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We investigated whether low melting point phase-change waxes could be incorporated into emergency drug transport bags to attenuate the known temperature extremes their contents can be exposed to. We exposed two custom-made hollow-walled drug containers placed within a pair of drug transport bags to three day/night cycles including periods of direct radiant sunlight. The wall cavities of one contained air, whereas those of the other contained a paraffin wax (melting point of 44-46 °C) with a high latent heat of fusion (until fully melted, its temperature does not increase further). We collected 25,920 temperature datasets at six locations. We found that 97.8% and 84.7% of ampoule temperatures within the wax and air cavity containers, respectively, were within a target range of 15-40 °C over the study duration (Levene statistic W = 4279.1; Levene's test for equality of variance, p0.001). Ampoule temperatures in the wax cavity container only exceeded 40 °C for 1.7% of the time. Even when they did so, their temperature was attenuated to 40.3 °C, despite an ambient air temperature of40 °C for 6.4% of the time (peak 46.9 °C) and a bag surface temperature of40 °C for 17.2% of the time (peak 64.4 °C). In contrast, the ampoule temperature in the air cavity container exceeded 40 °C for 17.1% of the time (peak 54.1 °C). The latent heat of fusion of phase-change materials may be exploited in the design of drug transport bags to mitigate any temperature changes in the drugs stored within them.

Background Talactoferrin alfa is an oral dendritic cell (DC)-mediated immunotherapy (DCMI). We tested whether talactoferrin was superior to placebo in advanced non-small-cell lung cancer (NSCLC). Patients and methods An FORTIS-M trial was an international, multicenter, randomized, double-blind comparison of talactoferrin (1.5g p.o. BID) versus placebo BID, in patients with stage IIIB/IV NSCLC whose disease had failed two or more prior regimens. Treatment was administered for a maximum of five 14-week cycles. The primary efficacy end point was overall survival (OS); secondary end points included 6- and 12-month survival, progression-free survival (PFS), and disease control rate (DCR). Results Seven hundred and forty-two patients were randomly assigned (2:1) to talactoferrin (497) or placebo (245). The median OS in the intent-to-treat (ITT) population was 7.66 months in the placebo arm and 7.49 months in the talactoferrin arm [hazard ratio (HR), 1.04; 95% CI, 0.873–1.24; P = 0.6602]. The 6-month survival rates were 59.9% (95% CI, 53.4% to 65.8%) and 55.7% (95% CI, 51.1% to 59.9%), respectively. The 12-month survival rates were 32.2% (95% CI, 26.3% to 38.2%) and 30.9% (95% CI, 26.8% to 35%), respectively. The median PFS rates were 1.64 months and 1.68 months, respectively (HR, 0.99; 95% CI, 0.835–1.16; P = 0.8073). The DCRs were 38.4 and 37.6%, respectively [stratified odds ratio (OR), 0.96; 95% CI, 0.698–1.33; P = 0.8336]. The safety profiles were comparable between arms. Conclusions There was no improvement in efficacy with talactoferrin alfa in patients with advanced NSCLC whose disease had failed two or more previous regimens.

Mutations in MECP2 cause the autism-spectrum disorder Rett syndrome. MeCP2 is predicted to bind to methylated promoters and silence transcription. However, the first large-scale mapping of neuronal MeCP2-binding sites on 26.3 Mb of imprinted and nonimprinted loci revealed that 59% of MeCP2-binding sites are outside of genes and that only 6% are in CpG islands. Integrated genome-wide promoter analysis of MeCP2 binding, CpG methylation, and gene expression revealed that 63% of MeCP2-bound promoters are actively expressed and that only 6% are highly methylated. These results indicate that the primary function of MeCP2 is not the silencing of methylated promoters.

[reaction: see text] L-Selectride is an efficient agent for the 3-O-demethylation of opioids and is known to cleave the least hindered methoxyl group in a molecule. The treatment of a 3,4-dimethoxymorphinan containing a 6-ketal with L-Selectride gave selective 4-O-demethylation, rather than cleavage of the less hindered 3-methoxyl. In contrast, a 3,4-dimethoxymorphinan lacking a 6-ketal gave selective 3-O-demethylation, suggesting that the regiochemistry of L-Selectride-mediated O-demethylation can be manipulated through altering the position of coordination of the lithium ion.

Small cell lung cancer remains an important malignancy with increasing lung cancer rates in many countries. It is important to distinguish between better and poorer prognostic patient groups in order to target therapy more effectively. Modern chemotherapy usually includes a platinum combination and in selected patient groups combined modality with thoracic and prophylactic cranial irradiation. For poorer prognostic groups, treatment is less well defined and less commonly researched. Nevertheless the integration of combined modality treatments and novel drugs beckons towards an exciting avenue for future research.

Lung cancer presents a major worldwide health problem, accounting for 10% of all cancers. The treatment for those patients with small cell lung cancer has been unsatisfactory, but combination chemotherapy administered with radiotherapy can effect a cure in a minority of patients. Intensification of chemotherapy can improve the cure rate, and there are now a variety of haemopoietic growth factors available to support this approach, as the principal toxicity of the drugs used today is bone marrow suppression. Strategies to protect the bone marrow are also in development and are likely to allow further dose intensification. Recently randomised trials have reported survival benefits for intensified treatments.

The overall median survival of patients with systemic metastasis from melanoma is about 6 months. Survival is dependent on the sites of first metastasis, the resectability of the metastases, and the number of metastases. Patients with non-visceral metastases at first relapse i.e. in skin, subcutaneous tissues, distant lymph nodes, and lung, have a better survival rate than patients with visceral (liver, bone, brain) metastases. Treatment of patients with systemic melanoma should include careful evaluation for the potential role of surgery, radiotherapy, and systemic therapy. The main use of chemotherapy in metastatic melanoma patients remains palliative. The chapter reviews the chemotherapeutic options available for the treatment of malignant melanoma including recent published works on new agents, multi-agent therapy, high dose chemotherapy with autologous bone marrow rescue, adjuvant chemotherapy and regional perfusion chemotherapy.

Objectives: Accelerometer-based, portable navigation instrumentation is a new method of achieving desired resection alignments in total knee arthroplasty (TKA). Methods: After randomisation and the application of exclusion criteria, 79 knees were analysed. 42 patients which underwent TKA using conventional intra-medullary (IM) alignment guides were compared to 37 patients with the use of accelerometer-based, portable navigation device (KneeAlign; OrthoAlign Inc, Aliso Viejo, California). Radiographic results were obtained from post-operative computer-tomography following the CT Perth Protocol. Results: In the IM cohort, 81.0% of patients had a coronal alignment within 3° of a neutral mechanical axis (vs 83.8% with KneeAlign, p=0.74), 81.0% had a femoral coronal alignment within 2° of perpendicular to the femoral mechanical axis (vs 89.2% with KneeAlign, p=0.31), and 92.9% had a tibial coronal alignment within 2° of perpendicular to the tibial mechanical axis (vs 81.1% with KneeAlign, p=0.12). Regarding sagittal alignment, the IM cohort had 90.5% of patients with femoral component alignment within 2° of optimum (vs 91.9% with KneeAlign, p=0.83) and 92.9% had a tibial component alignment within 2° of the optimal tibial slope (vs 89.2% with KneeAlign, p=0.57). The mean tourniquet time (from incision to completion of coronal bone resections) in the IM cohort was 16.5± 8.9 minutes vs 22.2 ± 7.6 minutes in the KneeAlign cohort (pConclusion: Accelerometer-based, portable navigation has a statistically similar outcome in alignment following TKA as IM guides. It is noted that using the portable navigation device does prolong surgical time compared to conventional IM surgery and this may be due to the learning curve.

The United Kingdom Coordinating Committee on Cancer Research represents the major organizations funding cancer research in the United Kingdom. The deliberations of a working party convened by the committee to evaluate recently expressed concerns that the changes in the NHS threaten research, especially clinical trials to evaluate new treatments, are reported. A survey of contributors to trials coordinated by the committee showed that half are now experiencing difficulties in continuing to participate in clinical trials. The two major problems identified were lack of time and of staff, especially for NHS staff in non-teaching hospitals. Recent changes in junior doctors' hours and proposed reductions in the length of time for training will exacerbate this. It is possible to identify the direct and indirect excess costs of conducting research in the NHS, but currently the mechanism does not exist to designate funds specifically for this purpose. Consultation with the regional directors of research and development confirmed that the service increment for teaching and research is not the solution for this. Proposals are made to secure future clinical research in the NHS, including finance, indemnity, the licensing of new drugs, the greater use of nurse counsellors, and the value of cancer registries.

The anticancer nucleoside analogue, Gemcitabine (2′, 2′-difluoro-2′-deoxycytidine) is an active nucleoside against solid tumours. Assessment of tumour and normal tissue concentration of drug in vivo provides pharmacokinetic data that can be related to response to treatment. Towards enabling PET imaging, the synthesis of a halogenated analogue has been undertaken for subsequent radiolabelling with 124I.

A database of 327 patients with advanced Renal Cell Carcinoma (RCC) has been analyzed in order to identify potential baseline prognostic factors predicting for survival, following recombinant Interleukin-2 treatment (rIL-2). All patients received a continuous infusion (CIV). Eligibility criteria were uniform across studies, and included patients with an ambulatory performance status (PS), measurable disease, no CNS metastases, and no major organ compromise. Multivariate analyses identified baseline PS (ECOG 0 vs. 1), time from diagnosis to treatment (DTI greater than 24 months vs. less than or equal to 24 months), and the number of metastatic sites (1 vs. greater than or equal to 2, where lung, bone and other sites are considered as separate sites) as important predictors for survival. Patients can be classified into 4 subgroups, which are a function of the number of risk factors present. Median survival for each subgroup is 28, 17, 10 and 5 months, respectively. The model was validated in an independent cohort of 125 patients with RCC treated with subcutaneous (s/c) rIL-2, and predicted for survival accurately. By determining in which risk group category patients may fall, treating physicians may be better equipped to decide on patient management. The model may also be of value in order to stratify patients in randomized clinical trials.

The tissue and cellular distribution of the DNA repair protein O6-alkylguanine-DNA-alkyltransferase (ATase) is an important question in relation to the response of tumour and normal tissues to chemotherapeutic regimes employing alkylating agents such as methyltriazenes and nitrosoureas. In order to examine this issue by immunostaining, we have raised a rabbit antiserum to apparently pure recombinant human enzyme. The antiserum is highly specific and sensitive, detecting a band at 24 kDa on western blots of crude extracts of ATase-expressing human lymphoblastoid cells, liver and melanoma. Adjacent sections of acetone or formalin fixed normal human liver and subcutaneous malignant melanoma were reacted with preimmune serum or antiserum and an immunoperoxidase detection system with silver enhancement was used to locate binding of the primary antibody to the antigen. In sections reacted with preimmune serum or with antigen-preadsorbed antiserum, only faint cytoplasmic and little or no nuclear staining was seen. In contrast, using antiserum, the reaction in positively staining cells was very intense and predominantly nuclear. In the liver, there was interindividual variation in the cellular distribution of reaction with staining present in all discernable cell types in most samples but confined to the hepatocytes and bile duct epithelial cells in others. In the melanoma sections, all discernable cell types showed mainly nuclear staining: the intensity of staining varied between tissue samples and there was evidence of a range of intermediate staining intensities with some melanoma cells showing no detectable reaction. Images Figure 1 Figure 2 Figure 3 p358-a Figure 4

Previous studies showed that 4-hydroxy-3-methoxyindolomorphinans had variable delta opioid affinity and selectivity. Herein, we show that the 3,4-dimethoxy analogs possessed similar low affinity, whereas the 3-hydroxy-4-methoxy analogs showed excellent delta opioid affinity and selectivity comparable with the parent indolomorphinans.

Background More than 85% of Rett syndrome (RTT) patients have heterozygous mutations in the X-linked MECP2 gene which encodes methyl-CpG-binding protein 2, a transcriptional repressor that binds methylated CpG sites. Because MECP2 is subject to X chromosome inactivation (XCI), girls with RTT express either the wild type or mutant MECP2 in each of their cells. To test the hypothesis that MECP2 mutations result in genome-wide transcriptional deregulation and identify its target genes in a system that circumvents the functional mosaicism resulting from XCI, we performed gene expression profiling of pure populations of untransformed T-lymphocytes that express either a mutant or a wild-type allele. Methods Single T lymphocytes from a patient with a c.473C>T (p.T158M) mutation and one with a c.1308-1309delTC mutation were subcloned and subjected to short term culture. Gene expression profiles of wild-type and mutant clones were compared by oligonucleotide expression microarray analysis. Results Expression profiling yielded 44 upregulated genes and 77 downregulated genes. We compared this gene list with expression profiles of independent microarray experiments in cells and tissues of RTT patients and mouse models with Mecp2 mutations. These comparisons identified a candidate MeCP2 target gene, SPOCK1, downregulated in two independent microarray experiments, but its expression was not altered by quantitative RT-PCR analysis on brain tissues from a RTT mouse model. Conclusion Initial expression profiling from T-cell clones of RTT patients identified a list of potential MeCP2 target genes. Further detailed analysis and comparison to independent microarray experiments did not confirm significantly altered expression of most candidate genes. These results are consistent with other reported data.

Mutations within the gene encoding methyl CpG binding protein 2 (MECP2) cause the autism-spectrum neurodevelopmental disorder Rett Syndrome (RTT). MECP2 recruits histone deacetylase to methylated DNA and acts as a long-range regulator of methylated genes. Despite ubiquitous MECP2 expression, the phenotype of RTT and the Mecp2-deficient mouse is largely restricted to the postnatal brain. Since Mecp2-deficient mice have a defect in neuronal maturation, we sought to understand how MECP2/Mecp2 mutations globally affect histone modifications during postnatal brain development by an immunofluorescence approach. Using an antibody specific to acetylated histone H3 lysine 9 (H3K9ac), a bright punctate nuclear staining pattern was observed as MECP2 expression increased in early postnatal neuronal nuclei. As neurons matured in juvenile and adult brain samples, the intensity of H3K9ac staining was reduced. Mecp2-deficient mouse and RTT cerebral neurons lacked this developmental reduction in H3K9ac staining compared to age-matched controls, resulting in a significant increase in neuronal nuclei with bright H3K9ac punctate staining. In contrast, trimethylated histone H3 lysine 9 (H3K9me3) localized to heterochromatin independent of MeCP2, but showed significantly reduced levels in Mecp2 deficient mouse and RTT brain. Autism brain with reduced MECP2 expression displayed similar histone H3 alterations as RTT brain. These observations suggest that MeCP2 regulates global histone modifications during a critical postnatal stage of neuronal maturation. These results have implications for understanding the molecular pathogenesis of RTT and autism in which MECP2 mutation or deficiency corresponds with arrested neurodevelopment.

Rett syndrome (RTT), caused by mutations in MECP2 (encoding methyl CpG binding protein 2), and Angelman syndrome (AS), caused by maternal deficiency of chromosome 15q11-13, are autism-spectrum neurodevelopmental disorders. MeCP2 is a transcriptional repressor of methylated genes, but MECP2 mutation does not directly affect the imprinted expression of genes within 15q11-13. We tested a potential role for MeCP2 in the homologous pairing of imprinted 15q11-13 alleles in human brain tissue and differentiated neurons by fluorescence in situ hybridization (FISH). FISH analysis of control cerebral samples demonstrated a significant increase in homologous pairing specific to chromosome 15 from infant to juvenile brain samples. Significant and specific deficiencies in the percentage of paired chromosome 15 alleles were observed in RTT, AS and autism brain samples when compared with normal controls. SH-SY5Y neuroblastoma cells also showed a significant and specific increase in the percentage of chromosome 15q11-13 paired alleles following induced differentiation in vitro. Transfection with a methylated oligonucleotide decoy specifically blocked binding of MeCP2 to the SNURF/SNRPN promoter within 15q11-13 and significantly lowered the percentage of paired 15q11-13 alleles in SH-SY5Y cells. These combined results suggest a role for MeCP2 in chromosome organization in the developing brain and provide a potential mechanistic association between several related neurodevelopmental disorders.

PURPOSE: Gemcitabine-platinum combination activity has been clearly established in a number of phase II studies. It has also been compared against other combinations in many phase III trials. It is generally believed that all such regimens have an equivalent impact on survival. This meta-analysis aims to quantify the treatment effect of gemcitabine plus a platinum agent in the treatment of advanced NSCLC and compare the combination to other regimens used globally. DESIGN: Data from a total of 4556 patients from 13 randomized trials investigating gemcitabine in combination with a platinum agent versus any other platinum-containing regimen were included in a meta-analysis of time-to-event outcomes. RESULTS: A significant reduction in overall mortality in favor of gemcitabine-platinum regimens was observed, hazard ratio (HR) 0.90 (95% CI: 0.84-0.96) with an absolute benefit at 1 year of 3.9%. Median survival was 9.0 months for the gemcitabine-platinum regimens and 8.2 months for the comparator regimens. Sub-group analysis of the first- and second-generation platinum-based comparator regimens also indicated a significant benefit for gemcitabine-platinum regimens, HR 0.84 (CI: 0.71-0.9985). Analysis of third-generation agent plus platinum regimens showed a non-significant trend favoring gemcitabine-platinum regimens, HR 0.93 (CI: 0.86-1.01). There was a significant decrease in the risk of disease progression in favor of gemcitabine-platinum regimens, HR 0.88 (CI: 0.82-0.93). An absolute benefit of 4.2% at 1 year was estimated. Median progression-free survival was 5.1 months for gemcitabine-platinum regimens compared with 4.4 months for the comparator regimens. Sub-group analysis indicated a statistically significant progression-free survival benefit for patients assigned to gemcitabine-platinum treatment compared to first- and second-generation platinum regimens, HR 0.85 (CI: 0.77-0.94), and third-generation agent plus platinum regimens, HR 0.89 (CI: 0.82-0.96).

Publisher Summary Rett syndrome (RTT) is characterized by mental retardation, deceleration of head growth, loss of purposeful hand movements, ataxia, loss of language, autistic features, seizures, and respiratory dysfunction. Autism, RTT, and Asperger syndrome (AS) are all characterized by loss or impairment of language, stereotyped behaviors, and a high frequency of seizures and sleep abnormalities. The combined phenotypic and genotypic evidence has set the stage for the emerging hypothesis that these three autism spectrum disorders share an overlapping molecular pathogenesis. This chapter discusses the evidence supporting this hypothesis by linking methyl CpG binding protein 2 (MeCP2), expression of genes within 15q11-13, and neuronal nuclear organization in an important molecular pathway that is defective in all three disorders. The MeCP2 binding sites and allele-specific methylation patterns across the entire 15q11-13 region, as well as the proposed chromatin loop structures, need to be defined. Another potential avenue to pursue would be genetic and environmental interactions on MeCP2 and 15q11-13 gene regulation. As MeCP2 expression is tightly linked to neuronal maturation, pharmacological agents and other compounds that either enhance or reduce neuronal stimulation, it could affect MeCP2 expression levels. In addition, environmental agents—such as mercury or pesticides that inhibit MeCP2 or gamma-aminobutyric acid receptor subunit beta-3 (GABRB3) function—could be involved in triggering autism in genetically susceptible individuals.

EDITORIAL COMMENT: We accepted this case report not only because readers could encounter a patient with Wegener granulomatosis, but also because the use of cyclophosphamide from 22 weeks gestation did not harm the fetus. We have asked the authors to provide readers with a 100–200 word summary of the aetiology, clinical features and complications of Wegener granulomatosis. Author's response: Wegener granulomatosis is a systemic necrotizing granulomatous vasculitis of unknown aetiology with a predilection for the kidney and upper respiratory tract, especially the nose and sinuses. It classically causes acute renal failure, epistaxis, and pulmonary haemorrhage. It can however affect any organ and untreated has an 82% 1-year mortality. It can occur at any age but is more common in elderly patients. It is very strongly associated with cytoplasmic antineutrophil cytoplasmic antibody (cANCA) which is directed against a serine proteinase 3 antigen and can be used to follow the course of the disease. Unless the patient has very localized disease, treatment always involves cyclophosphamide and high dose steroids ± plasmapheresis to obtain remission. This would then be followed by a prolonged course of azathioprine and steroid treatment.

The Western Australian (WA) Remote Area Dialysis Programme was developed in 1988 due to the cultural need to dialyze an increasing number of aboriginal patients in their own communities, rather than relocating them up to 3000 km away in Perth. The success of the program relies on remote area health services (RAHS), which have no prior experience in continuous ambulatory peritoneal dialysis (CAPD), providing consistent routine and emergency medical care to the patients. Our aim was to standardize the care of all CAPD patients in remote WA by providing the RAHS with an easy -to-follow manual. Although the RAHS received treatment protocols and in-service education, consistent care was not always provided. We confirmed this by: (1) examining the existing quality assurance tools, peritonitis and hospital admission rates, (2) discussion with remote area staff regarding patients, and (3) informal assessment of remote area staff receptiveness to in-service education by a CAPD nurse. We identified the causes of the inconsistent care to be: (1) high remote area staff turnover (six months average for a registered nurse), (2) the protocols were difficult to follow, and (3) confusion for the RAHS as to the appropriate contact person at our hospital. In 1994, the situation was exacerbated by the dramatic increase in the number of patients and RAHS involved (14 new patients, bringing the total to 20 patients in 12 centers) plus the introduction of a second treating hospital (with differing protocols). A team of two CAPD nurses and two nephrologists was established, to collaborate with two remote area hospitals and the second treating hospital to produce the “Remote Area CAPD Manual.” The manual is an easy-to-follow, stepby-step guide for the management of CAPD by nondialysis personnel. It has led to improved management of CAPD, improvement in communication with RAHS, and the increased confidence of remote area staff in the management of CAPD patients. In conclusion, RAHS can give consistent care if provided with clear, concise guidelines.

To delineate the differences between the structural requirements necessary for recognition at sigma-1 and sigma-2 receptors, a range of phenethyl- and phenylpropylpiperidines were evaluated in binding assays. Phenethylpiperidines were found to favor sigma-1 receptors, whereas phenylpropylpiperidines tend to favor sigma-2 receptors. It appears that phenylpropylamine is a potential pharmacophore for selective sigma-2 ligands.

The Ki-1 (CD 30) antigen has been identified as a marker of a subset of large cell anaplastic non-Hodgkin's lymphoma that may have a more favourable prognosis. We describe an immunosuppressed renal transplant patient who developed primary extranodal large cell anaplastic Ki-1 lymphoma of T-cell phenotype confined to the sigmoid colon. The patient was treated with local resection of the sigmoid colon and reduction of immunosuppression and at two year follow-up remains free of recurrence. It is recognised that long term immunosuppression in post transplant patients increases the risk of certain malignancies. Localised gut lymphoma may be amenable to local resection as a curative procedure. (Aust NZ J Med 1992; 22: 51–53.)

O6-Methylguanine-DNA methyltransferase (MGMT) is a major determinant of resistance to temozolomide. Its levels are depleted in lymphocytes after drug administration, but there is partial recovery by 24 hr, the usual time of subsequent dosing. Administering subsequent doses of temozolomide at the MGMT nadir could enhance its effectiveness, by increasing the amount of O6-methylguanine (O6-meG) in DNA. We evaluated the efficacy of such a schedule of temozolomide and determined the kinetics of MGMT depletion and O6-meG formation in DNA following treatment. Thirty patients with advanced malignant melanoma were treated with temozolomide 1,000 mg/m2 equally split into 5 doses over a 16 hr period every 28 days. O6-meG formation was determined in peripheral blood mononuclear cell (PBMC) DNA and, in a subset of patients, in tumor tissue during the first treatment cycle. MGMT levels fell rapidly with dosing, reaching a nadir in PBMCs of 18.0 +/- 2.26% of initial levels. O6-meG levels increased during the treatment period, peaking at 11.1 +/- 1.25 micromol/mol dG in PBMCs and at 4.25 +/- 0.79 micromol/mol dG in tumor biopsies. The main toxicities were grade IV thrombocytopenia in 12 patients (42.8%) and grade IV neutropenia in 11 patients (39.2%), associated with fever in 8 cases. There were 7 responses (1 complete), for an overall response rate of 23.3%; median overall survival was 6.1 months. The compressed schedule has activity against melanoma, with greater MGMT depletion and O6-meG formation than previously reported for O6-alkylating agent regimens. Myelosuppression precludes its wider application, but MGMT in PBMCs predicted the dose intensity of temozolomide that patients could sustain, suggesting a means by which individuals suitable for this approach might be identified.

This study was designed to assess the activity of oral topotecan (TPT) in patients with advanced non-small cell lung cancer previously untreated with chemotherapy. Eligible patients had inoperable stage III or stage IV non-small cell lung cancer and were chemotherapy-naive. Other inclusion criteria were Eastern Cooperative Oncology Group performance status 0, 1, or 2, adequate bone marrow, and renal and hepatic function. Of 30 patients, 29 were assessable for response. Oral TPT was administered for 5 days every 21 days for up to six cycles unless disease progression or unacceptable toxicity occurred. Patients received a dose of 2.3 mg/m2/day for the first cycle. Dose modification for subsequent cycles was based on tolerability. Patients completed symptom questionnaires every 3 weeks. Pharmacokinetics were evaluated in all patients during cycle 1. Three patients had radiological responses with a reduction in tumor size of 30-40%. No patients achieved complete or partial responses to treatment. Thirteen patients had a stable disease (43.3%), and the median survival was 39.9 weeks with a 1-year survival of 33.3%. At the time of analysis, 27 patients had died. Median time to progression was 12.3 weeks. Treatment was well tolerated. A total of 125 cycles of treatment were completed. Twelve patients (40%) experienced grade III/IV neutropenia. Five patients (16.6%) had grade III/IV anemia. There were two episodes of grade III/IV thrombocytopenia. The main nonhematological toxicities consisted of grade III nausea (13%) and grade III vomiting (13%). The most frequently reported disease-related symptoms at baseline were dyspnea, cough, and fatigue. There was a subsequent improvement in patient scores of dyspnea in 17% of patients, 31% showed improvement in cough, and 32% showed improvement in fatigue. The mean area under the curve of TPT following 2.3 mg/m2 p.o. was 51.6 ng.h/ml (%SD, 25%). The area under the curve of TPT on day 1 of the first cycle was correlated with the percentage fall in leukocytes. Although oral TPT at the applied dose and schedule showed modest activity as a single agent, almost one-half of the patients had a stable disease, and median time to progression was 12.3 weeks. The overall median survival was a promising 39.9 weeks, and useful palliation of symptoms was seen.

Tumour resistance to methylating agents is linked to expression of the DNA repair protein O(6)-alkylguanine-DNA alkyltransferase (ATase). There is considerable interest in improving the efficacy of O(6)-alkylating chemotherapy by prior depletion of ATase. We have tested the ability of a modified guanine base, O(6)-(4-bromothenyl)guanine (4BTG), to inactivate ATase and to enhance the anti-tumour effect of temozolomide in an animal model system. A375M human melanoma xenografts were established in the flanks of nude mice. ATase depletion after a single dose of 4BTG or O(6)-BG (20 mg/kg i.p.) was determined over a 24 hr period. Subsequently, we tested the effect of 4BTG (20 mg/kg i.p. daily) and/or temozolomide (80-175 mg/kg i.p. daily) over a 5-day schedule on tumour growth. 4BTG was an effective inactivator of ATase in tumour, producing complete depletion within 2 hr of dosing. Furthermore, it enhanced the tumour growth delay achieved with temozolomide, increasing the tumour quintupling time by 8.7 days (95% confidence interval 6.1-11.3 days, p0.0001). Whilst the delay in tumour growth was indistinguishable from that observed with O(6)-benzylguanine (O(6)-BG) and temozolomide, the 4BTG combination resulted in considerably less toxicity (0/9 vs. 2/9 deaths; 6.84% weight loss vs. 9.48%, p = 0.019). 4BTG is a potent inactivator of ATase and enhances the therapeutic ratio of temozolomide in this model system to a greater extent than O(6)-BG.

The effect of expression of an O 6 -benzylguanine (O 6 -beG)-resistant mutant (hATPA/GA) of human O 6 -alkylguanine-DNA alkyltransferase (ATase) on the in vivo toxicity and clastogenicity of the anti-tumour agent N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU) to murine bone marrow has been Investigated. When compared with control animals, the bipotent granulocyte-macrophage colony-forming (GM-CFC) progenitor population of the hATPA/GA transduced mice were somewhat more resistant to BCNU (1.4-fold, P = 0.047) and this effect was more significant In the presence of the ATase inactivator O 6 -beG (3.5-fold, P = 0.001). The polychromatic erythrocytes were also significantly protected against BCNU-induced clastogenicity both in the presence (P < 0.001) and absence of O 6 -beG (P < 0.05). The primitive, multipotent spleen colony-forming cells (CFU-S) in these animals also showed moderate (1.6-fold, P = 0.034) protection in the absence of O 6 -beG but in the presence of the inactivator they remained as sensitive to BCNU toxicity as those in the control animais (P 0.133). This result contrasts with previous findings demonstrating significant hATPA/GA-mediated, O 6 -beG-resistant protection against the toxicity and clastogeniclty of a number of O 6 -alkylating agents, including temozolomide, fotemustine and chlorozotocin. The possibility that our strategy for protective gene therapy may be highly agent and cell-type specific is unexpected and has possible implications for clinical trials of this approach using BCNU or related agents.

Retroviral transduction was used to introduce cDNAs encoding two mutants of human O6-alkylguanine-DNA alkyltransferase (hAT), one of which (hATPA) is 16 times more resistant to O6-benzylguanine (O6-beG), and the other (hATPA/GA) which is almost totally refractory to inactivation relative to the wild-type protein, into K562 human erythroleukaemic cells. A colony-forming assay was used to demonstrate significant protection (P < 0.001) against mitozolomide or temozolomide toxicity in K562 clones expressing either hAT mutant, as determined from an in vitro assay of activity. However, protection against these agents was reduced in hATPA expressing cells in the presence of 1 microM O6-beG and was lost in the presence of 20 microM O6-beG while cells expressing hATPA/GA retained protection even in the presence of 20 microM O6-beG (P < 0.001). Using primary human cord blood-derived CD34+ haemopoietic cells in which PCR analysis indicated that up to 70% of progenitors were transduced with retroviral constructs harbouring hATPA/GA, we observed significant protection of the granulocyte-macrophage colony-forming cells against mitozolomide (P < 0.05) and temozolomide (P < 0.001) induced toxicity in the presence of O6-beG. These findings indicate that retrovirus-mediated expression of hATPA/GA in primitive primary human haemopoietic cells is possible and does provide O6-beG-resistant protection for these cells. Using this strategy in patients may simultaneously permit attenuated myelosuppression and increased sensitivity of tumour cells to the effects of O6-alkylating agent chemotherapy. These data, taken together with the study reported by Chinnasamy et al in the accompanying article in this issue showing reduced toxicity and clastogenicity in murine haemopoietic progenitors, make a compelling case to test this strategy clinically.

Murine bone marrow cells were transduced ex vivo with a retrovirus encoding an O6-benzylguanine (O6-beG) insensitive, double mutant form of the human DNA repair protein O6-alkylguanine-DNA alkyltransferase (hATPA/GA). In animals reconstituted with the transduced bone marrow, about 50% of cells in the multipotent spleen colony-forming cells (CFU-S) and lineage restricted granulocyte–macrophage (GM-CFC) haemopoietic progenitor populations were found to be carrying the transgene and this correlated with the frequency of bone marrow cells and spleen colonies which stained positive for hATPA/GA by immunocytochemistry. Expression of hATPA/GA was associated with significant in vivo protection of both CFU-S (P = 0.001) and GM-CFC (P < 0.024) against the toxicity of the antitumour methylating agent, temozolomide, given in combination with o6-beG. Expression of hATPA/GA also led to a reduction in the frequency of combined O6-beG/temozolomide-induced micronuclei seen in polychromatic erythrocytes (P < 0.003). this study is the first to demonstrate in vivo protection of multipotent haemopoietic progenitors against the toxic and clastogenic effects of an o6-alkylating agent in the presence of O6-beG. It also represents the first report of reduced clastogenesis as a consequence of expression of an O6-beG-resistant ATase. In the accompanying article we report hATPA/GA-mediated resistance of human CD34+ haemopoietic progenitors to combined O6-beG/O6-alkylating agent toxicity. Together these two reports suggest that a gene therapy strategy whereby protection of normal haemopoietic tissue may be combined with O6-beG-mediated tumour sensitisation may be efficacious in achieving an increase in therapeutic index.

Patients with cancer have inappropriately low levels of endogenous erythropoietin for the degree of anemia and further suppression of erythropoiesis results from chemotherapy. Patients with lung cancer, in particular, require a high frequency of transfusions, as they are unable to tolerate the symptoms of anemia due to their underlying pulmonary disease and, often, their age. Data from phase I and II trials indicated that epoetin alfa could increase hemoglobin concentration and reduce transfusion requirements. The beneficial response was dose dependent. These findings were confirmed in a series of three double-blind, placebo-controlled, multicenter phase III trials. Clinical trial evidence indicates that 150 IU/kg epoetin alfa three times weekly effectively treats anemia and decreases transfusion requirements in most cancer patients after the first month of chemotherapy. Furthermore, epoetin alfa will reduce the degree of anemia and markedly reduce the need for transfusions, thereby preventing anemia in patients undergoing multiple cycles of platinum-based combination chemotherapy. Epoetin alfa is well tolerated and shows marked activity in preventing anemia and reducing blood transfusion requirements in patients undergoing cyclic chemotherapy.

Progression through the mammalian cell cycle is controlled by a series of cyclins, cyclin-dependent kinases (cdks) and cdk inhibitors. Cyclin D1, cdk4 and the tumour suppressors p16 and retinoblastoma protein (pRb) are thought to comprise a linked system governing cell passage through the G1 phase of the cell cycle. Extending an earlier study on cyclin D1 expression, a series of resectable non-small cell lung carcinomas (NSCLCs) was examined for defects in other elements of this control system. Forty-six of fifty-one NSCLC specimens exhibited at least one alteration of these cell-cycle regulators. Immunohistochemical analysis revealed that 33% and 47% of the tumours failed to express pRb and p16, respectively. Failure to detect pRb did not correlate with loss of heterozygosity at the RB1 locus. Eleven of 12 tumours showing positive (normal) pRb staining over-expressed nuclear localised cyclin D1, including 8 with amplification of the cyclin D1 gene (CCNDI). However, in a number of lesions (n = 5) where cyclin D1 was over-expressed but localised to the cytoplasm, pRb expression was undetectable. Sequencing of exons 1 and 2 of the p16 gene (CDKN2) revealed 3/51 tumours with somatic mutations (in addition to 1 case with a germ-line alteration). All of these lesions were positive for p16 protein. No clear homozygous deletions of CDKN2 were observed by multiplex PCR. As assessed by immunostaining using a p16 monoclonal antibody, there was an inverse correlation of pRb and p16 down-regulation. Whilst patients with tumours over-expressing cyclin D1 had a significantly lower incidence of local relapse, the group whose tumours failed to express pRb had a significantly greater risk of local relapse and tended to have shortened event-free survival. Our data show that alteration of at least one cell cycle-regulator gene occurs in the majority of resectable NSCLCs.

Recent studies have indicated that chemotherapy not only provides some survival benefit, but also reduces tumor-related symptoms and improves the performance status of patients receiving chemotherapy. Data from single-agent gemcitabine studies demonstrate improvements in a range of tumor symptoms, including cough, hemoptysis, pain, dyspnea, and anorexia, as well as increases in performance status. Indeed, more patients benefit from gemcitabine chemotherapy than suggested by the objective response rate. Surveys also have shown that patients are much more likely to accept chemotherapy for what is perceived by health care professionals as potentially small benefits. Gemcitabine has a role in the palliative treatment of patients with advanced non-small cell lung cancer.

The chiral title compound, C29H33NO4, crystallized from methanol. The absolute configuration was assigned by reference to an unchanging chiral center in the synthetic procedure.

Using Northern blotting and PCR analysis of cDNA derived from a range of cell lines and tissues, alternate splicing of the cyclin D1 gene (CCND1) mRNA has been demonstrated. The variant transcript shows no splicing at the downstream exon 4 boundary, encoding a protein with an altered carboxy-terminal domain. Investigation of mRNA extracted from mononuclear cells, lung tumour and normal tissue suggests that both transcripts are invariably expressed. However, splicing to produce the two forms of mRNA is modulated, in the heterozygote, by a frequent A/G polymorphism located within the splice donor region of exon 4. Preliminary analysis of patients with resectable non-small cell lung cancer suggests that genotype is associated with shortened event free survival and greater risk of local relapse.

Disseminated primary varicella zoster infection in renal transplant patients can result in severe and often fatal illness. The disease tends to be much more severe in adults with an 80% mortality in the only reported series (1). We report 3 cases of severe disseminated varicella zoster in adult renal transplant patients who all survived. Early diagnosis, institution of intravenous acyclovir 10 mg/kg tds, zoster immunoglobulin, cessation of azathioprine treatment and aggressive supportive care may improve an otherwise bleak prognosis.

Combination ifosfamide/carboplatin/etoposide (ICE) chemotherapy and ICE plus mid-cycle vincristine (VICE) are reviewed. Thoracic radiotherapy and prophylactic cranial irradiation given as single fractions in the majority of patients have been intercalated with VICE in the later studies. The patient populations have not been intensively staged with computed tomography, etc, but do have reasonable Karnofsky performance status ratings and biochemical screens. A policy of no dose reduction over six courses of VICE chemotherapy has been followed in three consecutive studies of 166 patients. The minimum length of follow-up is 26 months and the 2-year survival rate isor = 30%. Hematopoietic growth factor support in an attempt to overcome the considerable myelosuppression with VICE therapy is currently being evaluated.

Metastatic carcinoma of uncertain primary site (CUPS) is a common problem and has a poor prognosis. The intention of this study was to determine whether the addition of cisplatin and etoposide (PE) to vincristine, doxorubicin and cyclophosphamide (VAC) chemotherapy improves outcome.Fifty-seven consecutive patients with an initial diagnosis of CUPS were studied. The first 40 patients had received six or 10 cycles of VAC and 17 patients VAC alternating with PE for six cycles. Review of histology using immunohistochemical techniques where appropriate was performed in all cases.Histologic review resulted in six tumors reclassified as non-Hodgkin's lymphoma (NHL), one as hepatocarcinoma, and one as adenocarcinoma. Six of the 11 responses occurred in patients with a review diagnosis of NHL. If the six cases of NHL and the case of hepatocarcinoma were excluded, there was no difference in survival between VAC and VAC/PE treated patients. Five patients with true CUPS who responded to VAC or VAC/PE had poorly differentiated histology, and this group (n = 24) also had a significantly longer survival.Some patients thought to have CUPS actually have NHL, and it is this group which responds well to chemotherapy. True CUPS patients respond to chemotherapy only if the tumor is poorly differentiated; survival is significantly longer for this group of patients. An advantage for VAC/PE over VAC chemotherapy was not identified.

The effects of recombinant human interleukin-4 (rhu IL-4) on immunological parameters in patients receiving increasing doses of IL-4 in a Phase I trial were investigated. Peripheral blood mononuclear cells were phenotyped for a variety of lymphocyte markers, but no consistent effects were observed. However, increases in HLA Class II expression on monocytes were detected in four patients. NK and LAK activity were neither induced nor augmented by IL-4 treatment. Slight increases in proliferative responses to mitogens and cytokines were observed in some patients. The latter observations and other clinical studies suggest that a combination of IL-4 with IL-2 may be more effective in Phase II clinical trials.

The haematopoietic colony stimulating factors (CSFs) are glycoproteins that were found to stimulate and promote the proliferation of granulocyte and monocyte progenitor cells on semi-solid media in clonogenic assays (Table 1) [1]. The complex process of haematopoiesis is controlled by the diverse but intergrated actions of the CSFs, their target cells and stroma. Although CSFs are mainly noted for their proliferation and differentiation activities, other important aspects of their function include maintenance of cell viability, membrane integrity and functional stimulation of mature cells, e.g., granulocyte phagocytosis, superoxide production etc. (Table 2) [2]. Besides the CSFs a variety of other factors such as erythropoietin and interleukins (ILs) also have important effects on proliferation and differentiation of target cells.

Quality of life in patients with inoperable lung cancer receiving palliative treatment has become an important issue. As in other types of cancer, lack of standardization of quality of life data makes comparison between trials difficult, sometimes even impossible. Much effort is being given to the development of suitable instruments to measure quality of life and strategies to incorporate these measurements into clinical trials. The authors review studies in palliative lung cancer treatment where two crucial questions are being addressed: Do patients improve in performance status and symptom relief and does treatment toxicity outweigh disease palliation?

Two monoclonal antibodies used to investigate the expression of the epidermal growth factor receptor (EGF-R) in 45 lung cancers were compared. The R1 antibody recognises the extracellular domain portion of the receptor and the F4 is directed against the cytoplasmic portion. The reactivities of the two antibodies have been compared in fresh frozen tumour specimens. In addition the staining activity of the F4 antibody (the first to EGF-R which can be used in archival material) was compared using frozen and paraffin sections from the same series of tumours. Comparisons of the numbers of cells staining with the R1 and F4 antibody showed only slight discrepancy when fresh material was examined. The discrepancy that existed could be explained by the heterogeneity of the tumours. Very similar results were obtained using the F4 antibody on paraffin embedded and fresh non-small cell lung cancer material. We conclude that the expression EGF-R can be detected reliably by the F4 antibody in both the fresh frozen and formalin fixed, paraffin embedded material and could be useful in assessing the clinical importance of EGF-R in archival tumour material.