Unrecognized Heterogeneity Research Articles

The use of frailty hazard models for unrecognized heterogeneity that interacts with treatment: considerations of efficiency and power.

Increasingly, genetic studies of tumors of the same histologic diagnosis are elucidating subtypes that are distinct with respect to clinical endpoints such as response to treatment and survival. This raises concerns about the efficiency of using the simple log-rank test for analysis of treatment effect on survival in studies of possibly heterogeneous tumors. Furthermore, such studies, designed under the assumption of homogeneity, may be severely underpowered. We derive analytic approximations for the asymptotic relative efficiency of the simple log-rank test relative to the optimally weighted log-rank test and for the power of the simple log-rank test when applied to subjects with unobserved heterogeneity, as reflected in a continuous frailty, that may interact with treatment. Numerical studies demonstrate that the simple log-rank test may be quite inefficient if the frailty interacts with treatment. Further, there may be a substantial loss of power in the presence of the frailty with or without an interaction with treatment.

Intrahepatic Heterogeneity of Hepatic Venous Pressure Gradient in Human Cirrhosis Again

Background: The hepatic venous pressure gradient (HVPG) is used to evaluate portal hypertension. Methods: We measured HVPG in two separate liver veins in 169 liver vein catheterizations in 102 cirrhosis patients and in 27 patients with no liver disease (controls). Results: In the controls, the two measurements differed by 0.0 ± 1.8 mmHg (mean ± s , n = 27), upper 95% confidence limit 3.6 mmHg (mean + 2 s ). HVPG ranged from-0.1 to 8.3 mmHg, upper 95% confidence limit 6.7 mmHg. In cirrhosis, the two measurements agreed within ± 3.6 mmHg in 39%. In 61%, the measurements differed by 4-34 mmHg. In 35%, fluoroscopy demonstrated hepatic vein-to-hepatic-vein shunting in veins with low HVPG values. In some patients with HVPG measurements above 30 mmHg, Doppler ultrasound examination showed arterialization of the hepatic vasculature. Discussion: Our results demonstrate a hitherto unrecognized notable heterogeneity of the intrahepatic vasculature and HVPG measurements in cirrhosis. The presumption of interposition ...

Intrahepatic Heterogeneity of Hepatic Venous Pressure Gradient in Human Cirrhosis

Background: The hepatic venous pressure gradient (HVPG) is used to evaluate portal hypertension. Methods: We measured HVPG in two separate liver veins in 169 liver vein catheterizations in 102 cirrhosis patients and in 27 patients with no liver disease (controls). Results: In the controls, the two measurements differed by 0.0 ± 1.8 mmHg (mean ± s , n = 27), upper 95% confidence limit 3.6 mmHg (mean + 2 s ). HVPG ranged from-0.1 to 8.3 mmHg, upper 95% confidence limit 6.7 mmHg. In cirrhosis, the two measurements agreed within ± 3.6 mmHg in 39%. In 61%, the measurements differed by 4-34 mmHg. In 35%, fluoroscopy demonstrated hepatic vein-to-hepatic-vein shunting in veins with low HVPG values. In some patients with HVPG measurements above 30 mmHg, Doppler ultrasound examination showed arterialization of the hepatic vasculature. Discussion: Our results demonstrate a hitherto unrecognized notable heterogeneity of the intrahepatic vasculature and HVPG measurements in cirrhosis. The presumption of interposition of non-flowing blood between the catheter tip and the portal system for the measurement of HVPG may thus be violated in about one-third of the cirrhosis cases because of abnormal outlet into hepatic venous shunts and in a minor fraction because of abnormal arterial inlet. In 26%, one measurement was below 12 mmHg, the other measurement above. If the HVPG had been measured in only one liver vein, 13% of the cases would have been classified in a lower risk group than appropriate according to the 12 mmHg concept of risk of bleeding from oesophageal varices.

The Value of the Aggregate Data Approach in Meta-Analysis with Time-to-Event Outcomes

Summary Collecting individual patient data has been described as the ‘gold standard’ for undertaking meta-analysis. If studies involve time-to-event outcomes, conducting a meta-analysis based on aggregate data can be problematical. Two meta-analyses of randomized controlled trials with time-to-event outcomes are used to illustrate the practicality and value of several proposed methods to obtain summary statistic estimates. In the first example the results suggest that further effort should be made to find unpublished trials. In the second example the use of aggregate data for trials where no individual patient data have been supplied allows the totality of evidence to be assessed and indicates previously unrecognized heterogeneity.

Intratumoral variations in DNA ploidy and s-phase fraction in human breast cancer.

To study intratumoral DNA ploidy heterogeneity and S‐phase fraction (SPF) variability, we prospectively collected five different samples from 48 breast carcinomas and each sample was analysed separately by flow cytometry. Aneuploidy rate was 89.6% after analysis of four or five samples. DNA ploidy heterogeneity, i.e., different samples classified as either DNA euploid or DNA aneuploid in the same tumor was seen in 17%, and DNA index heterogeneity, i.e., tumor populations with different DNA indices (DIs) seen in different samples was 44%. A statistical model defining SPF heterogeneity is proposed. SPF heterogeneity as defined by us was 71%, and as expected the SPF heterogeneity rate increased significantly with increasing number of analysed samples. Four or more samples are needed to detect the most deviant (highest) SPF values. An unrecognized intratumor heterogeneity of DNA ploidy and SPF may partly explain the conflicting results reported in the literature on the above prognostic indicators.

Aggressive competition between males, female-controlled polygyny and sexual monomorphism in a Malagasy primate, Propithecus verreauxi

The evolutionary determinants of sexual dimorphism among primates continue to be debated. This paper uses field observations of a Malagasy primate, Propithecus verreauxi, to argue that one problem underlying this debates is the unrecognized heterogeneity of mating system categories used in comparative analyses investigating the importance of sexual selection in determining sexual dimorphism. It is suggested that at least one new mating system category warrants recognition, that of female-controlled polygyny, with predictions for sexual dimorphism that are in contrast to those of male-controlled polygyny.

Heterogeneity in the Xenopus ribosomal transcription factor xUBF has a molecular basis distinct from that in mammals

The Xenopus polymerase I transcription factor xUBF is an HMG-box protein which has been purified as two polypeptides of ∼82 and 85 kDa. Recently a cDNA sequence predicted an xUBF protein (xUBF1) of 677 amino acids (79 kDa) containing five tandem HMG-boxes. Here a second and distinct xUBF cDNA has been isolated and characterised. This cDNA codes an xUBF protein (xUBF2) of 701 amino acids (82 kDa), having 93% homology with xUBF1 but containing an insertion of 22 amino acids between HMG-boxes 3 and 4. In vitro translation of synthetic mRNAs derived from the xUBF1 and 2 cDNAs was used to show that the electrophoretic mobility of the gene products accounted for the major xUBF molecular weight heterogeneity noted in vivo. It is also shown that the Xenopus laevis genome contains 3 or 4 distinct loci which hybridise with xUBF coding sequences, leaving open the possibility of yet further unrecognised heterogeneity in xUBF.

Analytical isoelectric focusing with immobilized pH gradients of human apolipoprotein E from very low density lipoproteins and total plasma.

A method for analytical isoelectric focusing (IEF) of apolipoprotein E (apoE) in immobilized pH gradients (IPG) and immunodetection of the separated isoforms has been developed for use with either very low density lipoproteins (VLDL) or whole plasma. Both VLDL and plasma were sequentially delipidated with 1,4-dioxane, acetone-ethanol, and ether. Neuraminidase treatment preceded the delipidation when required. Using preformed plates, pH 5.0-6.0 (LKB, Bromma) after rehydration with 6 M urea and dextran T-10, the IPG focusing pattern of the common isoforms (E2, E3, E4) was found to be equivalent to conventional IEF with the added resolution of the E4 disialo form. The use of self-poured narrower gradients permitted the further resolution of the E4 monosialo form, a previously unrecognized heterogeneity of the E2, E3, and E4 monosialo isoforms and differentiation of the apoE2** mutant; all of these forms comigrate with the common isoproteins in conventional IEF. Finally, the conditions for IPG of whole plasma using apoE monoclonal antibodies and enzyme-conjugated anti-mouse IgG for detection were established. Thus, IPG focusing is shown to be a powerful method for resolution of the apoE sialoforms and apoE mutant forms. The method has important implications in accurate and diagnostic phenotyping. Moreover, it is a convenient method for phenotyping which requires only very small volumes of plasma.

Unusual Composition of Peptidoglycan in Bordetella pertussis

The composition of the peptidoglycan of Bordetella pertussis and the nature of its turnover products was determined by a new combination of analytical techniques: high performance liquid chromatography of an enzymatic peptidoglycan hydrolysate and fast atom bombardment mass spectrometry and fast atom bombardment collision-activated dissociation tandem mass spectrometry. Sixteen major components of the peptidoglycan were purified, and assignment of complete or partial chemical structures was achieved for nine and seven species, respectively. At this level of resolution, a previously unrecognized heterogeneity of monomeric (five new species; nine total) and dimeric species (five new species; five total) was detected. No species containing diaminopimelyl-diaminopimelic acid cross-links or lysyl-arginine substitutions were found. Previous estimates of total cross-linkage and average chain length were revised downward to 32% and 21 disaccharide residues, respectively. Detection of a chemically novel species, a disaccharide octapeptide monomer, in both the peptidoglycan hydrolysate and culture supernatant fluid, suggests that an N-acetyl-muramyl-L-alanine amidase acts on the intact peptidoglycan of Bordetella and participates in cell wall turnover. Five peptidoglycan turnover products were identified in the supernatant fluid of late logarithmic phase cultures, including the 1,6-anhydro monomeric species known as tracheal cytotoxin. Peptidoglycan turnover was detected at a low rate of approximately 10%/generation, a value sufficient to account for the generation of all tracheal cytotoxin found in culture supernatant fluids.

The effect of neuronal cells on kidney differentiation.

During embryonic growth, tissue interactions between dissimilar cells are the driving forces of morphogenesis. Although their importance has been well known for over the past 50 years, the molecular background of these interactions has remained unelucidated. The unrecognized heterogeneity of those mesenchymal cells that are involved in the epithelio-mesenchymal tissue interactions may be one reason for this. For example, studies of kidney differentiation show that the metanephric organ rudiment contains more cell-lines than previously thought. Identification of both neural crest- and mesoderm-derived cells in the nephrogenic mesenchyme helps in re-evaluating the biology of the tubule induction. The neural crest-derived cells of the nephric rudiment differentiate into neuronal cells, and later during differentiation some of them are found in the stroma. There is also experimental evidence for the role of these neuronal cells in the morphogenetic tissue interaction.

The colonic goblet cell and glycoprotein heterogeneity.

Mucin glycoproteins are an important 'nonspecific' host defense at the interface of mucosal surface and lumen. However insights into structural and functional features of these enormously complex and heterogeneous glycoproteins have been markedly limited. In recent studies, the presence of several distinct colonic mucin glycoproteins has been demonstrated and their oligosaccharide side chains extensively characterized through conventional structural analysis and utilization of monoclonal antibodies. Glycoprotein diversity has been found to arise from previously unrecognized functional heterogeneity of colonic goblet cells. Rates of biosynthesis and secretion of different HCM glycoproteins appear to vary. In addition, a selective reduction in HCM glycoprotein IV has been demonstrated in specific association with ulcerative colitis. Finally preliminary studies suggest that additional alterations in colonic glycoconjugates may be present in ulcerative colitis mucosa.

Cell-specific heterogeneity in sensitivity of phosphatidylinositol-anchored membrane antigens to release by phospholipase C

Cell surface antigens thought to be linked to the membrane via phosphatidylinositol (PI) are incompletely, and variably, released by treatment with phosphatidylinositol-specific phospholipase C (PI-PLC). The basis for this was investigated with cloned tumor cell lines and PI-PLCs isolated from two species of bacteria. Residual Thy-1 antigen, which was detectable by flow cytometry, remained on all thymoma cell lines after exposure to very high concentrations of either purified enzyme. A majority of the presumptive PI anchored molecules on all of the cell lines was sensitive to release by PI-PLC derived from Bacillus thuringiensis. However, cell lines differed dramatically in the ease with which PI-PLC from Staphylococcus aureus liberated the same surface antigens. This heterogeneity was determined at the single cell level because at least five different PI-anchored antigens exhibited similar behavior on a given cell line or transfected subclones of it. The two phospholipases differed with respect to molecular mass, serological cross-reactivity and sensitivity to inhibition by NaCl and detergents. These observations suggest that the two types of PI-PLC may have distinct substrate specificities or sensitivities to environmental conditions which account for the difference in their ability to act on PI-anchored proteins in particular cell types. Such enzymes should continue to be important tools for investigating the method and significance of attachment of lymphocyte surface glycoproteins. In particular, the S. aureus PI-PLC can be used to demonstrate and investigate a previously unrecognized heterogeneity in cells which express PI-anchored molecules.

Analysis of platelet von Willebrand factor antigen.

Platelet lysates were obtained from suspensions of normal washed platelets by freeze-thawing or Triton X-100 lysis. The resultant platelet lysates contained 0.34 +/- 0.15 U/10(9) platelets (n = 8) of von Willebrand factor antigen (vWf:Ag) as determined by radioelectroimmunoassay using a monospecific antibody to vWf:Ag. The vWf:Ag level was higher in platelet lysates prepared from freshly drawn blood than from outdated platelet packs. Platelet lysates from patients with severe von Willebrand's disease type I (n = 2) did not contain detectable vWf:Ag. When normal platelet lysates were analyzed by radiocrossed immunoelectrophoresis in agarose using a monospecific polyclonal antibody to plasma vWf:Ag, two immunochemically identical precipitin peaks were seen. One of the platelet vWf:Ag peaks corresponded in its electrophoretic mobility to plasma vWf:Ag, while the other peak, i.e. platelet vWf:Ag-peak II, migrated to a more anodal position. The presence of the platelet vWf:Ag-peak II suggests structural differences between plasma and platelet vWf:Ag and illustrates previously unrecognized heterogeneity of platelet vWf:Ag.

MONOCLONAL ANTI‐LWab AND ANTI‐D REAGENTS RECOGNIZE A NUMBER OF DIFFERENT EPITOPES

Monoclonal antibodies (mAbs) which detect antigens on human red cells are also suitable for testing cells of other species. Such studies may reveal previously unrecognized heterogeneity in antibodies which apparently detect the same antigen on the human red cell surface. Information is also provided on specificities shared amongst several species. Here three anti-LWab and a variety of Rh-related antibodies have been tested against the red cells of various primates. One monoclonal anti-LWab antibody, BS46, reacted with the red cells of gorillas and rhesus monkeys but not those of orang-utans, baboons or marmosets. In contrast, BS56 and NIM-M8 reacted with the cells of all these species. Chimpanzee cells, however, reacted only with NIM-M8. Use of primate cells has shown that all three monoclonal anti-LWab antibodies recognize different epitopes. These observations may explain early conflicting data concerning primate cells. The difference between the monoclonal anti-D, D4, and three other anti-D antibodies, 8G2, 8D6 and 7D10, has been confirmed. The D antigen is apparently confined to the red cells of apes and humans. D4 recognizes a polymorphism in chimpanzees and 8G2, 8D6 and 7D10 recognize a polymorphism in gorillas. Two Rh-related mAbs, R6A and K70, were also investigated. R6A fails to react with Rhnull cells and reacts more weakly with homozygous-D-cells than with cells of common Rh phenotypes. K70 reacts weakly with Rhnull and -D-/-D- cells. The antigen detected by R6A is confined to the red cells of humans, gorillas and chimpanzees, while the antigen detected by K70 shows a wider species distribution. In some primates LW antigens are expressed in the absence of the determinant recognized by R6A. This phenotype has never been known to occur in humans.

Immunochemical variants of HLA-B27.

Detailed study of HLA-B27 was prompted by the extremely strong associations between this antigen and spondyloarthropathies. Despite the relative homogeneity of this antigen when defined by alloantisera, B27 reactivity with the monoclonal antibody B27M2 suggests previously unrecognized heterogeneity. To define and confirm this heterogeneity on a molecular level, detergent extracts were prepared from B cell lines derived from individuals reactive (+) or unreactive (-) with the B27M2 antibody. Extracts were immunoprecipitated by specific allogeneic or monoclonal antibodies and analyzed by two-dimensional polyacrylamide gel electrophoresis. By this method the B27M2+ and B27M2- variants of HLA-B27 had different isoelectric points (pl) and could be distinguished from each other and from a different (Bw44) control alloantigen. Blockade of glycosylation by pretreatment of cells with tunicamycin did not alter pl but did reduce HLA antigens by approximately 3000 daltons. These data demonstrate that B27 antigens can be subdivided into subsets with different molecular composition. The effects of this heterogeneity upon the associations of B27 and disease are not yet known.

Heterogeneity in the rapidly exchanging metals of horse liver alcohol dehydrogenase

Substitution of the two rapidly exchanging zinc atoms of liver alcohol dehydrogenase by cobalt is biphasic; replacement by the first cobalt occurs at a rate ( t 1 2 = 15 minutes ) approximately ten times faster than substitution by the second cobalt atom. The hybrid enzyme containing one gram atom of cobalt has a characteristic visible absorption spectrum which is not perturbed by NADH or 1,10-phenanthroline. The fluorescence of NADH or ε-NAD bound to the hybrid is not quenched. These data indicate a previously unrecognized heterogeneity in the rapidly exchanging zinc atoms; one of the exchange labile zinc atoms is located at a structural metal binding site rather than an active site.