Analysis of multiple tumor data from a rodent carcinogenicity experiment

Rodent tumorigenicity experiments are conducted to determine the safety of substances for human exposure. The carcinogenicity of a substance is generally determined by statistical tests that compare the effects of treatment on the rate of tumor development at several body sites. The statistical analysis of such studies often includes hypothesis testing of the dose effect at each of the sites. However, the multiplicity of the significance tests may cause an excess overall false positive rate. In consideration of this problem, recent interest has focused on developing methods to test simultaneously for the treatment effect at multiple sites. In this paper, we propose a test that is based on the count of tumor-bearing sites. The test is appropriate regardless of tumor lethality or of treatment-related differences in the underlying mortality. Simulations are given which compare the performance of the proposed test to several other tests including a Bonferroni adjustment of site-specific tests, and the test is illustrated using the data from the large ED01 experiment.     

The companion dog as a model for human aging and mortality

Around the world, human populations have experienced large increases in average lifespan over the last 150 years, and while individuals are living longer, they are spending more years of life with multiple chronic morbidities. Researchers have used numerous laboratory animal models to understand the biological and environmental factors that influence aging, morbidity, and longevity. However, the most commonly studied animal species, laboratory mice and rats, do not experience environmental conditions similar to those to which humans are exposed, nor do we often diagnose them with many of the naturally occurring pathologies seen in humans. Recently, the companion dog has been proposed as a powerful model to better understand the genetic and environmental determinants of morbidity and mortality in humans. However, it is not known to what extent the age‐related dynamics of morbidity, comorbidity, and mortality are shared between humans and dogs. Here, we present the first large‐scale comparison of human and canine patterns of age‐specific morbidity and mortality. We find that many chronic conditions that commonly occur in human populations (obesity, arthritis, hypothyroidism, and diabetes), and which are associated with comorbidities, are also associated with similarly high levels of comorbidity in companion dogs. We also find significant similarities in the effect of age on disease risk in humans and dogs, with neoplastic, congenital, and metabolic causes of death showing similar age trajectories between the two species. Overall, our study suggests that the companion dog may be an ideal translational model to study the many complex facets of human morbidity and mortality.     

Late divergence of survival curves in cancer immunotherapy trials: interpretation and implications

Late divergence of survival curves of treated patients and controls is commonly seen in successful cancer immunotherapy trials. Although late survival curve divergence may be caused by a delayed action of therapy, it may also be related to early effects of the treatment. We suggest that late survival divergence most often reflects a specific benefit of therapy for patients who suffer from a comparatively slow progression of disease. The occurrence of delayed survival curve divergence has important implications for the statistical analysis of immunotherapy trials. Thus, it leads to non-proportional hazard ratios that make commonly used statistical tests, e.g., the logrank test, suboptimal. It is therefore suggested that the statistical analysis of immunotherapy trials primarily should be based on a test that compares the survival curves at or after a prespecified, fixed, late time point.     

Anti-TNF antibody treatment reduces mortality in experimental dengue virus infection

Here we describe a lethal mouse model infected with dengue virus type 2 with several similarities to human DEN-2 infection. Clinically animals demonstrated anemia, thrombocytopenia, pre-terminal paralysis and shock. The most impressive changes were seen with tumor necrosis factor (TNF)-alpha, which abruptly and steeply increased 24 h before the exitus (mean at day 6). Serum levels of IL-1beta, IL-6, IL-10, IL-1 receptor antagonist and soluble TNF receptor I continuously increased during the time of infection. A 100% mortality rate was noted in that group of animals. Treating animals with anti-TNF-alpha serum reduced mortality rate down to 40% (P<0.05). Our model supports the view that activation of innate immune response is at least partially responsible for mortality in DEN-2 infection, and in line with this concept, anti-TNF treatment significantly reduces mortality rates.     

Increasing incidence and mortality from myocardial infarction in Stockholm county.

A study was undertaken to examine trends in the incidence and mortality of myocardial infarction in Sweden. All cases (n = 19908) of myocardial infarction diagnosed in the population of Stockholm county during 1974-80 were identified by means of the cause of death register and the inpatient care register. Information on patients at risk was obtained from the civil registration system. The relative risk of developing, or dying of, myocardial infarction in one specific year, compared with the average for the whole period, was taken as the basis for describing the trends. For men in Stockholm the incidence as well as the mortality was appreciably increased; the annual increase in incidence was 3% and in mortality 4%. There were no signs of decreasing lethality. For women there was an appreciable increase in incidence; for mortality the result was less specific but was compatible with an increase. The observed increases in incidence and mortality appeared to be real and were probably not due to an increasing tendency for patients to seek hospital treatment or for doctors to make the diagnosis. The reason for the increase is unknown.     

Road mortality of pond-breeding amphibians during spring migrations in the Mazurian Lakeland, NE Poland

The effects of road traffic mortality on amphibian populations are in many cases hard to estimate and speculate, primarily due to the inaccuracy of the methods employed in studies. We have analyzed amphibian road mortality during two breeding seasons on selected road sections adjoining spawning ponds and have critically reviewed methods used for estimating the impact of road traffic on migrating amphibians. The mortality rates of particular amphibian species differed significantly and varied according to year, site, and traffic volume. The highest mortality was recorded for Bufo bufo and the lowest for Lissotriton vulgaris. Species-specific parameter estimates of mortality, evaluated on a daily basis counts of roadkills, were positively correlated with the mean body mass of the amphibian species. The share of a given species among carcasses found on the road, commonly used in such studies, proved to be an unreliable measure of mortality. We found that nearly 60% of amphibian roadkill victims had disappeared within 24?h of exposure, and the number of missing carcasses was inversely related to body mass.     

Predicting mortality from cervical cancer after negative smear test results.

OBJECTIVE--To assess the relative protection against death from cervical cancer after two or more negative smear test results and compare it with the protection against invasive cancer estimated by an International Agency for Research on Cancer (IARC) working group in an analysis of data from 10 large screening programmes. DESIGN--Comparison of risk of death from cervical cancer after two or more negative smear results with the risk in unscreened women by using a model constructed with data from the British Columbia screening programme. MAIN OUTCOME MEASURES--Mortality from and incidence of invasive cancer. RESULTS--In women with two negative smear results estimates of protection against cervical cancer were about 50% higher when lethal invasive cancer was used as the criterion rather than all invasive cancer. This difference was due to these women being more likely to attend for further tests at which invasive cancer could be detected: screen detected cancer has a better prognosis than clinically diagnosed cancer. Screening intervals could be longer than three years: screening women aged 35-64 every five years was predicted to result in a 90% reduction in mortality from cervical cancer. CONCLUSION--Because protection from mortality is higher than protection from disease and because of the high costs and negative side effects of frequent screening, screening intervals should be longer than three years.     

Enhancement of murine ascites tumor cell killing with x-irradiation by thiol binding agents

Female mice bearing the Ehrlich carcinoma or P388 lymphocytic leukemia tumors in ascites form were given sublethal doses of whole-body x-irradiation and the thiol binding agents N-ethylmaleimide, hydroxy-mercuribenzoate, or iodoacetamide, injected intraperitoneally prior to irradiation, as a single treatment. These compounds were found previously to sensitize mice to radiation lethality. Enhanced tumor cell killing was observed as measured by tumor cell count, along with slightly longer survival times of the host animal. Increasing the dose of either radiation or drug alone also caused an increase in tumor cell killing, but at the expense of earlier mortality of the host animal. At the doses employed the sensitizers examined appeared more effective on these two ascites tumors han on the host. The mechanism of enhancement of radiation killing of tumor cells by these drugs is not clear, although it appears not to be due to additive toxicity effects. Similar experiments with several cancer chemotherapy agents showed that those compounds did not act as radiosensitizers.     

Beyond the proportional frailty model: Bayesian estimation of individual heterogeneity on mortality parameters

Today, we know that demographic rates can be greatly influenced by differences among individuals in their capacity to survive and reproduce. These intrinsic differences, commonly known as individual heterogeneity, can rarely be measured and are thus treated as latent variables when modeling mortality. Finite mixture models and mixed effects models have been proposed as alternative approaches for inference on individual heterogeneity in mortality. However, in general models assume that individual heterogeneity influences mortality proportionally, which limits the possibility to test hypotheses on the effect of individual heterogeneity on other aspects of mortality such as ageing rates. Here, we propose a Bayesian model that builds upon the mixture models previously developed, but that facilitates making inferences on the effect of individual heterogeneity on mortality parameters other than the baseline mortality. As an illustration, we apply this framework to the Gompertz–Makeham mortality model, commonly used in human and wildlife studies, by assuming that the Gompertz rate parameter is affected by individual heterogeneity. We provide results of a simulation study where we show that the model appropriately retrieves the parameters used for simulation, even for low variances in the heterogeneous parameter. We then apply the model to a dataset on captive chimpanzees and on a cohort life table of 1751 Swedish men, and show how model selection against a null model (i.e., without heterogeneity) can be carried out.     

Likelihood-based tests of topologies in phylogenetics

Likelihood-based statistical tests of competing evolutionary hypotheses (tree topologies) have been available for approximately a decade. By far the most commonly used is the Kishino-Hasegawa test. However, the assumptions that have to be made to ensure the validity of the Kishino-Hasegawa test place important restrictions on its applicability. In particular, it is only valid when the topologies being compared are specified a priori. Unfortunately, this means that the Kishino-Hasegawa test may be severely biased in many cases in which it is now commonly used: for example, in any case in which one of the competing topologies has been selected for testing because it is the maximum likelihood topology for the data set at hand. We review the theory of the Kishino-Hasegawa test and contend that for the majority of popular applications this test should not be used. Previously published results from invalid applications of the Kishino-Hasegawa test should be treated extremely cautiously, and future applications should use appropriate alternative tests instead. We review such alternative tests, both nonparametric and parametric, and give two examples which illustrate the importance of our contentions.     

The application of the probit method to toxicity test data adjusted for mortality in the controls

When data from toxicity tests have to be adjusted for a mortality rate amongst untreated controls, three modifications to the usual method of probit analysis need to be considered. The full solution is more laborious than that ordinarily used, as it requires the estimation of this mortality rate from the whole of the data. When the rate is. not high (e.g. up to 20 %) a satisfactory approximation may be obtained by a simple alteration in die weighting coefficients, especially if the precaution is taken of using two or three times as many test organisms as controls as for any dosage of the poison, thus decreasing the relative value of the information on natural mortality contributed by the treated batches. The modified weighting coefficients are tabulated for values of the natural mortality from zero to 20 %. An example of the more complex calculations required for the full maximum likelihood solution is discussed in detail.     

In vitro efficacy of over-the-counter botanical pediculicides against the head louse Pediculus humanus var capitis based on a stringent standard for mortality assessment

Abstract.  Infestation of the head louse Pediculus humanus var capitis DeGeer (Phthiraptera: Pediculidae) is an important public health problem in Australia, with up to a third of children infested in some primary schools. Insecticide resistance and inadequate attention to the application instructions of topical pediculicides are common reasons for treatment failure. This study evaluated six popular Australian over-the-counter products against head lice, primarily comprised of different botanical extracts, and compared them with permethrin 1% (Quellada^®) and a non-treatment control in order to assess their in vitro efficacy. We also assessed commonly used criteria for evaluating pediculicide efficacy in vitro. All tested products failed to demonstrate high levels of efficacy with the exception of Tea Tree Gel^®, which outperformed 1% permethrin. Permethrin had a high level of efficacy, but using stringent criteria 18% of lice were not dead at 3 h, indicating some resistance to Quellada^®. Commonly used less stringent criteria were shown to overestimate mortality of head lice as a result of the protective phenomenon of stasis or sham death observed in exposed lice that may recover after some time. Using two different levels of stringency resulted in different rankings of efficacy for most products, with the exception of the first ranked product, Tea Tree Gel^®. Rankings of efficacy also varied over time, even within the different assessment criteria. Government regulatory agencies should require standard in vitro tests using stringent mortality criteria, with an observation period of ≥ 6 h, to determine the efficacy of new pediculicides, and only products that cause a minimum mortality rate (e.g. 80%) in head lice collected from the target population should be licensed for sale.     

A New Powerful Nonparametric Rank Test for Ordered Alternative Problem

We propose a new nonparametric test for ordered alternative problem based on the rank difference between two observations from different groups. These groups are assumed to be independent from each other. The exact mean and variance of the test statistic under the null distribution are derived, and its asymptotic distribution is proven to be normal. Furthermore, an extensive power comparison between the new test and other commonly used tests shows that the new test is generally more powerful than others under various conditions, including the same type of distribution, and mixed distributions. A real example from an anti-hypertensive drug trial is provided to illustrate the application of the tests. The new test is therefore recommended for use in practice due to easy calculation and substantial power gain.     

Optical motility test for the detection of trichothecenes using brine shrimps

A new optical bio-assay using brine shrimp larvae (Anemia salina L.) has been successfully applied for monitoring the presence of highly toxic trichothecenes. The motility of the swimming animals was determined instead of their lethality. This permits more objective and precise results. The inhibition of the motility can be seen earlier, than the death of animals in toxicity tests. The relationship between time and dose is indicated for two different concentrations. This test can also be applied in the control of other harmful compounds.     

Below-threshold mortality: implications for studies in evolution,ecology and demography

Evolutionary biologists, ecologists and experimental gerontologists have increasingly used estimates of age-specific mortality as a critical component in studies of a range of important biological processes. However, the analysis of age-specific mortality rates is plagued by specific statistical challenges caused by sampling error. Here we discuss the nature of this ‘demographic sampling error’, and the way in which it can bias our estimates of (1) rates of ageing, (2) age at onset of senescence, (3) costs of reproduction and (4) demographic tests of evolutionary models of ageing. We conducted simulations which suggest that using standard statistical techniques, we would need sample sizes on the order of tens of thousands in most experiments to effectively remove any bias due to sampling error. We argue that biologists should use much larger sample sizes than have previously been used. However, we also present simple maximum likelihood models that effectively remove biases due to demographic sampling error even at relatively small sample sizes.     

Studies of the mortality of A-bomb survivors. 8. Cancer mortality, 1950-1982

This study extends an earlier one by 4 years (1979-1982) and includes mortality data on 11,393 additional Nagasaki survivors. Significant dose responses are observed for leukemia, multiple myeloma, and cancers of the lung, female breast, stomach, colon, esophagus, and urinary tract. Due to diagnostic difficulties, results for liver and ovarian cancers, while suggestive of significant dose responses, do not provide convincing evidence for radiogenic effects. No significant dose responses are seen for cancers of the gallbladder, prostate, rectum, pancreas, or uterus, or for lymphoma. For solid tumors, largely due to sex-specific differences in the background rates, the relative risk of radiation-induced mortality is greater for women than for men. For nonleukemic cancers the relative risk seen in those who were young when exposed has decreased with time, while the smaller risks for those who were older at exposure have tended to increase. While the absolute excess risks of radiation-induced mortality due to nonleukemic cancer have increased with time for all age-at-exposure groups, both excess and relative risks of leukemia have generally decreased with time. For leukemia, the rate of decrease in risk and the initial level of risk are inversely related to age at exposure.     

Accelerated development of polyoma tumors and embryonic lethality: different effects of p53 loss on related mouse backgrounds.

Molecular evidence linking polyoma virus to p53 inactivation is thus far lacking, setting this highly oncogenic virus apart from other DNA tumor viruses. As a biological test for interaction, we studied the effects of p53 loss on development of virus-induced tumors. The absence of p53 led to more rapid tumor development on two different mouse backgrounds, indicating synergism between p53 loss and oncogenic pathways controlled directly by the virus. No effects of p53 on tumor type or frequency were noted. Polyoma tumor-derived cells in culture retained p53, and most of these showed induction of p21CIP1/WAF1 in response to DNA damage. These results indicate that p53 functions are not directly and fully impaired by the virus in the intact host. On one mouse background, it was discovered that loss of p53 resulted in complete embryonic lethality prior to 11 days of gestation. This lethality could be rescued by inclusion of gene(s) from a 129/SvJ background.     

Primary aldosteronism: the role of confirmatory tests

The high prevalence of primary aldosteronism among hypertensives justifies large scale screening by the aldosterone-renin ratio; however, this test is subject to several variables responsible for false-positive results. Functional tests to confirm autonomous aldosterone secretion are commonly used, with the fludrocortisone suppression test considered the gold standard, and saline infusion or captopril challenge, the most practical. However, each of these tests has sub-optimal sensitivity and specificity and none has been so far prospectively validated by comparing the results with the lateralization by adrenal vein sampling and the results of surgery. Their role in confirming the diagnosis of primary aldosteronism due to unilateral adenoma remains incompletely resolved.     

A note on the tests for clustered matched-pair binary data

McNemar's test is used to assess the difference between two different procedures (treatments) using independent matched-pair data. For matched-pair data collected in clusters, the tests proposed by Durkalski et al. and Obuchowski are popular and commonly used in practice since these tests do not require distributional assumptions or assumptions on the structure of the within-cluster correlation of the data. Motivated by these tests, this note proposes a modified Obuchowski test and illustrates comparisons of the proposed test with the extant methods. An extensive Monte Carlo simulation study suggests that the proposed test performs well with respect to the nominal size, and has higher power; Obuchowski's test is most conservative, and the performance of the Durkalski's test varies between the modified Obuchowski test and the original Obuchowski's test. These results form the basis for our recommendation that (i) for equal cluster size, the modified Obuchowski test is always preferred; (ii) for varying cluster size Durkalski's test can be used for a small number of clusters (e.g. K < 50), whereas for a large number of clusters (e.g. K ≥ 50) the modified Obuchowski test is preferred. Finally, to illustrate practical application of the competing tests, two real collections of clustered matched-pair data are analyzed.     

Statistical properties of affected sib-pair linkage tests

Genetic linkage analysis is a powerful tool for the identification of disease susceptibility loci. Among the most commonly applied genetic linkage strategies are affected sib-pair tests, but the statistical properties of these tests have not been well characterized. Here, we present a study of the distribution of affected sib-pair tests comparing the type I error rate and the power of the mean test and the proportion test, which are the most commonly used, along with a novel exact test. In contrast to existing literature, our findings showed that the mean and proportion tests have inflated type I error rates, especially when used with small samples. We developed and applied corrections to the tests which provide an excellent adjustment to the type I error rate for both small and large samples. We also developed a novel approach to identify the areas of higher power for the mean test versus the proportion test, providing a wider and simpler comparison with fewer assumptions about parameter values than existing approaches require.