Inguinal and Ilio-inguinal Lymphadenectomy in Management of Palpable Melanoma Lymph Node Metastasis: A Long-Term Prospective Evaluation of Morbidity and Quality of Life.

PURPOSE: Prospective data are lacking on long-term morbidity of inguinal lymphadenectomy including the influence of extent of surgery, use of radiotherapy, and patient factors. The aim of this study is to evaluate the effects of these factors on patient outcome, quality of life (QOL), regional symptoms, and limb volumes after inguinal or ilio-inguinal lymphadenectomy for melanoma. METHODS: Analysis of the subgroup of patients with inguinal lymph node field relapse of melanoma, treated by inguinal or ilio-inguinal lymphadenectomy in the ANZMTG/TROG randomized trial of adjuvant radiotherapy versus observation. RESULTS: Sixty-nine patients, 46 having undergone inguinal and 23 ilio-inguinal lymphadenectomy, with median follow-up of 73 months were analyzed. Mean limb volume increased rapidly after surgery (7% by 3 months) and continued to increase for at least another 18 months. Patients with body mass index (BMI) ≥ 25 kg/m2 had greater limb volume increase than normal-weight patients (13.3% versus 6.9%, P = 0.030). QOL improved over the first 18 months, but despite initial improvement, regional symptoms persisted long term. Type of surgery (inguinal or ilio-inguinal lymphadenectomy) had no demonstrably significant effect on limb volume (9.9% versus 13.4%, P = 0.35), QOL (P = 0.68), or regional symptoms (P = 0.65). There was no difference in overall survival between inguinal and ilio-inguinal lymphadenectomy [hazard ratio (HR) 0.75, 95% confidence interval (CI) 0.40-1.40, P = 0.43]. CONCLUSIONS: Inguinal lymphadenectomy for melanoma is a potentially morbid procedure with significant increases in limb volume. Patients report reasonable QOL but may have ongoing regional symptoms. Overweight/obesity is associated with poorer QOL, increased limb volume, and regional symptoms.

Phase I trial of docetaxel, cisplatin and concurrent radical radiotherapy in locally advanced oesophageal cancer.

BACKGROUND: Locally advanced oesophageal cancer (LAEC) is associated with poor survival and more effective treatments are needed. The aim of this phase I trial was to assess the maximum tolerated dose (MTD) of a novel weekly docetaxel and cisplatin regimen concurrent with radical radiotherapy. METHODS: Patients with unresectable, non-metastatic LAEC were eligible. Treatment comprised docetaxel 15-30 mg m(-2) per week and cisplatin 15-30 mg m(-2) per week in six planned dose levels (DLs) in 3-6 patient cohorts with 50 Gy radiotherapy in 25 fractions. Maximum tolerated dose was based on defined dose-limiting toxicities (DLTs) during therapy and 2 weeks post therapy. RESULTS: A total of 24 patients were enrolled. There were two DLTs: grade 3 fever in DL1 (docetaxel 15 mg m(-2), cisplatin 15 mg m(-2)) and grade 3 nausea in DL2 (20 mg m(-2), 15 mg m(-2)). These DLs were each expanded to six patients without further DLTs. The most common acute toxicity was grade 3 radiation oesophagitis (37.5%). There were no grade 4 toxicities, and haematologic toxicity was minimal. Cisplatin and docetaxel dose intensity was 100% at the highest dose level (DL6). A MTD was not reached in this trial. Tumour overall response rate was 50% (33% complete, 17% partial). CONCLUSION: Cisplatin and docetaxel each 30 mg m(-2) per week concurrent with 50 Gy radiotherapy is recommended for use in phase II clinical trials in oesophageal cancer.

Pharmacogenetics-based population pharmacokinetic analysis of efavirenz in HIV-1-infected individuals.

Besides CYP2B6, other polymorphic enzymes contribute to efavirenz (EFV) interindividual variability. This study was aimed at quantifying the impact of multiple alleles on EFV disposition. Plasma samples from 169 human immunodeficiency virus (HIV) patients characterized for CYP2B6, CYP2A6, and CYP3A4/5 allelic diversity were used to build up a population pharmacokinetic model using NONMEM (non-linear mixed effects modeling), the aim being to seek a general approach combining genetic and demographic covariates. Average clearance (CL) was 11.3 l/h with a 65% interindividual variability that was explained largely by CYP2B6 genetic variation (31%). CYP2A6 and CYP3A4 had a prominent influence on CL, mostly when CYP2B6 was impaired. Pharmacogenetics fully accounted for ethnicity, leaving body weight as the only significant demographic factor influencing CL. Square roots of the numbers of functional alleles best described the influence of each gene, without interaction. Functional genetic variations in both principal and accessory metabolic pathways demonstrate a joint impact on EFV disposition. Therefore, dosage adjustment in accordance with the type of polymorphism (CYP2B6, CYP2A6, or CYP3A4) is required in order to maintain EFV within the therapeutic target levels.

Stem cell factor and high-dose twice daily filgrastim is an effective strategy for peripheral blood stem cell mobilization in patients with indolent lymphoproliferative disorders previously treated with fludarabine: results of a Phase II study with an historical comparator.

Fludarabine exposure leads to impaired peripheral blood stem cell (PBSC) mobilization in indolent lymphoproliferative disorders (LPD). We previously reported that only 34% of fludarabine-exposed patients mobilized successfully using granulocyte-colony stimulating factor (G-CSF; median 10 microg/kg/day) with or without chemotherapy, with unpredictable kinetics and moderate infectious morbidity. Stem cell factor (SCF) plus high-dose twice daily (b.d.) G-CSF may improve mobilization in these patients. SCF 20 microg/kg/day subcutaneously was given from day 1, G-CSF 12 microg/kg b.d. subcutaneously from day 4, apheresis commenced from day 6. Previous study patients served as historical controls. Thirty five patients with indolent LPD were enrolled, median age was 54 years (range 31-66), 66% male, median cumulative prior fludarabine dose was 660 (405-900) mg. Overall, 22 patients (63%) collected >or= 2.0 x 10(6)/kg PBSC (success), compared to 34% controls (odds ratio (OR) 3.2; 95% confidence interval (CI) (1.2, 9.3); P=0.021). Median CD34(+) yield overall was 2.3 x 10(6)/kg (0.53-8.97) from median four (2-6) aphereses. Study patients >or= 50 years mobilized successfully more frequently than controls (58 versus 17%; P=0.0065). Adjusting for age, successful mobilization remained significantly higher in the current study (OR 4.2; 95% CI (1.4, 14.0); P=0.008). SCF/high-dose b.d. G-CSF improves PBSC mobilization efficacy after fludarabine exposure, over mobilization using G-CSF as the mobilizing cytokine. This combined growth factor strategy is a preferred mobilization method for fludarabine-exposed patients.

Prognostic features for response and survival in elderly patients with de novo acute myeloid leukemia treated with mitoxantrone and intermediate dose cytarabine.

Forty-three fit elderly patients with de novo acute myeloid leukemia (AML) received chemotherapy with mitoxantrone and intermediate dose cytarabine (MIDAC) in a phase II clinical trial conducted by the Australasian Leukaemia and Lymphoma Group. The main aim of the study was to evaluate the tolerability and efficacy of MIDAC in inducing durable remissions. While the chemotherapy was generally well tolerated, less than half the patients achieved complete remission (CR) after induction and many of those in CR could not receive planned consolidation cycles. The median overall survival for all patients was 6.5 months and the median disease-free survival for those achieving CR was 8.3 months. Only 2 patients survived beyond 4 years. Factors significantly associated with shorter survival were adverse cytogenetics, marrow dysplasia and increasing age. These results suggest that only selected elderly patients with AML are likely to benefit from aggressive chemotherapy and that novel therapies are required to improve the poor prognosis of this group.

Human placental nitric oxide synthase activity is not altered in diabetes.

Endothelial nitric oxide synthase (NOS) protein and mRNA have been identified and calcium-dependent NOS activity has been measured in human placentae during normal pregnancy. Recently, mRNA and protein for the inducible isoform of NOS have been detected in placentae of women with gestational diabetes. The aim of this study was to determine whether calcium-independent (ciNOS) and/or total (tNOS) NOS activities were increased in placentae obtained after vaginal delivery or Caesarean section from women assigned to the following groups according to standard obstetric criteria: gestational diabetes, diabetes before pregnancy and non-diabetic controls. tNOS and ciNOS were assessed by measuring the conversion of [3H]L-arginine to [3H]L-citrulline in the three groups. Michaelis-Menten constants (Km) and maximum velocities of reaction (Vmax) were calculated using Lineweaver-Burk analysis for tNOS. There were no significant differences in either ciNOS, Vmax or Km values between any of the three groups (normal, ciNOS 12.7+/-1.6%, Vmax 16.6+/-3.3 pmol.min-1.mg-1 protein, Km 15.30+/-2.6 micromol/l; gestational diabetes, ciNOS 15.4+/-1.4%, Vmax 14.8+/-5.2 pmol.min-1. mg-1 protein, Km 10.5+/-1.7 micromol/l; diabetes before pregnancy, ciNOS 13.4+/-1.1%, Vmax 14.9+/-3.4 pmol.min-1.mg-1 protein, Km 17. 7+/-2.2 micromol/l). The presence of macrosomia did not affect tNOS activity in those with diabetes before pregnancy, and glycosylated haemoglobin levels measured between weeks 27 and 39 were not correlated with ciNOS activity. The results from the present study do not provide evidence for increased placental tNOS or ciNOS activities in pregnancies complicated by gestational diabetes or diabetes present before pregnancy.

Reduction of placental nitric oxide synthase activity in pre-eclampsia.

1. The role of the potent vasodilator nitric oxide in the pathogenesis of pre-eclampsia is unclear. We have tested the hypothesis that placental activity of the enzyme which synthesizes nitric oxide (nitric oxide synthase) is reduced in pre-eclampsia. 2. Placentae were obtained after vaginal delivery or Caesarean section from women who had been assigned to the following groups according to standard obstetric criteria: term non-pre-eclamptic control, term pre-eclamptic, preterm non-pre-eclamptic control and preterm pre-eclamptic. Nitric oxide synthase activity of placental tissue homogenates was assessed by measuring conversion of [3H]L-arginine into [3H]L-citrulline in the presence of NADPH, FAD, tetrahydrobiopterin, calmodulin, CaCl2, magnesium acetate and a range of L-arginine concentrations. Michaelis Menton constants (K(m)) amd maximum velocities of reaction (Vmax) were calculated using Lineweaver-Burk analysis. 3. Vmax was significantly reduced in both term and preterm pre-eclamptic placentae compared with placentae from corresponding gestation-matched controls. There were no significant differences in the K(m) values for nitric oxide synthase between any of the four groups, nor were Vmax or K(m) values significantly influenced by mode of delivery. 4. These results provide evidence that human placental nitric oxide synthase activity is significantly reduced in pre-eclampsia. Such a reduction was evident at both term and preterm gestations. Reduced placental nitric oxide synthase activity may have an adverse effect on placental haemodynamic function in pre-eclampsia, and could be involved in the pathogenesis of this important and common obstetric complication.

Effects of ATP, ADP, thrombin, vasopressin and U46619 on human placental nitric oxide synthase activity.

The human placenta contains nitric oxide synthase (NOS) activity, which had been previously found to be localized in the syncytiotrophoblast and the endothelial cells of stem villous vessels. Nitric oxide produced by the syncytiotrophoblast could affect maternal platelet aggregation. The aim of the present study was to examine the effects of substances known to cause platelet aggregation (arginine vasopressin (AVP), the thromboxane A2 mimetic U46619, adenosine diphosphate (ADP) and thrombin) on NOS activity of human placental explants in vitro. Placentae were obtained at term from women with obstetrically uncomplicated deliveries. NOS activity was measured in explants by determining the conversion of [3H]L-arginine to [3H]L-citrulline during 60 min incubations. Either the presence of N-omega-L-arginine or the omission of calcium (in the presence of EDTA) significantly inhibited NOS activity. Adenosine triphosphate (ATP) at concentrations of 100-1000 microM significantly stimulated NOS activity, and was used as a positive control. ADP at a concentration of 1000 microM was found to significantly stimulate NOS activity, however, the other platelet aggregating substances investigated, thrombin (0.1, 10 U/ml), AVP (1, 10, 20 microM) and U46619 (3, 30, 300 nM), had no significant effect on NOS activity. These results show that ATP and ADP can stimulate human placental NOS activity, but provide no evidence that platelet aggregating agents other than ADP can affect the production of NO.

Determination of nitric oxide synthase activity in rat, pig and rabbit prostate glands.

The conversion of L-arginine to L-citrulline by nitric oxide synthase and other enzymes was studied in rat, pig and rabbit prostate glands by incubating preparations of the glands with [3H]L-arginine and measuring [3H]L-citrulline formation. The nitric oxide synthase inhibitor N(G)-nitro-L-arginine, (100 microM) reduced [3H]L-citrulline production in preparations from all three species. The arginase inhibitor L-valine (60 mM) inhibited [3H]L-citrulline production in rat and pig but not in rabbit prostate preparations. Omission of calcium or NADPH significantly reduced nitric oxide synthase-like activity in preparations from all three species but arginase-like activity was not significantly affected. The results indicate that the rabbit prostate contains the greatest amount of calcium-dependent nitric oxide synthase activity, the rat and pig prostates also have arginase-like enzymatic activity and the rat prostate contains an additional unidentified enzyme that converts L-arginine to L-citrulline.

Characterisation of excitatory and inhibitory transmitter systems in prostate glands of rats, guinea pigs, rabbits and pigs.

Excitatory and inhibitory transmitter systems were investigated in strips of prostate glands from rats, guinea pigs, pigs and rabbits. In strips from all species, electrical field stimulation (1 ms pulses at 1-30 Hz for 10 s) produced frequency-dependent contractions which were abolished by tetrodotoxin (1 microM). In strips from rats, guinea pigs and rabbits, contractions were reduced by prazosin (1 microM), guanethidine (10 microM) and atropine (2 microM), indicating the presence of noradrenergic and cholinergic mechanisms. However, the smooth muscle in the pig prostate appears to have a non-(nor)adrenergic non-cholinergic (NANC) excitatory innervation for which the transmitter was not identified. When noradrenergic and cholinergic mechanisms were blocked by guanethidine and atropine, respectively, and tone was raised with noradrenaline or methoxamine, field stimulation produced relaxations only in strips of rabbit prostate, and these were greatly reduced by N(G)-nitro-L-arginine methyl ester (L-NAME, 100 microM), providing functional evidence for a nitrergic relaxant innervation. In accord with this, nitric oxide (NO) synthase activity was considerably higher in rabbit than in rat or pig prostates.

Regulation of human placental fetal vessel tone: role of nitric oxide.

Factors affecting fetal vessel resistance have been studied in vitro in bilaterally perfused lobules of human placentae. Potent and efficacious constrictors in this preparation (in order of potency) include endothelin-1 > the thromboxane mimetic U46619 > endothelin-3 > prostaglandin F2 alpha. Inhibitors of eicosanoid synthesis did not affect fetal vessel basal perfusion pressure, nor did they potentiate the effects of the vasoconstrictor U46619. In contrast, the nitric oxide inhibitors N omega-nitro-L-arginine (NOLA), haemoglobin and methylene blue all increased fetal vessel basal perfusion pressure and also increased U46619-induced constriction. Similarly, NOLA markedly potentiated the constrictor effects of endothelin-1, angiotensin II, 5-hydroxytryptamine and bradykinin. These studies therefore provide evidence that NO is important in the maintenance of low basal fetal vessel impedance and also reduces the effects of a number of vasoconstrictor autacoids. Nitric oxide synthase (NOS) activity of human placental homogenates has been measured and shown to be mainly calcium-dependent. Human placental NOS activity was not affected by labour state but was reduced in pre-eclampsia. No evidence was found that in pre-eclampsia raised concentrations of the endogenous NOS inhibitor asymmetric dimethylarginine were responsible for the reduced placental NOS activity. Hence, these studies provide evidence that NO is an important endogenous dilator of the fetal vessels of the human placenta and that reduced NOS activity could contribute to the pathogenesis and/or effects of pre-eclampsia.

Human placental and fetal membrane nitric oxide synthase activity before, during and after labour at term.

The aim of this study was to determine whether any labour-associated changes in nitric oxide synthase (NOS) activity occur in human placenta and fetal membranes. NOS activity in amnion, choriodecidua, and placenta obtained from women before (at Caesarean section, not in labour), during (at Caesarean section, in labour) and after (spontaneous onset labour, normal vaginal delivery) labour was assessed by measuring conversion of radio-labelled L-arginine to L-citrulline. NOS activity, as judged by its inhibition by the specific NOS inhibitor N omega-nitro-L-arginine, was present in placental and amnionic tissues, but not in choriodecidual tissue specimens. Activity detected in choriodecidua was significantly blocked during incubation with a high concentration of valine, suggesting that L-arginine was being consumed by reactions other than NOS under the experimental conditions in that tissue. There were no significant differences among the labour groups in either amnion or placental NOS activities measured in the presence of 1 microM L-arginine. Amnion NOS activity was significantly less than that in placenta. Placental V(max) and Km values (determined after removal of endogenous L-arginine) did not differ significantly among the different labour groups.

Effect of asymmetric dimethyl arginine on nitric oxide synthase activity in normal and pre-eclamptic placentae.

An endogenous inhibitor of nitric oxide synthase (NOS), NG,NG dimethylarginine (asymmetric dimethylarginine, ADMA), which is present in human plasma and urine, has been reported to be elevated in the plasma of women with pre-eclampsia. As ADMA inhibition may contribute to reduced placental NOS activity observed in pre-eclampsia, the aim of this study was to compare the effects of ADMA on placental NOS activity from pre-eclamptic and normal pregnancies (gestational ages 38.4 +/- 0.9 and 38.3 +/- 0.3 weeks respectively). NOS activity was determined by measuring the conversion of [3H]L-arginine to [3H]L-citrulline in homogenates of normal and pre-eclamptic placentae in the absence and presence of increasing concentrations of ADMA (1-100 microM). The IC50 for ADMA for the pre-eclamptic placentae (22.1 +/- 2.1 microM, n = 6) was not significantly different from that for the normal placentae (18.8 +/- 1.4 microM, n = 6). When ADMA and L-arginine in homogenates was removed by ion exchange chromatography and exogenous L-arginine replaced (32 microM), the IC50 for the pre-eclamptic placentae (19.5 +/- 1.8 microM, n = 6) was not significantly different than that for the normal placentae (20.9 +/- 1.0 microM, n = 6), and NOS activity in the absence of endogenous and exogenous ADMA was still reduced in pre-eclamptic placentae. These results provide no evidence that the sensitivity of placental NOS to ADMA is affected by pre-eclampsia, or that placental ADMA contributes to the reduction of placental NOS in pre-eclampsia.

Role of uterine factors in the development of hypertension in SHR.

1. To examine whether the uterine environment plays a role in the development of hypertension in the spontaneously hypertensive rats (SHR), we have compared fetal weight, placental weight, and amniotic fluid composition of SHR and Wistar-Kyoto (WKY) rats after 20 days of gestation. 2. Pregnant SHR and WKY were anaesthetized at 20 days of gestation and the uterus and embryonic sacs removed. Fetal and placental weights were recorded and amniotic fluid collected for measurement of volume, osmolality and electrolyte composition. 3. No significant difference was found in litter size and placental weight between SHR and WKY. Total embryonic sac weight and fetal weight of SHR were significantly lower than WKY. Amniotic fluid volume, sodium concentration and osmolality of SHR were significantly higher than WKY, while amniotic fluid potassium concentration of SHR was significantly lower than WKY. 4. Thus, the SHR foetus was significantly underweight compared to the WKY and was bathed in amniotic fluid with a significantly higher osmolality and sodium concentration. As the mature foetus is known to drink amniotic fluid, it is hypothesized that the elevated Na/K ratio in SHR amniotic fluid may instigate or accelerate the hypertensive process.