Electrochemotherapy: an effective local treatment of cutaneous and subcutaneous melanoma metastases.

Treatment of multiple cutaneous and subcutaneous melanoma metastases is still represents a therapeutic challenge for both dermatologists and oncologists. Electrochemotherapy (ECT) is a promising therapeutic procedure, owing to its ability to improve the penetration of cytotoxic drugs into cancer cells by application of current electric pulses. The aim of our study is to evaluate efficacy, tolerability and long-term efficacy of ECT in the treatment of advanced metastatic melanoma. Thirty patients affected by a total of 654 cutaneous and subcutaneous melanoma metastatic nodules were recruited. All patients were treated after they had undergone to a mild general anesthesia. Intravenous Bleomicina solution was administered 8 minutes before the application of electric pulses, generated by a Cliniporator (TM) (the device validated for ECT). The objective response rate of 100% (67.28% complete response and 32.72% partial response) was observed. A total of 214 metastatic lesions from 24 patients received a second ECT session, among them 141 showed a further complete response. Twenty-four months later, the local tumor control rate was 72%. The results of this study seem to demonstrate that ECT is an effective and valid therapeutic tool for the treatment of cutaneous metastases from melanoma. ECT can be considered a first-line palliative treatment since it is able to alleviate pain and reduce the tumor's spontaneous bleeding with a significant improve of patients' quality of life.

Involvement of E-cadherin, beta-catenin, Cdc42 and CXCR4 in the progression and prognosis of cutaneous melanoma.

BACKGROUND: A key event in cancer metastasis is the migration of tumour cells from their original location to a secondary site. The development of melanoma may be viewed as a consequence of the disruption of homeostatic mechanisms in the skin of the original site. OBJECTIVES: To investigate whether dysregulation of cell motility (Cdc42 expression), escaping the control of cell-cell and cell-matrix interactions (E-cadherin, beta-catenin expression), enhances melanoma progression, and whether chemokine receptors (CXCR4) mediate cell migration and activation during invasion and metastasis development. METHODS: The immunohistochemical expression of Cdc42, E-cadherin, beta-catenin and CXCR4 was investigated in 30 patients with surgically treated nodular melanoma, 18 alive and disease free and 12 with a fatal outcome due to metastatic disease. RESULTS: E-cadherin expression was significantly reduced (P < 0.05) and cytoplasmic beta-catenin was increased in the patients who had died compared with disease-free individuals, while membrane expression of beta-catenin was similar in the two groups. Patients with fatal outcome had increased Cdc42 (P < 0.01) and CXCR4 (P < 0.05). In this group a positive correlation was found between melanocytic Cdc42 expression and Breslow thickness (r = 0.598, P < 0.05) and between CXCR4 expression and Breslow thickness (r = 0.583, P < 0.05). CONCLUSIONS: Findings suggest that primary cutaneous melanoma with a high Breslow thickness is characterized by tumour cells with high motility and invasion ability, in line with the hypothesis that low E-cadherin levels and overexpression of Cdc42 and CXCR4 could be prognostic markers of poor outcome.

Immunohistochemical study of apoptosis markers and involvement of chemokine CXCR4 in skin Merkel cell carcinoma.

BACKGROUND: Merkel cell carcinoma (MCC) is a rare, aggressive cancer of the skin that mainly affects elderly patients. Because of its rarity, there is no established treatment or proven markers to guide therapy or prognosis. Immunohistochemical expression of apoptosis proteins is considered a useful marker of both malignancy and tumour progression. Apoptosis plays a fundamental role in skin homeostasis, and apoptotic cells have been detected in normal and diseased skin. Chemokines possess a wide range of biological activities and CXCR4 is expressed in some cancer cells, where it plays an efficient role in metastasis formation. OBJECTIVE: To identify immunohistochemical parameters that can help clinicians select the most suitable therapy for skin MCC. DESIGN: Antibodies against ki67, bcl-2, p53, survivin, p16 and CXCR4 were tested to assess the usefulness of these antigens as indices of proliferation potential and predictors of prognosis. METHODS: Immunohistochemical detection of apoptosis inhibitors and CXCR4 was performed on tissue from 12 patients with primary MCC. After excision of the primary lesion, five survived and had no metastases, and seven experienced local recurrence or lymph node metastases. RESULTS: Expression of ki67 and survivin was increased in patients with local recurrence or metastasis (retrospectively classified as 'poor prognosis') compared with those with a 'good prognosis', and bcl-2 expression was significantly greater (P=0.003). P53 and p16 immunostaining was moderate in both groups. A positive correlation was observed between survivin and mutant p53 in the poor prognosis group (r=0.593, P=0.033; regression coefficient). High values of p53 were measured in patients with high levels of survivin and vice versa. CXCR4 was not detected at all. CONCLUSIONS: Our results show strong MCC cell apoptosis inhibition and a high cell proliferation capacity. The positive correlation between survivin and p53 may be a predictor of MCC spread via the lymphatic network. Absent CXCR4 expression may reflect a less aggressive form, with less efficient development of distant and non-organ-selective metastasis formation.

Spectrum and distribution of MECP2 mutations in 64 Italian Rett syndrome girls: tentative genotype/phenotype correlation.

We report a direct DNA sequencing analysis of the MECP2 gene undertaken on a further 64 Italian patients with Rett syndrome by using a LICOR 4200 Automated Sequencer. All of the girls entering the study had a consistent clinical diagnosis for this disorder. All coding regions and the flanking intronic splice site sequences were amplified as three non-overlapping fragments by using both forward and reverse primers. The results were then compared to the MECP2 reference sequences published in GenBank. Mutations of the MECP2 gene were identified in 64 of 75 (85.33%) unrelated sporadic Rett syndrome girls. Genotype/phenotype correlation studies, in particular in groups of patients with the same mutation, did not offer definitive and interesting data.

rhVEGF and experimental rat skin flaps: systemic or local administration and morphological characteristics.

Skin flap survival is a significant problem in skin surgery; in particular, inadequate arterial or venous blood supply results in necrosis of the distalmost portion. The aim of this study was to evaluate the ability of Vascular Endothelial Growth Factor (VEGF) of modifying the morphological features of skin flaps. Bilateral epigastric skin flaps were raised in 16 Wistar male rats. The epigastric artery and vein of the left flaps were clamped and then injected with rhVEGF (8 rats) or saline (8 rats). The right flaps were not clamped and received rhVEGF or saline systemically. The rats were euthanized on the seventh day and flap skin samples collected. Tissue fragments were subject to immunohistochemical (rhVEGF, VEGFr, VIII factor, CD34 antibodies), ultrastructural and morphostructural investigations. The results showed that rhVEGF improved the condition of flaps and that systemic administration was effective in promoting the development of an adequate vascular network.

Low dose interferon-alpha2b combined with PUVA is an effective treatment of early stage mycosis fungoides: results of a multicenter study. Cutaneous-T Cell Lymphoma Multicenter Study Group.

BACKGROUND AND OBJECTIVE: The early stages of mycosis fungoides (MF) can be treated but not cured by photochemotherapy (PUVA) alone; some recent studies of the effect of a combination of human interferon-alpha (IFN(alpha)) and PUVA reported a high degree of response. The aim of our study was to evaluate the activity of a low dose of IFN-alpha2b combined with PUVA. DESIGN AND METHODS: Twenty-five patients were included: 16 men and 9 women aged between 23-80 years; 19 patients ahd stage I and 6 stage II disease. In the induction phase, the dose of IFNalpha was gradually raised over 6-8 weeks to the target dose of 18 MU/week; in the maintenance phase, the combination with PUVA allowed IFNalpha to be reduced to a maximum dose of 6 MU/week; in this way the cumulative administration of IFNalpha and PUVA was considerably lower than in similar combination protocols. Treatment success was analyzed in terms of freedom from treatment failure (FFTF). RESULTS: After the induction phase 9/25 patients (36%) achieved complete remission (CR) and 15/25 (56%) achieved partial remission (PR). One to five months from the beginning of the maintenance phase, a CR was recorded in 19/25 patients (76%) and a PR in 5/25 patients (20%) accounting for an overall response rate of 96%. The median of FFTF was not reached; probability of FFTF was 82% at 12 months and 62% at 24 months. Disease free survival projected to 48 months was 75%. INTERPRETATION AND CONCLUSIONS: Even with low doses of IFNalpha plus PUVA it is possible to achieve excellent clinical responses,many of which are long-lasting, in patients with early MF.

Premature ovarian failure (POF) and fragile X premutation females: from POF to to fragile X carrier identification, from fragile X carrier diagnosis to POF association data.

Early menopause in the fragile X carriers has been well documented in several reports. All surveys demonstrated that 13-25% of fragile X carriers experienced premature ovarian failure (POF), defined as menopause before the age of 40 years. In 1995 we started screening two groups of subjects as a part of a Fragile X Research Program: 1) women previously diagnosed as fragile X carriers from the register of our center and 2) women with POF and without a family history of fragile X or other forms of mental retardation. In this study we report the preliminary data collected from 75 fragile X families; in 30 of them, POF was present in one or several subjects, all of whom had a fragile X premutation. None of the women with a full mutation experienced POF in our series of patients. We also identified 89 families without a family history of fragile X or mental retardation, and there were 108 subjects who experienced POF, of which 6.5% had a fragile X premutation. This is 70-fold higher than the background prevalence of fragile X premutation in the Italian population and suggests an association with POF. These data confirm the results of other surveys.

Advances in the understanding of malignant transformation of keratinocytes: an immunohistochemical study.

AIM: We set out to investigate the interactions between malignant transformation of keratinocytes, presence of oncoproteins and immunosurveillance in squamous cell carcinoma (SCC) and in a preneoplastic lesion, actinic keratosis (AK). METHODS: Samples of SCC, AK and normal skin (NS) were subjected to quantitative analysis using the following antibodies: anti-p53, Ki67, OKT6, OK-DR, B7/BB1, anti-CD54, anti-CD11, OKT3, OKT4, OKT8; positivity for ras-p21, EGFr and bcl-2 was evaluated by semiquantitative analysis. RESULTS: Oncoprotein alterations and increased keratinocyte proliferative activity were observed both in AK and SCC. The number of Langerhans cells (CD1a+ cells) was similar in the two lesions but lower in SCC compared to AK. The proportion of CD1a(+)-B7/BB1+ cells was slightly higher in AK and SCC than in NS. The Langerhans cells expressed the HLA-DR antigen in all groups. Values were highest in AK and NS, and quite low in SCC. Cytotoxic T lymphocytes were more numerous in SCC than in AK and NS. Interestingly, the total CD4/CD8 ratio was much lower in SCC than in AK and NS, which indicates an increase in the CD8+ subpopulation in samples of SCC. In the epithelia of SCC samples there were a considerable number of B7/BB1+ keratinocytes. CONCLUSIONS: We suggest that alterations in the immunodefence mechanisms have an important role in the transformation of AK into SCC, and that these changes affect not only lymphocytes, but also professional (i.e., Langerhans cells) and non-professional (i.e., keratinocytes) antigen presenting cells.

An experimental pilot study of tacalcitol activities during modulation of parakeratotic skin features.

Establishing guidelines and experimental models preclinical and clinical evaluations of new agents for treatment, and/or prevention of human diseases has become a task of crucial importance. Psoriasis is such one disease holding great interest for dermatology owing to its high rate of incidence and complexity of treatment. However the absence of psoriatic lesions in animals and the inability to induce them, calls for experimental techniques both in vitro and in vivo. The purpose of this study was to evaluate experimentally the effects of tacalcitol on cell proliferation and differentiation process. Thereafter a human pilot study on psoriatic patients has been developed.

Immunocytometric determination of the alpha and beta isoforms of human DNA topoisomerase II: influence of different fixatives.

The immunoreactivity of the alpha and beta isoforms (170 and 180 kDa, respectively) of human DNA topoisomerase II (topo II) toward specific monoclonal antibodies (MoAbs), was investigated in HeLa cells. The extent of antigen preservation achieved with the dehydrating fixatives, ethanol and acetone, was compared with that obtained with the cross-linking agent paraformaldehyde. Binding of each MoAb to the relative isozyme was detected by indirect immunofluorescence labelling and quantified by flow cytometry. The amount of antigen detectable was calculated by normalizing the mean fluorescence intensity of samples incubated with specific MoAbs, to the respective background fluorescence. For both isozymes, acetone provided the highest immunofluorescence/background ratio. A strong reduction in the immunoreactivity of the 180-kDa isoform was observed after ethanol, and to a minor extent after paraformaldehyde fixation, while reactivity of the 170-kDa isoform was not significantly affected. Cell cycle distribution of each protein was assessed by dual-parameter analysis of immunofluorescence vs. DNA content. No evident difference in the cell cycle distribution of each isozyme was found with each fixation protocol. At the morphological level, the pattern of immunofluorescence showed that the localization of each isozyme was very similar for all these fixatives. These results confirm the great sensitivity of the 180-kDa isoform to degradation, and indicate that fixation has to be carefully chosen in order to determine the relative amount of topo II isoforms with immunocytometric techniques.

[The effects of growth hormone in children with a GH deficiency: a report of 5 cases]. / Effetti dell'ormone della crescita nei bambini con deficit di GH: presentazione di 5 casi.

The authors describe 5 cases, 3 boys and 2 girls, with idiopathic growth hormone deficiency in prepubertal age, treated with human growth hormone. In four of five cases the response to treatment with GH was relevant. Only in one case (F1) the response was negative. The results of this study confirm that rhGH is a safe and effective therapy in children with GHD.

Reduced topoisomerase II activity in multidrug-resistant human non-small cell lung cancer cell lines.

Multidrug-resistant (MDR) cell lines often have a compound phenotype, combining reduced drug accumulation with a decrease in topoisomerase II. We have analysed alterations in topoisomerase II in MDR derivatives of the human lung cancer cell line SW-1573. Selection with doxorubicin frequently resulted in reduced topo II alpha mRNA and protein levels, whereas clones selected with vincristine showed normal levels of topo II alpha. No alterations of topo II beta levels were detected. To determine the contribution of topo II alterations to drug resistance, topo II activity was analysed by the determination of DNA breaks induced by the topo II-inhibiting drug 4'-(9-acridinylamino)methane-sulphon-m-anisidide (m-AMSA) in living cells, as m-AMSA is not affected by the drug efflux mechanism in the SW-1573 cells. The number of m-AMSA-induced DNA breaks correlated well (r = 0.96) with in vitro m-AMSA sensitivity. Drug sensitivity, however, did not always correlate with reduced topo II mRNA or protein levels. In one of the five doxorubicin-selected clones m-AMSA resistance and a reduction in m-AMSA-induced DNA breaks were found in the absence of reduced topo II protein levels. Therefore, we assume that post-translational modifications of topo II also contribute to drug resistance in SW-1573 cells. These results suggest that methods that detect quantitative as well as qualitative alterations of topo II should be used to predict the responsiveness of tumours to cytotoxic agents. The assay we used, which measures DNA breaks as an end point of topo II activity, could be a good candidate.

Dose-dependent zinc inhibition of DNA ladder in apoptotic HeLa cells regulates the activity of poly(ADP-ribose) polymerase and does not protect from death induced by VP-16.

Zinc ions exert an inhibitory effect on Ca(2+)Mg(2+)-dependent endonuclease which is supposed to be responsible for the fragmentation of DNA during apoptosis. In the experimental system we used, that is HeLa cells treated with VP-16, the protection from internucleosomal DNA degradation is modulated by Zn concentration and appears to be dependent on the time after treatment. This effect does not prevent cell death or occurrence of apoptotic parameters, suggesting that DNA ladder appearance is not a crucial event in apoptosis. The activation of poly(ADP-ribose)polymerase following the administration of VP-16, is not observed in cells in which DNA fragmentation has been abolished by zinc, supporting the hypothesis that this event is regulated by the appearance of small-sized DNA fragments.

Expression of the 170-kDa and 180-kDa isoforms of DNA topoisomerase II in resting and proliferating human lymphocytes.

The expression of the 170-kDa (alpha) and the 180-kDa (beta) isoforms of DNA topoisomerase II (topo II) was investigated with two specific monoclonal antibodies (MoAbs) in human peripheral blood lymphocytes (PBL), before and after phytohaemoagglutinin (PHA) stimulation. Binding of each MoAb was detected by indirect immunofluorescence labelling and quantified with flow cytometry. In resting PBL, the intensity of immunostaining was very low for both isozymes; however, topo IIbeta-associated immunofluorescence was about 2.5 times significantly higher (P<0.001) than that associated with the alpha isoform. Between 48 and 72 h of PHA stimulation, when the highest percentage of cells in S and G2+M phases was found, the levels of topo IIalpha and beta increased up to about 30 and 10 times the value measured in resting PBL, respectively. Thus, the two isoforms reached comparable immunofluorescence values. At longer stimulation periods (96-120 h), topo IIalpha immunofluorescence was not significantly changed, while that relative to topo IIbeta declined to about 50% of the peak value (P<0.02). At this time however, topo IIalpha-associated immunofluorescence was not significantly different from that related to the beta isozyme. These results suggest that in resting PBL topo IIalpha is required at levels lower than topo IIbeta, while in proliferating lymphocytes both isoforms are expressed to significantly higher levels.

Discrete localization of different DNA topoisomerases in HeLa and K562 cell nuclei and subnuclear fractions.

Monoclonal antibodies raised against DNA topoisomerase I and against topoisomerase II alpha and beta isoforms, which have been previously demonstrated to be highly specific and capable of detecting cell cycle-related variations of the topoisomerase II isoforms (Negri et al., 1992, Exp. Cell Res. 200, 452-459), have been utilized for a fine subcellular localization. Immunocytochemistry by confocal and electron microscopy have been used for a topological and quantitative evaluation of the fine distribution of the different topoisomerases in HeLa and K562 cells. Topoisomerase I and topoisomerase II alpha are present both in the nucleoplasm and in the nucleolus, though at different relative ratios, while topoisomerase II beta is exclusively present at the nucleolar level. This is further confirmed by immunoblotting and immunocytochemical quantitative evaluations performed on purified nuclear matrix fractions obtained from K562 cells. In fact, the amount of topoisomerase I and topoisomerase II alpha present in the whole cell nuclei is partly lost in isolated nuclei but, while topoisomerase I is further significantly reduced in nuclear matrix preparations, the topoisomerase II alpha content is only slightly decreased. On the other hand, the great majority of topoisomerase II beta is retained in the nuclear matrix and can be detected exclusively in association with the nucleolar remnant. These results are consistent with specific functional roles hypothesized for the different topoisomerase types.

The effect of the chemotherapeutic drug VP-16 on poly(ADP-ribosylation) in apoptotic HeLa cells.

We have studied the effect of the chemotherapeutic drug VP-16 (etoposide) on the metabolism of HeLa cells by analysing different cellular parameters; in particular we have focused on changes in cellular morphology that are considered as markers of apoptosis. By immunofluorescence experiments we have shown that VP-16 causes the complete disruption of nucleoli and induces chromatin margination and fragmentation. Agarose gel electrophoresis of DNA from cells treated with 10-100 microM VP-16 showed the appearance of a characteristic ladder due to the internucleosomal DNA cleavage. The effect of etoposide on DNA integrity was not prevented by preincubation of cells with the protein synthesis inhibitor cycloheximide. These results provide experimental evidence indicating that the typical features of apoptosis are visible in HeLa cells exposed to VP-16. In this experimental system we have investigated whether the ADP-ribosylation process could be regulated by the presence of DNA fragments. By means of the activity gel technique, which allows the direct evaluation of automodified poly(ADP-ribose)polymerase, we have observed that in extracts from cells where etoposide-induced DNA fragmentation occurred, the autoribosylated form of the enzyme is greatly increased. Ribosylated poly(ADP-ribose)polymerase has been isolated by affinity chromatography on boronate column from cells permeabilized and labelled with [32P]NAD. Drug exposure caused a strong augmentation of modified enzyme. These observations suggest that activation of ADP-ribosylation process occurs in cells that show the typical features of apoptosis.

The sensitivity to DNA topoisomerase inhibitors in L5178Y lymphoma strains is not related to a primary defect of DNA topoisomerases.

DNA topoisomerase-targeting antitumor drugs are potent inducers of protein-concealed strand breaks in mammalian cells and act by trapping DNA topoisomerases on chromosomal DNA in the form of drug-enzyme-DNA cleavable complexes. It has been proposed that the cleavable complex is an unusual form of DNA damage that elicits cellular responses analogous to those caused by DNA damaging agents. The relationship between topoisomerase-targeting drug-induced damage and radiation-induced damage has been investigated by analyzing the properties of DNA topoisomerases in mouse L5178Y lymphoma strains that are cross-sensitive to topoisomerase I-II inhibitors and to UV light or X-ray irradiation. The strains are LY-R, isolated from L5178Y cells on the basis of increased resistance to ionizing radiation, and strain LY-S, isolated from LY-R cells following a spontaneous increase in the sensitivity to ionizing radiation. LY-S cells, deficient in the rejoining of DNA double-strand breaks, show enhanced sensitivity to topoisomerase II-targeting inhibitors, whereas LY-R cells have an increased sensitivity to UV radiation and to the topoisomerase I inhibitor, camptothecin. The cellular availability of DNA topoisomerase I and II and the sensitivity of the enzymes to their specific inhibitors have been measured in the two related strains. In the LY-R strain, we found a 30% decrease in topoisomerase I content but no difference in camptothecin sensitivity, while no quantitative or qualitative differences were observed for the topoisomerase II. The results indicate that variations in sensitivity of the L5178Y strains to topoisomerase inhibitors are unlikely to be related to primary defects of the target enzymes, and thus it is possible that common pathways exist for processing of topoisomerase- and radiation-induced damage.

Antibodies to topoisomerase II in idiopathic pulmonary fibrosis.

Antibodies to DNA topoisomerase II (anti-topoisomerase II) were detected by ELISA in the sera of 18 out of 41 (44%) patients with idiopathic pulmonary fibrosis (IPF). Follow-up sera were also obtained from 19 of the patients. DNA topoisomerase II binding remained constantly high or low in the majority of follow-up sera, but 2 out of the 8 positive cases became negative while 3 out of the 11 negative cases became positive during follow-up. No association was found between occurrence of anti-topoisomerase II antibodies and any indices of disease severity. Furthermore, individual patient follow-up did not show any correlation between changes in topoisomerase II binding and deterioration or improvement of clinical status. In conclusion our study shows that although anti-topoisomerase II are detectable in a large fraction (approximately 50%) of IPF patients and are useful for diagnostic purposes, they do not provide a measure of clinical activity.

DNA topoisomerase II beta: stability and distribution in different animal cells in comparison to DNA topoisomerase I and II alpha.

DNA topoisomerase I and the isoforms alpha and beta of DNA topoisomerase II were analyzed in different animal cells using a panel of monoclonal antibodies (MoAbs). The beta isoform is a most unstable enzyme. We investigated conditions to stabilize beta isoform because its variability changes according to the derivation of cells. We describe two MoAbs specific to DNA topoisomerase I: the first one recognizes the enzyme in all the species tested including fish; the second one, in contrast, recognizes an epitope present only in mammalian cells. We also found that eight of eight MoAbs against DNA topoisomerase II alpha and five of six against the beta isoform recognize the respective enzymes in all the species tested excluding fish. In addition, MoAbs to the alpha isoform are specific to epitopes not present in the carboxyl third of the enzyme.