What do we need to obtain high quality circulating tumor DNA (ctDNA) for routine diagnostic test in oncology? - Considerations on pre-analytical aspects by the IFCC workgroup cfDNA.

The analysis of circulating cell free DNA is an important tool for the analysis of tumor resistance, tumor heterogeneity, detection of minimal residual disease and detection of allograft rejection in kidney or heart transplant patients. The proper use of this technique is important, and starts with considering pre-analytic aspects. The current paper addresses some important technical considerations to ensure the proper and harmonized use of cfDNA techniques.

Psychological and adjustment problems due to acquired brain lesions in childhood: a comparison between post-traumatic patients and brain tumour survivors.

OBJECTIVE: To define and differentiate psychological and adjustment problems due to brain injury or brain tumour in children and adolescents. METHODS: Two groups of patients with acquired brain lesions (24 post-traumatic patients and 22 brain tumour survivors), ranging in age between 8-15 years, received a psychological evaluation, including the Child Behaviour Checklist for Ages 4-18 (CBCL) and the Vineland Behaviour Adaptive Scales (VABS). RESULTS: Both groups showed psychological and social adjustment problems. Post-traumatic patients were more impaired than brain tumour survivors. Social adjustment problems were associated to externalizing problems in post-traumatic patients and internalizing problems in brain tumour surviving patients. CONCLUSIONS: These differences in psychological and behavioural disorders between the two groups must necessarily be considered when developing psychological treatment, rehabilitation plan and social re-entry.

Extracorporeal photochemotherapy reduces the severity of Lewis rat experimental allergic encephalomyelitis through a modulation of the function of peripheral blood mononuclear cells.

Extra corporeal photochemotherapy (ECP) is an immunomodulating procedure used in several nonneurological diseases which, similarly to multiple sclerosis, are likely to be due to T-cell-mediated autoimmunity and it is probable that ECP can modulate the normal activity of peripheral blood mononuclear cells (PBMC). Using the Lewis rat experimental allergic encephalomyelitis (EAE) model of human multiple sclerosis (MS) we examined the effect of extracorporeal UV-A irradiation on psoralen-activated PBMC. In our experiment the comparison between the two groups of animals (ECP or sham-treatment) evidenced that the ECP treatment reduced the severity of EAE on clinical grounds and this result was confirmed by the pathological examination. The changes in the titers of anti-myelin antigen antibodies typical of EAE were also modulated by the procedure. Ex vivo examination evidenced a significant reduction in tumor-necrosis factor-alpha (TNF-alpha) released by PBMC after lipopolysaccharides (LPS) stimulation in culture. We conclude that ECP modifies the normal activity of PBMC during the course of EAE and it is possible that one of the anti-inflammatory mechanisms of action of ECP is correlated to a down-regulation of T-helper 1 lymphocytes activity.

Pixantrone (BBR2778) reduces the severity of experimental allergic encephalomyelitis.

Pixantrone is less cardiotoxic and is similarly effective to mitoxantrone (MTX) as an antineoplastic drug. In our study, pixantrone reduced the severity of acute and decreased the relapse rate of chronic relapsing experimental allergic encephalomyelitis (EAE) in rats. A marked and long-lasting decrease in CD3+, CD4+, CD8+ and CD45RA+ blood cells and reduced anti-MBP titers were observed with both pixantrone and MTX. In vitro mitogen- and antigen-induced T-cell proliferation tests of human and rodents cells evidenced that pixantrone was effective at concentrations which can be effectively obtained after i.v. administration in humans. Cardiotoxicity was present only in MTX-treated rats. The effectiveness and the favorable safety profile makes pixantrone a most promising immunosuppressant agent for clinical use in multiple sclerosis (MS).

A CAV3 microdeletion differentially affects skeletal muscle and myocardium.

BACKGROUND: Caveolin-3 is the muscle-specific protein product of the caveolin gene family and an integral membrane component of caveolae. Mutations in the gene encoding caveolin-3 (CAV3) underlie four distinct disorders of skeletal muscle: the autosomal dominant form of limb-girdle muscular dystrophy type 1C (LGMD-1C), rippling muscle disease (RMD), sporadic and familial forms of hyperCKemia, and distal myopathy. OBJECTIVE: To characterize a multigenerational Italian family affected by an autosomal dominant myopathic disorder and to assess the expression of caveolin-3, dystrophin, dystrophin-associated glycoproteins, and neuronal nitric oxide synthase in the myocardium of an affected patient. METHODS: Clinical analysis involved 15 family members. Skeletal muscle expression of sarcolemmal proteins was evaluated by immunohistochemistry and western blot analysis in three affected individuals. Caveolar structures were analyzed through electron microscopy in muscle biopsies and in one heart biopsy. RESULTS: CAV3 genetic analysis showed a heterozygous 3-bp microdeletion (328-330del) in affected individuals, resulting in the loss of a phenylalanine (Phe97del) in the transmembrane domain. In the skeletal muscle, the mutation was associated with severe caveolin-3 deficiency and caveolar disorganization, whereas the expression of the other analyzed muscle proteins was unaltered. Remarkably, caveolin-3 was expressed in myocardium at a level corresponding to about 60% of that of control individuals and was correctly localized at the myocardial cell membranes, with preservation of cardiac myofiber caveolar structures. Clinical analysis revealed the concomitant presence in this family of the following phenotypes: RMD, LGMD, and hyperCKemia. CONCLUSIONS: Intrafamilial phenotypic heterogeneity is associated with caveolin-3 Phe97 microdeletion. The molecular network interacting with caveolin-3 in skeletal muscle and heart may differ.

Circulating nerve growth factor level changes during oxaliplatin treatment-induced neurotoxicity in the rat.

BACKGROUND: Oxaliplatin neurotoxicity represents a clinically-relevant problem and its etio-pathogenesis is still unknown. We explored the possible role of some neuronal growth factors ("neurotrophins") during the course of oxaliplatin sensory neuronopathy. MATERIALS AND METHODS: In our rat model two different doses of oxaliplatin were used (2 and 3 mg/kg i.v. twice weekly for 9 times). The neurotoxicity of the treatment was assessed with neurophysiological and pathological methods and serum neurotrophin levels were measured by ELISA. RESULTS: Both oxaliplatin-treated groups showed the neurophysiological and neuropathological changes which mimic the chronic effects of oxaliplatin administration in humans, e.g. reversible sensory impairment due to dorsal root ganglia neuron damage. These changes were associated with a significant and dose-dependent reduction only in the circulating level of nerve growth factor (NGF), which returned to normal values after neurophysiological and pathological recovery. CONCLUSION: This specific association between neurological impairment and NGF modulation indicates that NGF impairment has a role in the neurotoxicity of oxaliplatin.

p53, cyclin-D1, PCNA, AgNOR expression in squamous cell cancer of the lip: a multicenter study.

BACKGROUND: The development of squamous cell carcinoma of the lower lip is an interesting model of photocarcinogenesis because of the structural and topographic characteristics of the lips. The purpose of this study was to evaluate the expression of immunohistochemical markers on the lips of patients with lower lip squamous cell carcinoma (LLSCC), compared with a control population. METHODS: Of the 98 subjects involved in the study, 58 were suffering from squamous cell carcinoma of the lower lip. The remaining 40 acted as a control. The case studies were taken from six university and hospital dermatology and plastic surgery departments. Questionnaires were administered to assess the risk factors for LLSCC. The cases involving squamous cell carcinoma underwent surgical excision and punch biopsy specimens were obtained from 20 control patients. Tissues were prepared in 5-microm-thick sections to carry out the following immunohistochemical study: PCNA, p53, AgNOR, cyclin-D1, bcl-2. RESULTS: The lower lip was the predominant location of squamous cell carcinoma, with the following factors playing important roles: chronic sun exposure, history of smoking, alcohol use and familial risk of cutaneous tumors. The male/female ratio in our survey was 5:1. The p53 protein was positive in approximately 50% of SCC cases and in 20% of controls. This protein is mostly associated with chronically photoexposed skin areas. AgNOR positivity increased with the loss of cellular differentiation; a progressive increase in size and a poorly defined shape were evident in poorly differentiated carcinomas. CONCLUSIONS: The results of this multicenter study showed that there is a noticeable difference in the expression of PCNA, p53, cyclin-D1, and AgNOR in tissues from patients with LLSCC and controls.

Inhibition of basophil histamine release by tyrosine kinase and phosphatidylinositol 3-kinase inhibitors.

It has been demonstrated that tyrosine kinase (TK) and phosphatidylinositol 3-kinase (PI3-K) are involved in IgE-mediated stimulation of human basophils; conversely, little is known about the biochemical pathways activated by IL-3 and GM-CSF. The aim of this study was to evaluate the effects of TK and PI3-K inhibitors on basophil histamine release induced by anti-IgE, IL-3 and GM-CSF. Since IL-3 and GM-CSF cause histamine release from normal human basophils only when the inhibitory effect of extracellular Na(+) has been removed, peripheral blood leukocytes were suspended in isotonic solutions containing either 140 mM NaCl or 140 mM N-methyl-D-glucamine(+). After stimulation with anti-IgE, IL-3 or GM-CSF, histamine release was measured by an automated fluorometric method. The effects of preincubation with four different TK inhibitors (AG-126, genistein, lavendustin A, tyrphostin 51) and one PI3-K inhibitor (wortmannin) were evaluated. AG-126, genistein and lavendustin A exerted a significant dose-dependent inhibitory effect on basophil histamine release induced by anti-IgE (either in high or in low Na(+) medium), IL-3 and GM-CSF. Among the TK inhibitors, lavendustin A exerted the most potent activity, followed by AG-126 and genistein. Tyrphostin 51 caused a weak inhibition of histamine release induced by IL-3, GM-CSF and anti-IgE in a low Na(+) medium, but not in a physiological Na(+)-containing medium. The PI3-K inhibitor wortmannin exerted the most effective inhibitory activity on the histamine release induced by the three agonists. The combined effects of lavendustin A and wortmannin were less than additive, suggesting that TK and PI3-K are involved in the same activation pathway in human basophils. These results suggest a possible role of TK and PI3-K in basophil histamine release induced by anti-IgE, IL-3 and GM-CSF. TK and PI3-K are indeed potential therapeutic targets for antiallergic drugs.

Resveratrol, map kinases and neuronal cells: might wine be a neuroprotectant?

Alcohol is noxious to the brain and peripheral nervous system. However, wine contains substances that may have positive biological and pharmacological effects. Resveratrol is the most studied and probably the most active of these substances. This naturally occurring compound, which is present in wine and grapes, reduces oxidative stress in neuronal-like cell cultures. We have shown that resveratrol induces phosphorylation of the mitogen-activated protein (MAP) kinase family members, extracellular regulated kinase 1 (ERK1) and ERK2, in the human neuroblastoma SH-SY5Y cells in vitro at much lower concentrations than those found in the plasma of rats after oral wine administration. MAP kinases are involved in numerous different aspects of signal transduction in the cells. In particular, phosphorylation of ERK2 has been related to the synaptic changes at the basis of memory and learning processes. These findings, together with our own, on resveratrol-induced activation of MAP kinases in human neuronal-like cells, and previously published epidemiological studies which have demonstrated an inverse relationship between moderate wine intake and dementia, suggest that wine (not alcohol) may have a positive effect on nervous cells.

Clinical correlations in 16 patients with total or partial laminin alpha2 deficiency characterized using antibodies against 2 fragments of the protein.

BACKGROUND: Many patients with classic congenital muscular dystrophy have been found to have partial or total deficiency of the alpha2 chain of laminin 2 (merosin). This deficiency has mostly been studied using only 1 antibody against a fragment of the protein. OBJECTIVES: To characterize the expression of laminin alpha2 in the skeletal muscle of patients with laminin alpha2 deficiency using antibodies against 2 different portions of the protein and to correlate the immunochemical findings with clinical phenotype. METHODS: We studied 4 patients with total lack of laminin alpha2 and 12 with partial laminin alpha2 deficiency with immunohistochemical techniques and Western blot analysis. We used antibodies recognizing an 80-kd fragment toward the C-terminus and a 300-kd fragment toward the amino-terminal. Patient characteristics examined were functional compromise, magnetic resonance imaging or computed tomography of the brain, electromyography, evoked potentials, and creatine kinase levels. RESULTS: In 4 patients, immunohistochemical analysis revealed no reactivity to either antibody; in 2 patients, the 300-kd fragment alone was partially expressed; in 2 patients, the 80-kd fragment alone was partially expressed; and in 8 patients, both fragments were partially expressed. Immunoblot analysis revealed bands of reduced intensity and normal molecular weight generally corresponding to the immunohistochemical findings. Absence of both fragments or of one with reduction of the other always produced a severe clinical phenotype, while a milder clinical phenotype was observed when both fragments were partially expressed. CONCLUSIONS: Extent of laminin alpha2 deficiency in most cases correlates with clinical phenotype but not with peripheral and central white matter abnormalities. Skin biopsy specimens may reveal laminin alpha2 deficiency in patients who have normal laminin alpha2 levels in muscle biopsy specimens.