF, c‐met, and u‐PA mRNA expression occurs during hepatocarcinogenesis. Particularly, up‐regulation of c‐met and u‐PA mRNA transcription appears to be coordinately regulated, and their levels of expression are inversely correlated with survival; they must therefore play an important role in the development and progression of human HCC and may also be relevant prognostic markers. Int. J. Cancer 87:644–649, 2000.

Relations Between Cardiac and Vascular Structure in Patients With Primary and Secondary Hypertension

BACKGROUND Data on cardiac and vascular structure in secondary hypertension are generally scarce, and no data on the interrelations between cardiac mass and structural characteristics of the vessel wall, both in large and in small resistance arteries, are presently available. OBJECTIVES The aim of this study was to investigate the relation between structural changes in subcutaneous small arteries, left ventricular mass and wall thickness of the common carotid artery in patients with primary and secondary hypertension. METHODS Seventy-four subjects were included in the study: 11 patients with pheochromocytoma, 14 with primary aldosteronism (PA), 19 with renovascular hypertension (RVH), 18 with essential hypertension (EH) and 12 normotensive (NT) control subjects. All subjects were submitted to a biopsy of subcutaneous fat. Morphologic characteristics of subcutaneous small resistance arteries (relaxed diameter <300 microm) were directly evaluated using a micromyographic technique. All subjects were submitted to calculation of left ventricular mass index (LVMI) and common carotid artery intima-media thickness (CCIMT), using ultrasound technique. RESULTS The correlation coefficients between the media to lumen ratio in subcutaneous small arteries (M/L) and LVMI or between M/L and CCIMT were closer in RVH than in pheochromocytoma, EH or NT; in PA the correlation coefficients were slightly less close than those in RVH. An excess prevalence of carotid plaques in RVH was observed. CONCLUSIONS A close relation between small resistance artery morphology and cardiac or carotid artery structure may be observed in those hypertensive patients in whom the renin-angiotensin-aldosterone system is activated. In constrast, in NT, EH and pheochromocytoma no significant correlation between M/L and LVMI or CCIMT was observed.

Endothelial dysfunction in small resistance arteries of patients with non-insulin-dependent diabetes mellitus

Objective Arterial hypertension is frequently associated with the presence of endothelial dysfunction in human subcutaneous small resistance arteries, as evaluated by responses to acetylcholine or bradykinin; however it is not known whether patients with diabetes mellitus show similar alterations. Therefore, we have investigated endothelial function in subcutaneous arteries of normotensive subjects (NT), of patients with essential hypertension (EH), of patients with non-insulin-dependent diabetes mellitus (NIDDM), as well as of patients with both essential hypertension and non-insulin-dependent diabetes mellitus (NIDDM + EH). Patients and methods All subjects were submitted to a biopsy of the subcutaneous fat. Small arteries were dissected and mounted on a micromyograph. The media to lumen ratio (M/L) was calculated. A concentration-response curve to acetylcholine, to bradykinin as well as to the endothelium-independent vasodilator sodium nitroprusside were performed. We also evaluated the contractile response to endothelin-1. Intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) plasma levels were also measured. Results The vasodilatation to acetylcholine and bradykinin (but not to sodium nitroprusside) was significantly and similarly reduced in EH, in NIDDM, and in NIDDM + EH compared with NT. The contractile response to endothelin-1 was similarly reduced in EH, in NIDDM and in NIDDM + EH. Plasma ICAM-1 and VCAM-1 concentrations were higher in EH, NIDDM and NIDDM + EH than in NT. Conclusions An evident endothelial dysfunction was detected in patients with NIDDM, and the simultaneous presence of EH did not seem to exert an additive effect. The contractile responses to endothelin-1 were reduced possibly as a consequence of ETA receptor down-regulation.