Cancer-related pain: a pan-European survey of prevalence, treatment, and patient attitudes.

BACKGROUND: The European Pain in Cancer survey sought to increase understanding of cancer-related pain and treatment across Europe. PATIENTS AND METHODS: Patients with all stages of cancer participated in a two-phase telephone survey conducted in 11 European countries and Israel in 2006-2007. The survey screened for patients experiencing pain at least weekly, then randomly selected adult patients with pain of at least moderate intensity occurring several times per week for the last month completed a detailed attitudinal questionnaire. RESULTS: Of 5084 adult patients contacted, 56% suffered moderate-to-severe pain at least monthly. Of 573 patients randomly selected for the second survey phase, 77% were receiving prescription-only analgesics, with 41% taking strong opioids either alone or with other drugs for cancer-related pain. Of those prescribed analgesics, 63% experienced breakthrough pain. In all, 69% reported pain-related difficulties with everyday activities; however, 50% believed that their quality of life was not considered a priority in their overall care by their health care professional. CONCLUSIONS: Across Europe and Israel, treatment of cancer pain is suboptimal. Pain and pain relief should be considered integral to the diagnosis and treatment of cancer; management guidelines should be revised to improve pain control in patients with cancer.

Expression of a distinctive BCR-ABL oncogene in Ph1-positive acute lymphocytic leukemia (ALL).

The Philadelphia chromosome (Ph1) is a translocation between chromosomes 9 and 22 that is found in chronic myelogenous leukemia (CML) and a subset of acute lymphocytic leukemia patients (ALL). In CML, this results in the expression of a chimeric 8.5-kilobase BCR-ABL transcript that encodes the P210BCR-ABL tyrosine kinase. The Ph1 chromosome in ALL expresses a distinct ABL-derived 7-kilobase messenger RNA that encodes the P185ALL-ABL protein. Since the expression of different oncogene products may play a role in the distinctive presentation of Ph1-positive ALL versus CML, it is necessary to understand the molecular basis for the expression of P185ALL-ABL. Both P210BCR-ABL and P185ALL-ABL are recognized by an antiserum directed to BCR determinants in the amino-terminal region of both proteins. Antisera to BCR determinants proximal to the BCR-ABL junction in CML immunoprecipitated P210BCR-ABL but not P185ALL-ABL. Nucleotide sequence analysis of complementary DNA clones made from RNA from the Ph1-positive ALL SUP-B15 cell line, and S1 nuclease protection analysis confirmed the presence of BCR-ABL chimeric transcripts in Ph1-positive ALL cells. In Ph1-positive ALL, ABL sequences were joined to BCR sequences approximately 1.5 kilobases 5' of the CML junction. P185ALL-ABL represents the product of a BCR-ABL fusion gene in Ph1-positive ALL that is distinct from the BCR-ABL fusion gene of CML.

The tumour-suppressor Scribble dictates cell polarity during directed epithelial migration: regulation of Rho GTPase recruitment to the leading edge.

Altered expression of human Scribble is associated with invasive epithelial cancers, however, its role in tumour development remains unclear. Mutations in Drosophila Scribble result in loss of polarity, overproliferation and 3D-tumourous overgrowth of epithelial cells. Using complementation studies in Drosophila we recently demonstrated that expression of human Scribble can also regulate polarity and restrict tissue overgrowth. Here, we have undertaken a detailed study of human Scribble function in the polarized mammary cell line, MCF10A. We show that although Scribble does not seem to be required for apical-basal polarity or proliferation control in MCF10A cells, Scribble is essential for the control of polarity associated with directed epithelial cell migration. Scribble-depleted MCF10A cells show defective in vitro wound closure and chemotactic movement. The cells at the wound edge fail to polarize, show reduced lamellipodia formation and impaired recruitment of Cdc42 and Rac1 to the leading edge. Furthermore, we show that this function is relevant in vivo as Scribble mutant mice show defective epidermal wound healing. This data identifies an essential role for mammalian Scribble in the regulation of the polarity specifically involved in directed epithelial migration.

Clonogenic assay for Wilms' tumor: improved technique for obtaining single-cell suspensions and evidence for tumor cell specificity.

Wilms' tumors from seven patients were dissociated by mechanical and enzymatic means; this technique resulted in single-cell suspensions for five specimens and a few aggregates for two. By dye exclusion, cell viability ranged from 56 to 100% (median, 92%). All seven preparations produced more than five colonies/2 x 10(5) cells plated. Forty-three colonies grown from cells of a glucose-6-phosphate dehydrogenase heterozygote were of the same glucose-6-phosphate dehydrogenase isoenzyme type as the original tumor, indicating that the assay is specific for tumor cells. We attribute the high rate of colony formation to an improved method of cell preparation (combined mechanical and enzymatic dissociation of tumors) which may be applicable to other primary human tumors assayed in the soft agar system.

Comparative cytotoxic and cytokinetic effects of the epipodophyllotoxins 4'-demethylepipodophyllotoxin-9-(4,6-O-2-ethylidene-beta-D-glucopyranoside) and 4'-demethylepipodophyllotoxin-9-(4,6-O-2-thenylidene-beta-D-glucopyranoside) and their metabolites on human leukemic lymphoblasts.

We compared the effects of the epipodophyllotoxins 4'-demethylepipodophyllotoxin-9-(4,6-O-2-ethylidene-beta-D-glucopyranoside) (VP-16-213) and 4'-demethylepipodophyllotoxin-9-(4,6-O-2-thenylidene-beta-D-glucopyranoside) (VM-26) and several of their derivatives on cell cycle progression and viability of human leukemic lymphoblasts (CCRF-CEM). The cis-(picro)-lactone derivatives, like both VP-16-213 and VM-26, produced G2-phase arrest and cytotoxicity, but only at concentrations 100 times greater than required with the parent compounds. The epiaglycone derivative showed potent cytotoxicity: at 100 to 250 nM, it reduced cloning efficiency by 50%, an effect requiring 25 to 40 nM VM-26 and 340 to 425 nM VP-16-213. In contrast to VM-26 and VP-16-213, the epiaglycone arrested cells in M, consistent with evidence that it, like podophyllotoxin, is an inhibitor of microtubule assembly. At concentrations resulting in 50% cell kill and an increase of cells in M, however, the epiaglycone produced little change in the proportion of cells in G1 or early S phase, while podophyllotoxin produced a shift of cells to mid- and late S. The hydroxy acid derivatives, although found in detectable quantities in patients' urine and serum, were inactive in vitro. Structural differences among the compounds account for their different biochemical and cell kinetic effects and, hence, different cytotoxic activities. Because the epiaglycone is a potent compound and combines activities of both the podophyllotoxins and 4'-demethyl derivatives, further studies of its cytotoxicity are indicated.

Phase I clinical trial of orally administered 4-demethoxydaunorubicin (idarubicin) with pharmacokinetic and in vitro drug sensitivity testing in children with refractory leukemia.

Fifteen children with acute leukemia in relapse, refractory to conventional therapy, were treated with idarubicin administered orally for 3 consecutive days in dosages ranging from 30 to 50 mg/m2 per day at 19- to 21-day intervals. Gastrointestinal complications, including nausea, vomiting, abdominal pain, diarrhea and stomatitis, were the major forms of dose-limiting toxicity, affecting the majority of patients at all levels of idarubicin dosage. Two patients who had received total-body irradiation for bone marrow transplantation developed life-threatening gastrointestinal toxicity suggestive of a radiation "recall" phenomenon. Echocardiographic evidence of depressed cardiac function, without clinical symptoms or signs, was noted in six of 11 patients, although the changes were judged to be significant in only one child. The maximal tolerated oral dose of idarubicin was 40 mg/m2 per day. The medium terminal plasma half-life of idarubicin was 9.2 h (range, 6.4-25.5 h). Both idarubicin and its metabolite, idarubicinol, accumulated during the 3 days of therapy. Among the five patients with acute nonlymphoblastic leukemia whose cells were tested for drug sensitivity in vitro, the idarubicin concentration resulting in 50% inhibition (IC50) of cluster and colony formation ranged from 1.6 x 10(-10) M to 5 x 10(-7) M. There was no obvious relationship between the IC50 for idarubicin and that for epirubicin or daunorubicin. Oral idarubicin produced definite antileukemic effects, clearing blast cells from the circulation in 13 of the 14 evaluable patients. Future studies should define an optimal dose schedule to circumvent the limiting gastrointestinal complications associated with this agent.

Unexpectedly severe toxicity from intensive early treatment of childhood lymphoblastic leukemia.

In early 1984, we treated 13 consecutive patients with acute lymphoblastic leukemia (ALL) using an induction regimen of rapidly rotated combinations of prednisone, vincristine, asparaginase, teniposide (VM-26), cytosine arabinoside, and high-dose methotrexate (MTX) followed by leucovorin rescue. The intent of this clinical trial, designated Total Therapy Study XI, is to test the hypothesis that greater initial leukemia cell kill will decrease opportunities for the development of drug-resistant mutants, with resultant improvement in the length of disease-free survival. Five patients experienced life-threatening gastrointestinal toxicity within three weeks of the start of treatment. One died. Three other patients had severe abdominal pain, abdominal distention, diarrhea, and weight loss, but not gastrointestinal bleeding. In the remaining five patients, toxicity was rapidly reversible, and each child was able to complete the planned course of chemotherapy. The study was then amended to switch high-dose MTX from the induction phase to the consolidation phase, allowing at least one week for mucosal recovery. Among the next 28 patients who were treated, none showed evidence of severe gastrointestinal toxicity. Patients now receive high-dose MTX alone as consolidation therapy and are tolerating it adequately. Drug timing should be examined critically when intensified multiple-agent regimens are being devised for initial treatment of ALL.

Childhood monosomy 7 syndrome: clinical and in vitro studies.

The clinical and cell growth characteristics of 11 children with monosomy 7 presenting as preleukemia (eight cases) or acute nonlymphoblastic leukemia (three cases) were studied. Anemia was common to all patients, with nine showing leukocytosis, seven thrombocytopenia, and one thrombocytosis. There was a striking predominance of males (M/F ratio, 10:1) and a young median age (3 years). Preleukemia evolved to acute nonlymphoblastic leukemia in five patients and to myelofibrosis in one. In vitro studies of bone marrow progenitor cells cultured in leukocyte feeder-stimulated agar revealed abnormal cell proliferative patterns, most often an increased number of small clusters, for all 11 subjects. The cells of some preleukemic patients showed increased growth even in the absence of an exogenous source of colony-stimulating factor, suggesting autonomous growth or possibly autocrine stimulation. Combination chemotherapy or bone marrow transplantation failed to induce complete remission in the seven patients who were treated. Our findings in these 11 cases confirm the poor prognosis of monosomy 7 presenting as preleukemia in children. The in vitro studies suggest an association between altered cell growth in vitro and clinical evolution to frank leukemia.

Oxidation of a critical methionine modulates DNA binding of the Drosophila melanogaster high mobility group protein, HMG-D.

HMG-D is a major high mobility group chromosomal protein present during early embryogenesis in Drosophila melanogaster. During overexpression and purification of HMG-D from E. coli, a key DNA binding residue, methionine 13, undergoes oxidation to methionine sulfoxide. Oxidation of this critical residue decreases the affinity of HMG-D for DNA by three-fold, altering the structure of the HMG-D-DNA complex without affecting the structure of the free protein. This work shows that minor modification of DNA intercalating residues may be used to fine tune the DNA binding affinity of HMG domain proteins.

Illusion processing in hemispatial neglect.

Twelve patients with hemispatial neglect and two control groups were tested to examine the effects of the Müller-Lyer and Judd illusions on bisection behaviour. The studies were designed to investigate whether neglect patients were indeed unaware of the left sides of the illusory figures. In Experiment 1, participants were asked to describe the illusory figures prior to bisection, whereas in Experiment 2, they compared two illusions whose fins, in the critical condition, differed on the left and then performed the bisection. It was found that the illusions worked equally well in all three groups. Interestingly, apart from one exception, almost all neglect patients explicitly reported the left-sided fins in Experiment 1. Only five patients failed to do so but only on an average of 16% of trials. In Experiment 2, six patients made errors in the comparison task but four of these patients did not neglect any left-sided fins in Experiment 1 (with the exception of three overall trials for LC and EdR). This finding seems a good indication that the two tasks differ in their requirements. The comparison task may be perceived as harder as it requires discrimination rather than detection and thus lead to more neglect type errors than the bisection task. In one neglect patient, the illusions consistently failed to work. This patient presented with an occipito-temporal and basal ganglia lesion and the mechanisms responsible for the processing of simple visual features might have possibly been impaired in her case.

A new approach to template purification for sequencing applications using paramagnetic particles.

A new method using traditional hybridization methodology, coupled with the new magnetic particle technology, has been developed for DNA purification, specifically for sequencing applications. The method is similar to the reverse hybridization blot system; however, a specific oligonucleotide probe was attached to the paramagnetic particle instead of a sheet membrane. The target DNA containing the complementary sequence of the probe hybridizes to the probe that is attached to the bead and is then magnetically removed from solution, washed and collected. This system eliminates the need of organic extractions and precipitation/concentration steps. The entire hybridization-purification system can be done in a 1.5-ml microcentrifuge tube making the method ideal for automation. M13 phage clones were purified with this method, both by manual means and by using the CATALYST 800 Molecular Biology LabStation fitted with a prototype magnetic station, and then sequenced. DNA sequencing results obtained with this system were reproducible and gave excellent length of read with low background.

Heterozygote and mutation detection by direct automated fluorescent DNA sequencing using a mutant Taq DNA polymerase.

We describe a method for direct cycle sequencing of PCR fragments amplified from genomic DNA or cDNA. DNA sequencing template is amplified using PCR and oligonucleotide primers flanking the region of interest. The amplified fragment is directly cycle sequenced using fluorescent sequencing primers, Sanger dideoxy sequencing chemistry and an enzyme mixture of a mutant Taq DNA polymerase and thermostable pyrophosphatase. The sequence ladders produced are analyzed on a real-time, automated four-color sequencing system. The method produces sequence ladders from unpurified PCR fragments of sufficiently high quality such that heterozygotes can be reproducibly detected and identified by software that recognizes signal-strength patterns indicative of mixed-base positions.

Juvenile chronic myelogenous leukemia: surface antigen phenotyping by monoclonal antibodies and cytogenetic studies.

Cells from three children with juvenile chronic myelogenous leukemia were studied using culture in semisolid media, cytogenetic analysis, and surface staining with the monocyte-specific monoclonal antibodies 61D3 and 63D3. The percentage of bone marrow mononuclear cells that were 61D3- and 63D3-positive was markedly increased in all three patients. Bone marrow and peripheral blood mononuclear cells exhibited exceptionally bright immunofluorescence with these antibodies. The presence of monocyte-specific antigens on the surface of juvenile chronic myelogenous leukemia cells suggests that they are derived from a precursor with monocytic characteristics. A specific chromosomal abnormality (47,XY+21) was present in fresh bone marrow cells from one patient; in contrast, 50 metaphases from phytohemagglutinin-stimulated peripheral blood contained a normal karyotype. The chromosomal abnormality was also identified in myeloid colonies grown in vitro from this patient. Granulocytic elements were demonstrated in tissue sections and in cultured myeloid colonies from this child. Our data suggest that malignant transformation in juvenile chronic myelogenous leukemia involves a myeloid progenitor population capable of differentiation in vitro to cells with monocytic or granulocytic characteristics.

HLA-B alleles associated with the B15 serologically defined antigens.

Cells expressing HLA molecules in the B15 family were identified by serologic typing in routine testing of volunteer donors of various ethnic backgrounds for a bone marrow registry. DNA sequencing was used to identify HLA-B15 alleles associated with each serologic type and to examine the diversity within the B15 antigen family. Alleles which appeared predominantly in each B15 serologic cluster included: B15 with no defined serologic subdivision (B*1501), B62 (B*1501), B63 (B*1516, B*1517), B75 (B*1502, B*1521), and B76/77 (B*1513). Other B*15 alleles were also found associated with the serotypes and some of these alleles (e.g., B*1501 and B*1516) were found in two or more serologic clusters illustrating the complexity of this family. The B15 unsplit and B75 groups were the most complex exhibiting 16 and 7 alleles, respectively, within each serotype. Five new B*15 alleles (B*1530, B*1531, B*1533, B*1534, B*1535) and 5 other new HLA-B alleles (B*38022, B*3910, B*4010, B*51012, and B*5108) were also identified.

A generic sequencing based typing approach for the identification of HLA-A diversity.

Sequencing Based Typing (SBT) is a generic approach for the identification of HLA-A polymorphism. This approach includes the high resolution typing of the HLA-A broad reacting groups, HLA-A subtypes and will identify new alleles directly. The SBT approach described here uses a locus specific amplification of DNA from exon 1 to exon 5. The resulting 2,022 bp PCR product serves as a template for the subsequent sequencing reactions. Amplification is followed by direct sequencing of exons 2, 3 and 4 in both orientations with fluorescently labeled primers to define all polymorphic positions leading to a high resolution typing result. In this study the sequence of exons 2 and 3 of a panel of 49 cell lines was determined. In addition, the exon 4 region of 35 cell lines was also sequenced to evaluate the exon 4 polymorphism. The HLA-A type of most of the cells could be identified by sequencing only exons 2 and 3. However, the sequence of exon 4 was required to discriminate A*0201 from A*0209 and A*0207 from A*0215N. In this panel, an identical new "HLA-A*0103" was identified in two Caucasian samples.

Analysis of HIV type 1 protease and reverse transcriptase in antiretroviral drug-naive Ugandan adults.

We analyzed plasma HIV-1 from 27 antiretroviral drug-naive Ugandan adults. Previous subtype analysis of env and gag sequences from these samples identified subtypes A, C, D, and recombinant HIV-1. Sequences of HIV-1 protease and reverse transcriptase (RT) were obtained with a commercial HIV-1 genotyping system. Subtypes based on protease sequences differed from gag subtypes for 5 of 27 samples, demonstrating a high rate of recombination between the gag and pol regions. Protease and RT sequences were analyzed for the presence of amino acid polymorphisms at positions that are sites of previously characterized drug resistance mutations. At those sites, frequent polymorphisms were detected at positions 36 and 69 in protease and positions 179, 211, and 214 in RT. Subtype-specific amino acid motifs were identified in protease. Most of the subtype A sequences had the amino acids DKKM at positions 35, 57, 69, and 89, whereas most subtype D sequences had the amino acids ERHL at those positions. Detection of those polymorphisms may provide a useful approach for rapid identification of subtype A and D isolates in Uganda. This analysis significantly increases the number of Ugandan protease and RT sequences characterized to date and demonstrates successful use of a commercial HIV-1 genotyping system for analysis of diverse non-B HIV-1 subtypes.

Comparison of two clonogenic assays for determining cell growth patterns in childhood acute myeloid leukemia: relationships to clinical findings and prognosis.

Bone marrow cells from 99 patients with acute myeloid leukemia were cloned in either agar stimulated by leukocyte feeder layers (AG/F) or methylcellulose supplemented with medium conditioned by phytohemagglutinin stimulation of leukocytes (MC/P). Although cell growth in the two systems was correlated (r = 0.74, p less than 0.0001), there was increased formation and size of clusters and colonies in AG/F, suggesting that the clonogenic cells from children with AML are more readily assayed in AG/F. The number and size of clones in either system did not show a relationship to the morphologic subtype of leukemia. Depending on the scoring system used, increased growth in MC/P was related to abnormal karyotype. Also dependent on scoring system, the ability of leukemic cells to form small clusters in AG/F was associated with resistance to induction therapy: cells of patients with resistant disease were more likely to produce small clusters (p = 0.02). Our results suggest that clonogenic cells from children with AML grow more readily in AG/F than in MC/P, but that neither culture system supports the growth of cells from all patients. Depending on scoring criteria, in-vitro growth patterns in AG/F correlate with response to induction therapy.

Grip force scaling after hemispatial neglect.

Spatial neglect has been explained as an impairment in the representation of extrapersonal space. One account suggests that representations of extrapersonal space are spatially compressed following neglect. In support of this view it has been demonstrated that neglect patients systematically underestimate the size of stimuli presented in their left hemifield. In the current study we investigated this phenomena by obtaining an indirect measure of perceived object size - the scaling of grip force during prehension. We demonstrate for the first time that neglect patients show increased levels of grip force for objects presented in their left hemifield. This finding is discussed with reference to a proposed distinction between visual processing used for object recognition, and visual processing used to guide action.

Pharmacokinetics of Teniposide (VM26) and etoposide (VP16-213) in children with cancer.

The clinical pharmacokinetics of VM26 and VP16-213 were assessed in 15 children (median age 10 years) with acute leukemia, using a new high-performance liquid chromatography-electrochemical assay. Pharmacokinetic parameters were calculated by both model-dependent and compartment model-independent methods. These studies demonstrated substantial differences in the central volumes of distribution (VDc), steady-state volumes of distribution (VDss) and systemic clearances (Cls) of VM26 and VP16-213; with the VDc, VDss, and Cls all being smaller for VM26, Systemic clearances determined by model-independent methods were 5.2 +/- 1.0 ml/min/m2 (mean +/- SD) for VM26 and 17.8 +/- 11.2 ml/min/m2 for VP16-213. The major metabolites. detected in serum and urine were the hydroxy acids. Low levels of the picro-lactone isomers were detected in some patients while the aglycones were not detected in the serum or urine of any patients.

The epidemiology and therapy of airflow limitation in the elderly.

Airways disease is a frequent problem poorly recognised in older people. Many have reversible airway limitation and do not receive appropriate therapy. As in younger patients, pulmonary function tests are essential as a baseline and in relation to formal trials of treatment in both the diagnosis and management. Preferably, the assessment of all patients with airflow limitation should include a corticosteroid trial to correctly identify all patients who need long term prophylactic therapy. Many older patients have difficulty using the metered dose inhalers and the addition of volume spacer devices, though cumbersome, has many further advantages. In some patients, airflow limitation may be complicated by the presence of cardiac failure, arrhythmias and arterial hypoxia, and these problems also need to be reviewed.