Missing quality of life data in cancer clinical trials: serious problems and challenges

Measurement of quality of life (QOL) in cancer clinical trials has increased in recent years as more groups realize the importance of such endpoints. A key problem has been missing data. Some QOL data may unavoidably be missing, as for example when patients are too ill to complete forms. Other important sources are potentially avoidable and can broadly be divided into three categories: (i) methodological factors; (ii) logistic and administrative factors; (iii) patient-related factors. Logistic and administrative factors, for example, staff oversights, have proven to be most important. Since most QOL measurements require patient self-report, it is usually not possible to rectify the failure to collect baseline data or any follow-up assessments. There is strong evidence that such data are not 'missing at random', and cannot be ignored without introducing bias. Although several approaches to the analysis of partly missing data have been described, none is entirely satisfactory. Prevention of avoidable missing data is better than attempted cure. In July 1996, an international conference on missing QOL data in cancer clinical trials reported the experience of most major groups involved. This paper will serve as an introduction to the problem and provide an estimation of its magnitude, and approaches to its prevention and solution.     

Effects of vasodilators and perfusion pressure on coronary flow and simultaneous release of nitric oxide from guinea pig isolated hearts

OBJECTIVE: The aims were to validate the use of a direct reading NO electrode, to compare the effects of diverse acting drugs on altering coronary flow (CF) and NO release, and to examine the effects of altered perfusion pressure on flow-induced changes in NO concentration [NO] in the hemoglobin free effluent of guinea pig isolated hearts. METHODS: Hearts were isolated and perfused initially at a constant perfusion pressure (55 mmHg) with a modified Krebs-Ringer's solution equilibrated with 97% O2 and 3% CO2 at 37 degrees C. Heart rate, left ventricular pressure, CF, and effluent pH, pCO2, pO2, and NO generated current were monitored continuously on-line. Effluent was sampled for L-citrulline. Percent O2 extraction and O2 consumption were calculated. [NO] was quantitated with a sensitive amperometric sensor (sensitivity > or = 1 nmol/l approximately 3 pA) and a selective gas permeable membrane. RESULTS: The electrode was not sensitive to changes in solution pO2, flow, or pressure. The electrode was sensitive to pCO2 (-0.50 nmol/l/mmHg) and temperature (+24.5 nmol/l/degree C), so coronary effluent pCO2 was measured to compensate for a small decrease in pCO2 that occurred with an increase in coronary flow, and effluent temperature was rigidly controlled. Serotonin, bradykinin, and nitroprusside increased NO release along with CF, whereas nifedipine, butanedione monoxime, zaprinast, and bimakalim comparably increased CF but did not increase [NO] or NO release. Increases in CF (ml/g/min) and NO release (pmol/g/min), respectively, were 5.0 +/- 1 and 100 +/- 17 for 1 mumol/l serotonin, 7.5 +/- 1 and 148 +/- 18 for 100 nmol/l bradykinin, and 7.8 +/- 1 and 173 +/- 28 for 100 mumol/l nitroprusside. The increases in effluent NO by bradykinin were proportional to the increases in L-citrulline. Tetraethylammonium decreased CF, but did not change NO release, indomethacin changed neither CF nor NO release, and NG-nitro-L-arginine methyl ester (L-NAME) reduced CF by 2.6 +/- 1 ml/g/min and NO release by 25 +/- 8 pmol/g/min. An increase of CF of 8.0 +/- 0.3 ml/g/min, produced by increasing perfusion pressure from 25 to 90 mmHg, increased [NO] by 30 +/- 4 nmol/l; L-NAME but did not reduce the pressure-induced increase in CF, but reduced the increase in [NO] to 10 +/- 5 nmol/l. CONCLUSIONS: This study demonstrates in intact hearts real-time release of NO by several vasodilator drugs and by pressure-induced increases in flow (shear stress) and attenuation of these effects by L-NAME.     

Anterior-posterior patterning within the Caenorhabditis elegans endoderm

The endoderm of higher organisms is extensively patterned along the anterior/posterior axis. Although the endoderm (gut or E lineage) of the nematode Caenorhabditis elegans appears to be a simple uniform tube, cells in the anterior gut show several molecular and anatomical differences from cells in the posterior gut. In particular, the gut esterase ges-1 gene, which is normally expressed in all cells of the endoderm, is expressed only in the anterior-most gut cells when certain sequences in the ges-1 promoter are deleted. Using such a deleted ges-1 transgene as a biochemical marker of differentiation, we have investigated the basis of anterior-posterior gut patterning in C. elegans. Although homeotic genes are involved in endoderm patterning in other organisms, we show that anterior gut markers are expressed normally in C. elegans embryos lacking genes of the homeotic cluster. Although signalling from the mesoderm is involved in endoderm patterning in other organisms, we show that ablation of all non-gut blastomeres from the C. elegans embryo does not affect anterior gut marker expression; furthermore, ectopic guts produced by genetic transformation express anterior gut markers generally in the expected location and in the expected number of cells. We conclude that anterior gut fate requires no specific cell-cell contact but rather is produced autonomously within the E lineage. Cytochalasin D blocking experiments fully support this conclusion. Finally, the HMG protein POP-1, a downstream component of the Wnt signalling pathway, has recently been shown to be important in many anterior/posterior fate decisions during C. elegans embryogenesis (Lin, R., Hill, R. J. and Priess, J. R. (1998) Cell 92, 229-239). When RNA-mediated interference is used to eliminate pop-1 function from the embryo, gut is still produced but anterior gut marker expression is abolished. We suggest that the C. elegans endoderm is patterned by elements of the Wnt/pop-1 signalling pathway acting autonomously within the E lineage.     

Insulin-like growth factors in plasma and afferent mammary lymph of lactating cows deprived of feed or treated with bovine somatotropin

The galactopoietic actions of bovine somatotropin are both direct and indirect. Indirect actions are apparently mediated by the insulin-like growth factor (IGF) system. The objective of this study was to compare systemic (plasma) versus local (lymph) concentrations of IGF. Afferent mammary lymph most nearly represents the extracellular fluid that is bathing mammary tissue. Catheters were surgically implanted in the jugular vein and the superficial afferent mammary lymph vessels of four lactating cows. A crossover design was utilized to evaluate the effects of bovine somatotropin (bST). After 2 d of basal sampling, either bST (40 mg/d) or excipient was administered daily for 5 d. After the conclusion of the bST study, a second study was conducted in which cows were deprived of feed for 36 h. Blood and lymph were simultaneously sampled at least every 6 h throughout both studies. Milk yield was increased by bST, and concentrations of IGF-I were increased in plasma and lymph. The relationship between plasma and lymph concentrations for IGF-II, bST, insulin, glucose, urea nitrogen, and nonesterified fatty acids were similar during bST treatment. Milk yield was reduced 76% by 36 h of feed deprivation. Feed deprivation also caused a reduction of IGF-I in plasma, but concentrations of IGF-I in lymph were not altered. In contrast, changes in IGF-II, bST, insulin, glucose, urea nitrogen, and nonesterified fatty acids that were caused by feed deprivation followed similar patterns in plasma and lymph. Clearly, if IGF-I mediates the mammary actions of bST, then concentrations of IGF-I in plasma correlate with milk yield responses as well as, if not better than, concentrations in lymph.     

The CYP2 family: models, mutants and interactions

1. The construction of three-dimensional models of mammalian cytochromes P450 from the CYP2 family is reported based on protein sequence alignment with CYP102, a bacterial P450 of known crystal structure. 2. The homology models of CYP2 family enzymes appear to show self-consistency with the currently accumulated information from site-directed mutagenesis and chemical modification of amino acid residues known to affect redox partner interactions. 3. The generation of these models from the recently reported crystal structure of substrate-bound CYP102 enables the exploration of likely active site contacts with specific substrates of CYP2 family isozymes.     

Addressing the tertiary structure of human parathyroid hormone-(1-34)

Parathyroid hormone (PTH) regulates mineral metabolism and bone turnover by activating specific receptors located on osteoblastic and renal tubular cells and is fully functional as the N-terminal 1-34 fragment, PTH-(1-34). Previously, a "U-shaped" conformation with N- and C-terminal helices brought in close proximity by a turn has been postulated. The general acceptance of this hypothesis, despite limited experimental evidence, has altered the direction of the design of PTH-analogs. Examining the structure of human PTH-(1-34) under conditions that encompass the different environments the hormone may experience in the approach to and interaction with the G-protein-coupled receptor (including benign aqueous and saline solutions and in the presence of dodecylphosphocholine), we observe no evidence for a U-shape conformation or any tertiary structure. Instead, the N- and C-terminal helical domains, which vary in length and stability depending on the conditions, are separated by a highly flexible region of undefined conformation. These observations are in complete accord with recent conformational studies of PTH-related protein analogs containing lactams (Mierke, D. F., Maretto, S., Schievano, E. , DeLuca, D., Bisello, A., Mammi, S., Rosenblatt, M., Peggion, E., and Chorev, M. (1997) Biochemistry 36, 10372-10383) or a model amphiphilic alpha-helix (Pellegrini, M., Bisello, A., Rosenblatt, M., Chorev, M., and Mierke, D. F. (1997) J. Med. Chem. 40, 3025-3031). Reliable structural data from different environmental conditions are absolutely requisite for the next step in the design of non-peptide PTH analogs.     

trans-encapsidation of a poliovirus replicon by different picornavirus capsid proteins

A trans-encapsidation assay was established to study the specificity of picornavirus RNA encapsidation. A poliovirus replicon with the luciferase gene replacing the capsid protein-coding region was coexpressed in transfected HeLa cells with capsid proteins from homologous or heterologous virus. Successful trans-encapsidation resulted in assembly and production of virions whose replication, upon subsequent infection of HeLa cells, was accompanied by expression of luciferase activity. The amount of luciferase activity was proportional to the amount of trans-encapsidated virus produced from the cotransfection. When poliovirus capsid proteins were supplied in trans, >2 x 10(6) infectious particles/ml were produced. When coxsackievirus B3, human rhinovirus 14, mengovirus, or hepatitis A virus (HAV) capsid proteins were supplied in trans, all but HAV showed some encapsidation of the replicon. The overall encapsidation efficiency of the replicon RNA by heterologous capsid proteins was significantly lower than when poliovirus capsid was used. trans-encapsidated particles could be completely neutralized with specific antisera against each of the donor virus capsids. The results indicate that encapsidation is regulated by specific viral nucleic acid and protein sequences.     

Epoxidation of olefins by cytochrome P450: evidence from site-specific mutagenesis for hydroperoxo-iron as an electrophilic oxidant

P450 cytochromes (P450) catalyze many types of oxidative reactions, including the conversion of olefinic substrates to epoxides by oxygen insertion. In some instances epoxidation leads to the formation of products of physiological importance from naturally occurring substrates, such as arachidonic acid, and to the toxicity, carcinogenicity, or teratogenicity of foreign compounds, including drugs. In the present mechanistic study, the rates of oxidation of model olefins were determined with N-terminal-truncated P450s 2B4 and 2E1 and their respective mutants in which the threonine believed to facilitate proton delivery to the active site was replaced by alanine. Styrene epoxidation, cyclohexene epoxidation and hydroxylation to give 1-cyclohexene-3-ol, and cis- or trans-butene epoxidation (without isomerization) and hydroxylation to give 2-butene-1-ol were all significantly decreased by the 2B4 T302A mutation. Reduced proton delivery in this mutant is believed to interfere with the activation of dioxygen to the oxenoid species, as shown earlier by decreased hydroxylation of several substrates and enhanced aldehyde deformylation via a presumed peroxo intermediate. Of particular interest, however, the T303A mutation of P450 2E1 resulted in enhanced epoxidation of all of the model olefins along with decreased allylic hydroxylation of cyclohexene and butene. These results and a comparison of the ratios of the rates of epoxidation and hydroxylation support the concept that two different species with electrophilic properties, hydroperoxo-iron (FeO2H)3+ and oxenoid-iron (FeO)3+, can effect olefin epoxidation. The ability of cytochrome P450 to use several different active oxidants generated from molecular oxygen may help account for the broad reaction specificity and variety of products formed by this versatile catalyst.     

Dietary oxysterols are incorporated in plasma triglyceride-rich lipoproteins, increase their susceptibility to oxidation and increase aortic cholesterol concentration of rabbits

Early fatty streaks and advanced lesions are characterized by the deposition of cholesterol and cholesterol oxidation products (oxysterols). Oxysterols have been shown to be cytotoxic and pro-atherogenic compared to cholesterol and are found in cholesterol-rich processed foods. The consumption of dietary oxysterols may be significant in the onset and development of vascular disease. In order to study the short term effects of low levels of ingested dietary oxysterols on lipoprotein and aortic cholesterol and oxysterol levels, rabbits were fed either standard chow, chow supplemented with 1.0% oxidized cholesterol (containing 6% oxysterols), or 1.0% purified cholesterol (control). To determine the distribution and uptake of oxysterols after a 2-week dietary period, triglyceride-rich plasma lipoproteins, low density lipoproteins and aorta were analyzed by GC-MS. The concentration of 7beta-hydroxycholesterol was similar in all groups but the oxidized cholesterol-fed animals showed five times the concentration of 5alpha,6alpha-epoxycholesterol and double the level of 7-ketocholesterol in triglyceride-rich lipoproteins compared to the purified cholesterol-fed animals. The presence of 7-ketocholesterol in LDL was exclusive to animals fed the oxidized cholesterol diet. In addition, oxidation of triglyceride-rich lipoproteins was significantly greater in rabbits fed oxidized cholesterol compared to the pure cholesterol-fed animals. The oxidized cholesterol-fed animals also had a 64% increase in total aortic cholesterol, despite lower plasma cholesterol levels compared to the pure cholesterol control animals. Taken together these results suggest that dietary oxysterols may substantially increase the atherogenicity of lipoproteins.