Andrea Mezzetti

EGFR Mutations in Non–Small-Cell Lung Cancer: Analysis of a Large Series of Cases and Development of a Rapid and Sensitive Method for Diagnostic Screening With Potential Implications on Pharmacologic Treatment

PURPOSE It has been reported that EGFR mutations in lung carcinomas make the disease more responsive to treatment with tyrosine kinase inhibitors. We decided to evaluate the prevalence of EGFR mutations in a large series of non-small-cell lung carcinomas (NSCLCs) and to develop a rapid and sensitive screening method. PATIENTS AND METHODS We examined 860 consecutive NSCLC patients for EGFR mutations in exons 18, 19, and 21 using a dual technical approach--direct sequencing of polymerase chain reaction (PCR) products and PCR single-strand conformation polymorphism (SSCP) analysis. Moreover, all lung adenocarcinomas were analyzed for K-ras mutations at codon 12 by allele-specific oligoprobe hybriditations. RESULTS There were no EGFR mutations in 454 squamous carcinomas and 31 large cell carcinomas investigated. Thirty-nine mutations were found in the series of 375 adenocarcinomas (10%). Mutations were present in 26% of 86 bronchioloalveolar carcinomas (BACs) and in 6% of 289 conventional lung adenocarcinomas; P = .000002. EGFR mutations and K-ras mutations were mutually exclusive. A multivariable analysis revealed that BAC histotype, being a never smoker, and female sex were independently associated with EGFR mutations (odds ratios: 4.542, 3.632, and 2.895, respectively). The SSCP analysis was accurate and sensitive, allowing identification of mutations that were undetectable (21% of cases) by direct sequencing. CONCLUSION Mutations in the EGFR tyrosine kinase domain define a new molecular type of lung carcinoma, more frequent in particular subsets of patients. The SSCP assay is a rapid and reliable method for the detection of EGFR kinase domain mutations in lung cancer.

In Vivo Formation of 8-Iso-Prostaglandin F2α and Platelet Activation in Diabetes Mellitus Effects of Improved Metabolic Control and Vitamin E Supplementation

Background—Diabetes mellitus (DM) is associated with enhanced lipid peroxidation and persistent platelet activation. We tested the hypothesis that the in vivo formation of the F2-isoprostane 8-iso-prostaglandin (PG)F2α, a bioactive product of arachidonic acid peroxidation, is enhanced in DM and contributes to platelet activation. Methods and Results—Urine samples were obtained from 85 diabetic patients and 85 age- and sex-matched healthy subjects for measurement of immunoreactive 8-iso-PGF2α and 11-dehydro-thromboxane B2 (TXM), an in vivo index of platelet activation. Sixty-two had non–insulin-dependent (NID)DM, and 23 had insulin-dependent (ID) DM. Vitamin E supplementation, metabolic control, and cyclooxygenase inhibitors were used to investigate the mechanisms of formation of 8-iso-PGF2α in this setting. Urinary 8-iso-PGF2α excretion was significantly higher (P=0.0001) in NIDDM patients (419±208 pg/mg creatinine; range 160 to 1014) than in age-matched control subjects (208±92; 41 to 433). Urinary 8-iso...

In Vivo Formation of 8-Epi-Prostaglandin F2α Is Increased in Hypercholesterolemia

Abstract F2-isoprostanes are bioactive prostaglandin (PG) -like compounds that are produced from arachidonic acid through a nonenzymatic process of lipid peroxidation catalyzed by oxygen free-radicals. 8-Epi-PGF2α may amplify the platelet response to agonists, circulates in plasma, and is excreted in urine. We examined the hypothesis that the formation of 8-epi-PGF2α is altered in patients with hypercholesterolemia and contributes to platelet activation in this setting. Urine samples were obtained from 40 hypercholesterolemic patients and 40 age- and sex-matched control subjects for measurement of immunoreactive 8-epi-PGF2α. Urinary excretion of 11-dehydro-thromboxane (TX) B2, a major metabolite of TXA2, was measured as an in vivo index of platelet activation. Low-dose aspirin, indobufen, and vitamin E were used to investigate the mechanism of formation and effects of 8-epi-PGF2α on platelet activation. Urinary 8-epi-PGF2α was significantly ( P =.0001) higher in hypercholesterolemic patients than in control subjects: 473±305 versus 205±95 pg/mg creatinine. Its rate of excretion was inversely related to the vitamin E content of LDL and showed a positive correlation with urinary 11-dehydro-TXB2. Urinary 8-epi-PGF2α was unchanged after 2-week dosing with aspirin and indobufen despite complete suppression of TX metabolite excretion. Vitamin E supplementation was associated with dose-dependent reductions in both urinary 8-epi-PGF2α and 11-dehydro-TXB2 by 34% to 36% and 47% to 58% at 100 and 600 mg daily, respectively. We conclude that the in vivo formation of the F2-isoprostane 8-epi-PGF2α is enhanced in the vast majority of patients with hypercholesterolemia. This provides an aspirin-insensitive mechanism possibly linking lipid peroxidation to amplification of platelet activation in the setting of hypercholesterolemia. Dose-dependent suppression of enhanced 8-epi-PGF2α formation by vitamin E supplementation may contribute to the beneficial effects of antioxidant treatment.

The Receptor RAGE as a Progression Factor Amplifying Arachidonate-Dependent Inflammatory and Proteolytic Response in Human Atherosclerotic Plaques Role of Glycemic Control

Background—RAGE (receptor for advanced glycation end products [AGEs]) plays a role in diabetic atherosclerosis. Recently, we have demonstrated enhanced expression of cyclooxygenase-2 and PGE synthase-1 (COX-2/mPGES-1) in human symptomatic plaques, and provided evidence that it is associated with metalloproteinase (MMP)-induced plaque rupture. However, the specific transmembrane signaling pathway(s) influencing plaque COX-2/mPGES-1 expression is unknown. The aim of this study was to characterize RAGE expression in human plaques and to correlate it with the inflammatory infiltration, COX-2/mPGES-1 and MMP expression, and with clinical evidence of diabetes. Methods and Results—Plaques obtained from 60 patients undergoing carotid endarterectomy were divided into diabetic and nondiabetic according to clinical evidence of type 2 diabetes. Plaques were subjected to analysis of RAGE, NF-&kgr;B, COX-2/mPGES-1, MMP-2 and MMP-9, lipid and oxidized LDL (oxLDL) content, and collagen content by immunohistochemistry and Western blot, whereas zymography was used to detect MMP activity. Immunohistochemistry was used to identify CD68+ macrophages, CD3+ T-lymphocytes, smooth muscle cells (SMCs), and HLA-DR+ inflammatory cells. Diabetic plaques had more (P <0.0001) macrophages, T-lymphocytes, and HLA-DR+ cells, more (P <0.0001) immunoreactivity for RAGE, activated NF-&kgr;B, COX-2/mPGES-1, and MMPs, increased (P <0.0001) gelatinolytic activity, reduced (P <0.0001) collagen content, and increased (P <0.0001) lipid and oxLDL content. Interestingly, RAGE, COX-2/mPGES-1, and MMP expression was linearly correlated with plasma level of HbA1c. Conclusions—In conclusion, this study demonstrates in humans that RAGE overexpression is associated with enhanced inflammatory reaction and COX-2/mPGES-1 expression in diabetic plaque macrophages, and this effect may contribute to plaque destabilization by inducing culprit metalloproteinase expression.

Intermittent antegrade warm blood cardioplegia

Intermittent antegrade warm blood cardioplegia has been used routinely at our institution over the last 3 years. We report here a comparison between the first 250 consecutive patients undergoing elective coronary artery bypass grafting in which intermittent antegrade warm blood cardioplegia was used (group A) and the last 250 consecutive patients who received intermittent antegrade cold blood cardioplegia, during bypass grafting (group B). There were no differences in sex, age, number of grafts, and functional status between the two groups; left ventricular ejection fraction was lower in group A. The overall mortality rate in group A was 0.8% versus 3.6% in group B (p < 0.05). There was no in-hospital mortality among high-risk patients (ejection fraction < or = 0.35) in group A (0/53) versus two deaths in group B (2/28) (p < 0.05). No patient in group A needed circulatory assistance; 4 patients in group B received intraaortic balloon pumping. Only 1 patient in group A required inotropic support versus 20 patients in group B (p < 0.0005), and 5 patients in group A received lidocaine hydrochloride for ventricular arrhythmias versus 18 in group B (p < 0.01). The rates of myocardial infarction and stroke were not different between the two groups. The peak concentration of the myocardial-specific isoenzyme of creatine kinase were higher in group B in absolute value (51 +/- 30 IU/L) than in Group A (38 +/- 38 IU/L) (p < 0.0005) and in percent of total creatine kinase (8.2% +/- 4.1% versus 6.2% +/- 2.9%, respectively).

Association between enhanced soluble CD40L and prothrombotic state in hypercholesterolemia : Effects of statin therapy

Background—Hypercholesterolemia is associated with inflammation and the prothrombotic state. CD40-CD40 ligand (CD40L) interactions promote a prothrombotic response in nucleated cells. The aim of this study was to characterize the in vivo expression of soluble CD40L (sCD40L) in hypercholesterolemia, to correlate it with the extent of the prothrombotic state, and to investigate whether it may be modified by statins. Methods and Results—We studied 80 hypercholesterolemic patients and 80 matched healthy subjects. Hypercholesterolemic subjects had enhanced levels of sCD40L, factor VIIa (FVIIa), and prothrombin fragment 1+2 (F1+2) compared with healthy subjects. sCD40L correlated with total cholesterol and LDL cholesterol. Moreover, sCD40L was positively associated with in vivo platelet activation, as reflected by plasma P-selectin and urinary 11-dehydro-thromboxane B2, and with procoagulant state, as reflected by FVIIa and F1+2. Inhibition of cholesterol biosynthesis by pravastatin or cerivastatin was associated with comparable, significant reductions in sCD40L, FVIIa, and F1+2. Conclusions—This study suggests that sCD40L may represent the molecular link between hypercholesterolemia and the prothrombotic state and demonstrates that statin therapy may significantly reduce sCD40L and the prothrombotic state.

Vitamins E, C and lipid peroxidation in plasma and arterial tissue of smokers and non-smokers

An imbalance between pro-oxidants and antioxidants is operative in atherosclerosis. Cigarette smoke is a major risk factor of atherosclerosis and has been reported to contain large amounts of oxidants. We assessed arterial (internal mammary artery) and plasma levels of vitamins E and C and lipid peroxides in 48 male patients, 24 smokers and 24 non-smokers, undergoing coronary bypass surgery. Lipid peroxidation was studied using fluorescent products of lipid peroxidation (FPLs). Tissue vitamins E and C levels were significantly lower and FPLs significantly higher in smokers than in non-smokers (P < 0.0006, 0.0005 and 0.0005, respectively). This pattern was associated with lower vitamin C and higher lipid peroxide plasma levels in smokers than in non-smokers (P < 0.0002 and 0.0005, respectively). Vitamins E and C plasma levels were strongly related to their tissue content both in smokers (r = 0.60, P < 0.005 and r = 0.57, P < 0.01) and in non-smokers (r = 0.42, P < 0.05 and r = 0.46, P < 0.05). Moreover, vitamin E content was significantly related to that of vitamin C only in the arterial tissue of both groups, pointing to the existence of a functional interaction between these antioxidants. In both groups, FPLs were significantly and inversely related to vitamin C in plasma and to vitamin E in tissue, suggesting the antioxidant primary of vitamin C and vitamin E in the plasma and arterial tissue compartments, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Glutathione-Related Antioxidant Defenses in Human Atherosclerotic Plaques

BACKGROUND Oxidative stress, resulting from an antioxidant/prooxidant imbalance, seems to be crucial in atherogenesis. Recent evidence has emerged, however, of a surprisingly high content of low-molecular-weight antioxidants in human atherosclerotic plaques, although other antioxidant systems have not been investigated in these lesions. METHODS AND RESULTS We studied glutathione-related antioxidant defenses (which play a key role in tissue antioxidant protection) in carotid atherosclerotic plaques of 13 patients subjected to endarterectomy and in normal internal mammary arteries of 13 patients undergoing coronary artery bypass surgery. Selenium-dependent glutathione peroxidase activity was undetectable in the plaques of 7 patients; the other 6 patients with plaques showed a mean enzymatic activity approximately 3.5-fold lower than that of mammary arteries. Glutathione reductase activity was also markedly lower in the plaques than in the arteries. Glutathione transferase instead had comparable activity in the two tissues. Remarkably, 5 of the 7 patients with an undetectable selenium-dependent glutathione peroxidase activity but none of the 6 with a detectable one were characterized by multivascular atherosclerotic involvement (3 patients) or stenosis of the contralateral carotid artery (2 patients). CONCLUSIONS A weak glutathione-related enzymatic antioxidant shield is present in human atherosclerotic lesions. Although the cause of this phenomenon remains to be determined, the present data suggest that a specific antioxidant/prooxidant imbalance operative in the vascular wall may be involved in atherogenic processes in humans.

Increased Levels of Soluble P-Selectin in Hypercholesterolemic Patients

BACKGROUND Hypercholesterolemia is considered a major risk factor for the development of atherosclerosis. Enhanced lipid peroxidation and persistent platelet activation can be observed in vivo in hypercholesterolemic patients and may have pathophysiological implications in the occurrence of cardiovascular events. P-selectin may play an important role in the pathogenesis of multicellular events, including atherosclerosis. We studied the impact of hypercholesterolemia and oxidative stress on plasma levels of P-selectin. METHODS AND RESULTS Plasma levels of P-selectin were measured by means of an enzyme immunoassay in 20 hypercholesterolemic patients with no clinical evidence of cardiovascular disease and in 20 sex- and age-matched normocholesterolemic subjects. Hypercholesterolemic patients had higher levels of P-selectin compared with that of control subjects (98+/-61 versus 56+/-14 ng/mL; P=.001). They also displayed increased von Willebrand Factor (vWF) levels (176+/-22 versus 119+/-12%; P=.0001). A direct correlation was observed between P-selectin and LDL cholesterol levels (p=.453). Administration of vitamin E (600 mg/d for 2 weeks) to hypercholesterolemic patients significantly reduced plasma P-selectin (40%), and an inverse correlation was observed between vitamin E and P-selectin plasma levels (p=-.446). CONCLUSIONS Hypercholesterolemia is associated with elevated plasmatic P-selectin. Altered oxidative processes leading to endothelial dysfunction and persistent platelet activation may contribute to increased soluble P-selectin levels. P-selectin may be proposed as a marker of endothelial dysfunction in hypercholesterolemic patients.

Oxidative stress and cardiovascular complications in diabetes: isoprostanes as new markers on an old paradigm

Long-term vascular complications still represent the main cause of morbidity and mortality in diabetic patients. Although randomized long-term clinical studies comparing the effects of conventional and intensive therapy have demonstrated a clear link between hyperglycemia and the development of complications of diabetes, they have not defined the mechanism through which excess glucose results in tissue damage. Evidence has accumulated indicating that oxidative stress may play a key role in the etiology of diabetic complications. Isoprostanes are emerging as a new class of biologically active products of arachidonic acid metabolism of potential relevance to human vascular disease. Their formation in vivo seems to reflect primarily, if not exclusively, a nonenzymatic process of lipid peroxidation. Enhanced urinary excretion of 8-iso-PGF(2alpha) has been described in association with both type 1 and type 2 diabetes mellitus, and correlates with impaired glycemic control. Besides providing a likely noninvasive index of lipid peroxidation in this setting, measurements of specific F(2) isoprostanes in urine may provide a sensitive biochemical end point for dose-finding studies of natural and synthetic inhibitors of lipid peroxidation. Although the biological effects of 8-iso-PGF(2alpha) in vitro suggest that it and other isoeicosanoids may modulate the functional consequences of lipid peroxidation in diabetes, evidence that this is likely in vivo remains inadequate at this time.