Differences in clinicopathological features and distribution of risk factors in Italian melanoma patients.

BACKGROUND: No studies are available in the literature on the distribution of different melanoma features and risk factors in the Italian geographical areas. OBJECTIVE: To identify the differences in clinical-pathological features of melanoma, the distribution of risk factors and sun exposure in various Italian macro-areas. METHODS: Multicentric-observational study involving 1,472 melanoma cases (713 north, 345 centre, 414 south) from 26 referral centres belonging to the Italian Multidisciplinary Group for Melanoma. RESULTS: Melanoma patients in northern regions are younger, with thinner melanoma, multiple primaries, lower-intermediate phototype and higher counts of naevi with respect to southern patients; detection of a primary was mostly connected with a physician examination, while relatives were more involved in the south. Northern patients reported a more frequent use of sunbeds and occurrence of sunburns before melanoma despite sunscreen use and a lower sun exposure during the central hours of the day. CONCLUSIONS: The understanding of differences in risk factors distribution could represent the basis for tailored prevention programmes.

Melanoma detection in Italian pigmented lesion clinics.

AIM: Accuracy in melanoma detection is important to recognize early curable melanomas and to minimize the unnecessary excision of benign lesions. The aim of this paper was to evaluate melanoma screening accuracy of Italian pigmented lesion clinics in terms of number needed to excise (NNE), melanoma thickness, and number of melanomas diagnosed during patient follow-up. METHODS: Information on all skin tumors excised in 2011 were extracted from the databases of the participating centers. Information whether the lesion was excised at the baseline examination or during patient follow-up was recorded, as well as the overall number of patients examined in each center in 2011. RESULTS: After e-mail solicitation, 22 of 40 centers agreed to participate. A total of 8229 excised lesions were collected. The overall number of examined patients was 86.564, thus 9.5% of screened patients had a lesion removed. Of the excised lesions, 866 were diagnosed as melanoma (1% of examined patients) and 5311 (88.9%) were melanocytic nevi. Three NNE were calculated giving values of 7.9 excised lesions to find 1 melanoma, 7.1 melanocytic lesions to find 1 melanoma, and 3.7 lesions to find 1 skin malignancy. The median melanoma thickness was 0.6 mm, with only 15.1% of melanomas ≥ 1 mm of thickness. Melanomas detected over time were 96 (11.1%; mean thickness, 0.3 mm), with 15.6% of lesions excised after short-term follow-up and 84.4% after long-term follow-up. CONCLUSION: The NNE values comparable to those achieved in specialized clinical settings and the high number of early melanomas diagnosed at the baseline examination or during patient follow-up indicate a high level of accuracy in melanoma screening achieved by Italian pigmented lesion clinics.

Extragenital lichen sclerosus and atrophicus treated with topical steroids and retinoids in a child with vitiligo.

Lichen sclerosus and atrophicus (LSA) most commonly affects the anogenital region. Extragenital involvement is rare, and women are reported to be affected 6 to 10 times more often than men. The aetiology of LSA is unclear, but genetic, physiological and environmental factors are thought to be involved. Several lines of evidence support the hypothesis of an autoimmune basis for LSA; an increased incidence of tissue-specific antibodies and an association with autoimmune disorders such as vitiligo, alopecia areata, thyroid disease and pernicious anaemia have been reported. We describe a paediatric patient with extragenital LSA associated with vitiligo who was successfully treated with topical steroids and retinoids.

Impetigo herpetiformis occurring during N-butyl-scopolammonium bromide therapy in pregnancy: case report.

Impetigo herpetiformis (IH) is a rare dermatosis arising during the third trimester of pregnancy which is generally considered as a form of pustular psoriasis of unknown aetiology. Clinically it is characterized by erythematous plaques surrounded by sterile pustules associated with fever, diarrhea, sweating and increasing risk of stillbirth for placental insufficiency. We describe a case of developed erythematous plaques surrounded by pustules localised initially to the trunk of a 35-year-old woman at the 34th week of gestation after 5 days of treatment with N-Butyl-Scopolammonium, and which later involved the upper and lower limbs. Skin histology confirmed the diagnosis of generalised pregnancy pustular psoriasis (impetigo herpetiformis). IH is reported to be associated with hypocalcemia, hypoparathyroidism, use of oral contraceptives and bacterial infections. This is the first report suggesting the potential role of drugs other than oral contraceptives in the pathogenetic mechanism of this disease. In this case an adverse cutaneous reaction to BB could be the cause of the development of Koebner isomorphism.

Membrane transport influences the rate of accumulation of cytosine arabinoside in human leukemia cells.

The role of membrane transport in the cellular accumulation of 1-beta-D-arabinofuranosylcytosine (ara-C) was studied in freshly isolated human acute leukemia cells. Patient cells had low rates for ara-C transport as compared with human and murine experimental cells and correspondingly low binding capacities for the nucleoside transport inhibitor, nitrobenzylmercaptopurine riboside (NBMPR). At 1 microM ara-C, the rate of net cellular accumulation was close to the membrane transport rate, and NBMPR inhibited transport and accumulation to the same extent. The rate of ara-C accumulation was half maximal at only 3-5 microM, a level much lower than that required for murine cells (67-85 microM). At concentrations below 1 microM the rate of ara-C accumulation was determined primarily by the transport rate, but at higher concentrations above 10 microM, phosphorylation capacity was the principal determinant of the net uptake rate. This difference in the role of transport at high and low ara-C concentrations may explain, in part, the efficacy of high-dose ara-C in patients refractory to standard dose protocols.

Effect of uracil arabinoside on metabolism and cytotoxicity of cytosine arabinoside in L5178Y murine leukemia.

Pretreatment of L5178Y murine leukemia cells with uracil arabinoside (ara-U) enhances the cytotoxicity of cytosine arabinoside (ara-C). This effect is mediated by the cytostatic effect of ara-U, which causes a delay of cell progression through S-phase. Consequently, the specific activity of enzymes that peak during S-phase increases, and deoxycytidine kinase increases 3.6-fold over untreated controls. This allows enhanced anabolism of ara-C to nucleotides, as well as increased incorporation into DNA with ultimate synergistic cytotoxicity. It is postulated that the systemic metabolism of high-dose ara-C to sustained high levels of ara-U in patients with acute leukemia may enhance the activity of subsequent doses of ara-C, and thus contribute to a means for pharmacologic self-potentiation, contributing to the unique therapeutic activity of high-dose ara-C.

Cutaneous manifestations in celiac disease.

Celiac disease (CD) is an autoimmune gluten-dependent enteropathy characterized by atrophy of intestinal villi that improves after gluten-free diet (GFD). CD is often associated with extra-intestinal manifestations; among them, several skin diseases are described in CD patients. The present review reports all CD-associated skin manifestations described in the literature and tries to analyze the possible mechanisms involved in this association. The opportunity to evaluate the possible presence of CD in patients affected by skin disorders is discussed.

A critical role for uridine nucleotides in the regulation of deoxycytidine kinase and the concentration dependence of 1-beta-D-arabinofuranosylcytosine phosphorylation in human leukemia cells.

The intracellular concentration of 1-beta-D-arabinofuranosylcytosine (ara-C) for half-maximal phosphorylation by leukemic blasts obtained directly from patients was 2.1 +/- 2.5 microM (median, 1.3 microM, N = 25), and the rate of ara-C accumulation actually declined at concentrations above 20 microM in 35% of these cell populations. These apparent Km values for cellular phosphorylation were an order of magnitude lower than the Km of deoxycytidine (dCyd) kinase for ara-C with ATP as phosphate donor. dCyd kinase was purified from human leukemia cells and assayed for [3H]ara-C kinase activity with a mixture of 7 nucleotides at their approximate cellular concentrations or with a single nucleotide deleted. At low or high ara-C concentrations, ATP, GTP, CTP, or dTTP could be eliminated without significantly altering the rate. The only potential phosphate donor that was clearly important was UTP, since its deletion reduced the rate to only 25% of that with the complete mix. As anticipated, eliminating dCTP, the end product of this salvage pathway, moderately increased the rate by 50% at 0.4 microM ara-C or by 26% at 40 microM ara-C. At 40 microM ara-C, deleting UDP from the mix increased the rate more than deleting dCTP. dCTP was less inhibitory against 1 mM UTP (50% inhibitory concentration, 26 microM) than against 4 mM ATP (50% inhibitory concentration, 2.2 microM). In kinetic assays with 4 mM ATP and variable ara-C, UDP was a potent uncompetitive inhibitor with a Ki of 4 microM; the Ki for ADP was 1000-fold higher. Direct fit of kinetic data to the Michaelis equation yielded a Km for ara-C of 49 microM with 4 mM ATP as the phosphate donor; however, there was evidence of negative cooperativity with a Hill coefficient of 0.7. High ara-C Km values were also obtained with GTP and CTP, but with no evidence of cooperativity. With 1 mM UTP, the Km was 1.5 microM with moderate substrate inhibition; thus the kinetic data with UTP were similar to those for ara-C phosphorylation by intact cells. UDP was less potent versus UTP than versus ATP. It lowered the Vmax and enhanced the ara-C substrate inhibition without altering the Km. When 1 mM UTP and 4 mM ATP were mixed, the kinetic pattern was similar to that for UTP alone. The Km for UTP with [3H]dCyd as the phosphate acceptor of 0.8 microM was 25-fold lower than the Km for ATP of 20 microM.(ABSTRACT TRUNCATED AT 400 WORDS)

1-beta-D-arabinofuranosylcytosine-diphosphate-choline is formed by the reversal of cholinephosphotransferase and not via cytidylyltransferase.

In an effort to identify the pathway leading to the formation of 1-beta-D-arabinofuranosylcytosine-diphosphate (ara-CDP)-choline from 1-beta-D-arabinofuranosylcytosine (ara-C) treatment of cultured cells, as well as of cells obtained from leukemia patients, we probed the enzymatic steps involved in the CDP-choline pathway for phosphatidylcholine biosynthesis. Ara-C-triphosphate was not a substrate for CTP:phosphocholine cytidylyltransferase activity under the conditions employed, whereas CTP and dCTP were utilized to form CDP-choline and dCDP-choline, respectively. When presented together, ara-C-triphosphate and CTP inhibited the enzymatic conversion of CTP to CDP-choline in the presence of phosphocholine, with a Ki of 6 mM. Since CTP:phosphocholine cytidylyltransferase did not appear to be responsible for the increased levels of ara-CDP-choline, we next studied the other enzyme in the pathway for phosphatidylcholine synthesis that could form ara-CDP-choline, CDP-choline:1,2-diacylglycerol cholinephosphotransferase. CDP-choline:1,2-diacylglycerol cholinephosphotransferase activity present in microsomes isolated from L5178Y murine leukemia cells exhibited a reversal of its normal catalytic activity, using CMP and 1-beta-D-arabinofuranosylcytosine-monophosphate (ara-CMP) along with phosphatidylcholine to produce either CDP-choline or ara-CDP-choline, plus diradylglycerol. The Vmax and Km values for CMP were 0.78 +/- 0.04 nmol/min/mg and 340 +/- 20 microM, respectively, whereas the Vmax and Km for ara-CMP were 0.22 +/- 0.06 nmol/min/mg and 1410 +/- 540 microM, respectively. A Ki value of 3 mM was obtained for ara-CMP under the cell-free assay conditions used. These results indicate that ara-CDP-choline most likely arises from a reversal of the CDP-choline:1,2-diacylglycerol cholinephosphotransferase utilizing ara-CMP, rather than from the catalysis of ara-C-triphosphate plus phosphocholine to ara-CDP-choline by CTP:phosphocholine cytidylyltransferase. It is speculated that this mechanism may explain, in part, the rapid cellular lysis observed with high dose ara-C therapy.

Schedule-dependent synergy and antagonism between high-dose 1-beta-D-arabinofuranosylcytosine and asparaginase in the L5178Y murine leukemia.

The effect of schedule of drug administration on the biochemical and therapeutic effects of the combination of 1-beta-D-arabinofuranosylcytosine (ara-C) and asparaginase was investigated in vivo and in vitro using the murine leukemia L5178Y. Treatment of cells in vitro with either ara-C (10(-6) M) or asparaginase (0.5 IU/ml) for 8 hr resulted in 45 and 24% viability, respectively; simultaneous exposure to both drugs resulted in 25% viability, a subadditive effect. Sequential 8-hr in vitro treatments with asparaginase preceding ara-C or ara-C preceding asparaginase resulted in 43 and 8% viability, respectively, indicating strong schedule dependency. Recovery from drug-induced inhibition of cell growth in vivo suggested an optimal interval of 120 hr. Treatment of leukemic mice with asparaginase, ara-C, or both drugs simultaneously 3 days after inoculation of 10(6) cells resulted in mean survival times of 16, 21, and 18 days, respectively (control mean survival time, 10 days). With a 120-hr interval between the two drugs, treatment with ara-C followed by asparaginase resulted in 20 of 24 sixty-day survivors. In contrast, when asparaginase preceded ara-C, there was a mean survival time of only 23 days with no 60-day survivors. Maximal weight loss with either combination was only 10%. Mechanisms for the pharmacological antagonism include asparaginase-induced decreased cellular uptake and incorporation of ara-C into macromolecules. The apparent synergy is related to the timing of asparaginase treatment, the "optimal therapeutic effect" occurring when sequential asparaginase is administered before the cells recover from the ara-C effect. Since both drugs are probable components of antileukemic combinations, understanding of such drug-drug interactions would optimize clinical therapy.

Methotrexate cytotoxicity for L5178Y/Asn- lymphoblasts: relationship of dose and duration of exposure to tumor cell viability.

To determine the relative contribution of dose and duration of exposure to methotrexate (MTX) cytotoxicity, suspension cultures of L5178Y/Asn- murine leukemic cells were exposed to 0.1 to 100 microM MTX for 3 to 42 hr. Viability was determined by cloning in soft agar. While there was a linear relationship between dose and MTX cytotoxicity for exposure duration of 3 and 6 hr (r = -0.66), there was a pronounced flattening of this curve at exposure durations of 18 to 42 hr (r = -0.48). Furthermore, there was an excellent correlation (r = -0.85) between MTX cytotoxicity and durations of exposure for 6 to 42 hr (dose range, 1 to 100 microM). Using the linear least-squares method, a best-fit equation for the kinetics of MTX cytotoxicity was determined to be: log viability = 2.25 = 1.76 (log duration) - 0.31 (log dose). In practice, this equation predicts that a 1-log increase in duration of exposure results in almost a 2-log increase in cytotoxicity, whereas a 1-log increase in dose results in only a 0.3-log increase in cytotoxicity. The clinical utility of these data suggest that protracted infusions of lower doses of MTX would be equally as useful as or more useful than short-term high-dose infusions.

Kinetics of phototoxicity of Fischer's medium for L5178Y leukemic cells.

The uncontrolled exposure of Fischer's medium to cool white fluorescent (CWF) light or other sources emitting near-ultraviolet or visible light absorbance by riboflavin is a crucial random variable in experiments which utilize L5178Y cells and this medium. The radiation effects of CWF light result in the rapid development of toxic photoproducts in the medium which are cytostatic at lower doses of radiation and cytotoxic at higher doses. After a 24-hr suspension in medium irradiated for 3 or 48 hr, the cloning efficiencies of cells subsequently plated in light-protected medium were 87 and 3%, respectively. The corresponding near-ultraviolet doses for these periods of exposure to CWF light were 0.22 x 10(4) for a 3-hr exposure and 3.47 x 10(4) J/sq m for a 48-hr exposure. Cells incubated in lightly irradiated medium resumed growth at nearly normal rates following a 24- to 48-hr period in which no increase in cell numbers occurred. Exposure of medium containing riboflavin, but not tryptophan or tyrosine, to CWF light also produces toxic medium. Tryptophan enhances riboflavin-induced phototoxicity, whereas tyrosine diminishes this effect. As photosusceptibility of this system is very high, Fischer's medium must be fully protected from all sources of light absorbable by riboflavin.

Proliferation-dependent cytotoxicity of methotrexate in murine L5178Y leukemia.

The basis for the proliferation-dependent cytotoxicity of methotrexate has been investigated in mice bearing the L5178Y ascites leukemia. Methotrexate at 60 mg/kg i.p. reduced the viability of logarithmically growing ascites cells (55% active S phase cells) to 28% of control, whereas the viability of the slowly growing cells (18% active S phase) was decreased to only 59% of control. Log phase tumor cells accumulated 8-fold higher levels of methotrexate polyglutamates compared to cells that had approached the stationary phase. However, no differences between log phase and slowly growing tumor cells were observed in the cellular levels of unmetabolized methotrexate. Intestinal mucosa and bone marrow from non-tumor-bearing mice resembled slowly growing tumor cells and had markedly lower levels of methotrexate polyglutamates than logarithmically growing cells. The greater accumulation of methotrexate polyglutamates in the logarithmically growing tumor cells was consistent with an increased synthesis of methotrexate polyglutamates in these cells. The enhanced methotrexate polyglutamylation in log phase versus slowly growing cells was not related to changes in the rates of either cellular methotrexate transport, transmembrane efflux of methotrexate, or hydrolysis of methotrexate polyglutamates. Thymidylate synthase activity measured in situ and in extracts from log phase cells was 4- and 2-fold higher, respectively, than in the more slowly growing cells. Methotrexate produced a 2.4-fold greater depletion of poly-gamma-glutamyl derivatives of 5,10-methylenetetrahydropteroylglutamate in log phase cells compared to slowly growing cells, and this was a function of both the increased methotrexate polyglutamate accumulation and thymidylate synthase activity in the rapidly proliferating cells. These results provide further evidence that the selectivity of methotrexate for tumors with a high growth fraction is a consequence of the rapid rates of both cellular methotrexate polyglutamate synthesis and oxidation of 5,10-methylenetetrahydropteroyl polyglutamates by thymidylate synthase.

L-asparaginase-induced modulation of methotrexate polyglutamylation in murine leukemia L5178Y.

The modulation of methotrexate polyglutamylation by L-asparaginase has been examined in mice bearing sublines of leukemia L5178Y that have different sensitivities to asparaginase. A single i.p. injection of 200 IU/kg of asparaginase completely inhibited ascites tumor cell growth in the parental L5178Y/S+ tumor for 120 h compared to 72 and 30 h in the L5178Y/S and L5178Y/S+/- sublines, respectively. Similarly, DNA and protein synthesis were completely inhibited by asparaginase for 96 h in L5178Y/S+ cells, but only for 72 and 24 h in L5178Y/S and L5178Y/S+/- cells. In each tumor the temporal patterns of depletion and recovery of S-phase cells were similar to the patterns of suppression and recovery of DNA and protein synthesis observed in that tumor. When methotrexate was administered at either 96 or 24 h after asparaginase during the asparaginase-induced S-phase nadirs of L5178Y/S+ and L5178Y/S+/- cells, respectively, subsequent methotrexate polyglutamylation was inhibited 83 and 92% compared to tumor cells exposed to methotrexate only. Recovery of methotrexate polyglutamylation in both tumors following L-asparaginase pretreatment coincided in time with the return in the fraction of S-phase cells towards the pretreatment values. The inhibition of methotrexate polyglutamate accumulation by asparaginase was associated with decreased retention of methotrexate in tumor cells. In contrast, asparaginase had no significant effect on methotrexate polyglutamate accumulation and methotrexate retention when administered after methotrexate. These data indicated that the asparaginase-induced modulation of methotrexate polyglutamylation in mice was directly related to the time course of inhibition and recovery of tumor cell proliferation by asparaginase, and thus varied with the intrinsic sensitivity of the individual tumor to the enzyme.

Modulation of the metabolism and pharmacokinetics of 1-beta-D-arabinofuranosylcytosine by 1-beta-D-arabinofuranosyluracil in leukemic mice.

The interaction between high concentrations of 1-beta-D-arabinofuranosyluracil (HiCAU) and 1-beta-D-arabinofuranosylcytosine (ara-C) was investigated in vivo with emphasis on cell kinetics, pharmacokinetics, and drug metabolism. Mice bearing L5178Y leukemia were given a 48-h s.c. infusion of high-dose ara-U (HiDAU) to achieve a plasma level of 0.5 to 1 mM. A total dose of 7.35 g/kg/day for 2 days was nontoxic; the mean survival of control (saline treated) leukemic mice was 12.2 +/- 1.8 days and 11.7 +/- 2.0 days for the HiDAU-treated leukemic mice. Using flow cytometry, cell cycle progression of L5178Y ascites cells was monitored during HiDAU infusion. At 48 h, the proliferative index (PI) percentage of the leukemic cells is significantly different (P less than 0.001) in HiDAU-treated leukemic mice (mean = 50.8) versus control (mean = 45.6). A higher PI percentage is associated with accumulation of cells in S phase. This effect was highly variable in the ara-U-treated mice, and the ara-U "perturbed" group was defined as those mice whose cells had an increase in the PI to greater than or equal to 50%. The higher PI percentage in HiDAU-treated mice correlated with HiCAU in ascites fluid, leukemic cells, and kidney of perturbed mice. HiCAU in the "ara-U-perturbed" group altered the plasma pharmacokinetics of high-dose ara-C (HiDAC, 1 g/kg), increased the cellular metabolism of ara-C to 1-beta-D-arabinofuranosylcytidine triphosphate (ara-CTP) (3-fold), and increased ara-C-DNA synthesis (3-fold). In mice bearing the L5178Y leukemia, a 48-h infusion of ara-U followed by a 24-h s.c. infusion of 40 mg/kg resulted in a 260% increase in life span and seven 90-day survivors among 16 treated mice. In contrast, ara-U or ara-C alone had a negligible therapeutic effect. ara-U-induced alterations in the systemic pharmacokinetics of ara-C are the result of inhibition of cytidine deaminase activity by HiCAU in liver and kidneys. This results in a decrease in ara-C catabolism and prolongs the plasma half-life of ara-C. The dual alteration of the pharmacokinetics of ara-C and cytokinetics of the leukemia cells by HiCAU results in enhanced survival of leukemic mice. These results may help explain the clinical utility of HiDAC treatment programs for patients with acute leukemia.

Controlled trial of methotrexate and Bacillus Calmette-Guérin therapy for advanced head and neck cancer.

Thirty-eight patients with advanced, inoperable squamous cell carcinoma of the head and neck were randomized to receive methotrexate alone or methotrexate with Bacillus Calmette-Guérin. The response rates with methotrexate (3 of 19) and methotrexate plus B. Calmette-Guérin (4 of 16) were similar, as was the duration of response and survival of the two groups. The results of in vitro immunological studies of lymphocytes were assessed. Marked weight loss, poor performance status, and distant metastases were the most important prognostic factors. The presence of anergy was significantly correlated with weight loss. This study also indicated that a large tumor burden is a frequent occurrence in advanced head and neck cancer and may account for the lack of efficacy of B. Calmette-Guérin.

Ether-linked phosphoglyceride content of human leukemia cells.

The glycerolipids of most cells are characterized by a specific proportion of ether linkages at the sn-1 position of the glycerol backbone. A number of tumors are known to have altered concentrations of ether-linked lipids compared to normal tissues. However, no through examination of the ether-lipid content of human leukemia cells has been reported despite the importance of these lipids in normal leukocyte function. In the present study samples were obtained from adults with acute myelogenous leukemia (AML), chronic granulocytic leukemia in blast crisis, and acute lymphocytic leukemia and from healthy human donors. The cellular lipids were extracted, the individual phospholipid classes were isolated, lipid phosphorus content was determined, and the lipids were converted to diglyceride benzoate derivatives for separation and quantitation of the subclasses by high performance liquid chromatography. The data indicate that all the leukemic cells analyzed have an altered phospholipid composition compared to their respective normal leukocytes. Furthermore, among the AML patients both the percentage of the choline-containing phosphoglyceride fraction (PC) which is alkyl linked and the nmoles alkyl-PC/10(6) cells differ significantly by FAB subtype. A positive correlation between the levels of alkyl-PC and the degree of cellular differentiation is observed. Although no differences are observed between chronic granulocytic leukemia in blast crisis and AML lipids, the leukemic cells contain dramatically lower levels of alkyl-linked PC than do normal polymorphonuclear leukocytes. In contrast, no differences are observed between the alkyl-PC content of normal and leukemic lymphocytes. In light of the relations among ether-lipids, protein kinase C, and cell differentiation, these data suggest the ether-linked lipids are important in myeloid cell function and differentiation.