Redefining high-risk patients with stage II colon cancer by risk index and microRNA-21: results from a population-based cohort.

BACKGROUND: The aim of the present study was to analyse the prognostic value of microRNA-21 (miRNA-21) in patients with stage II colon cancer aiming at a risk index for this group of patients. METHODS: A population-based cohort of 554 patients was included. MicroRNA-21 was analysed by qPCR based on tumour tissue. An index was created using the coefficients obtained from a collective multiple Cox regression. The entire procedure was cross-validated (10-fold). The performance of the index was quantified by time-dependent receiver operating characteristics curves. RESULTS: High miRNA-21 expression was associated with an unfavourable recurrence-free cancer-specific survival (RF-CSS), hazard ratio 1.35 (95% confidence interval, 1.03-1.76) (P=0.028). The generated RF-CSS index divided the traditional high-risk patients into subgroups with 5-year RF-CSS rates of 87% and 73%, respectively (P<0.001). The overall survival (OS) index identified three different subgroups (P<0.001). Cross-validated 5-year OS rates were 88%, 68%, and 50%, respectively. CONCLUSIONS: This population-based study supports miRNA-21 as an additional prognostic biomarker in patients with stage II colon cancer. Furthermore, the introduction of a risk index may guide the use of postoperative adjuvant treatment in a more appropriate way compared with current practice.

Association of extracolonic manifestations of familial adenomatous polyposis with acetylation phenotype in a large FAP kindred.

Familial adenomatous polyposis coli (FAP) has been shown to be associated with germline mutations of the adenomatous polyposis gene (APC) on chromosome 5. Extra-colonic manifestations also occur in FAP and include desmoid tumors, epidermoid cysts and osteomas. The combination of FAP with extracolonic symptoms is commonly referred to as Gardner's syndrome. It remains difficult, however, to predict which patients may have a propensity to develop extracolonic manifestations. The rapid acetylation phenotype is believed to be associated with an increased likelihood of sporadic colorectal cancer, whereas the slow acetylation phenotype is recognized as a predisposing factor for bladder cancer. The slow acetylation phenotype is caused by mutant alleles of the cytosolic enzyme N-acetyltransferase (NAT2). In this study, we determined the NAT2 genotype in members of one large FAP family and three smaller ones all of which had been shown to harbor the same germline APC gene mutation. We observed a significant correlation between slow acetylation genotypes and extracolonic manifestations of the disease. Rapid acetylation genotypes were not overrepresented in colorectal cancer cases in this family as compared to the frequency of this genotype in the normal Caucasian population.

Comparison of the mutagenic responses of mismatch repair-proficient (TK6) and mismatch repair-deficient (MT1) human lymphoblast cells to the food-borne carcinogen PhIP.

Heterocyclic amines are ubiquitously present in cooked meats and fish. They represent an important class of food-borne carcinogens. We describe the cytotoxic, apoptotic, and mutagenic responses of mismatch repair-proficient (TK6) and mismatch repair-deficient (MT1) human lymphoblastoid cells to PhIP, the most abundant heterocyclic amine. Dose-dependent increases in cytotoxicity, in apoptosis, and in mutant fractions at the hprt locus were observed following PhIP treatment. We present a statistical method that is useful for comparing two populations. With this method, we show that the data fitted a model that assumes that the PhIP-induced mutation rate is dependent on the cell line. Estimated rates of increase of 22.8 x 10(-6) and 2.2 x 10(-6) mutation per cell per microg PhIP were found in MT1 and TK6, respectively, showing that MT1 is hypermutable to PhIP. MT1 also exhibited lower PhIP-induced apoptosis. We conclude from these results that mismatch repair-deficient cells are hypermutable to the food-borne carcinogen PhIP and that the PhIP-DNA adducts, when not eliminated by apoptosis, can be transformed into mutations.

Variance estimation in single-cell mutation assays: comparison to experimental observations in human lymphoblasts at 4 gene loci.

The performance of mutation assays with single cells involves a series of separate steps beginning with the induction of mutant cells and ending with the counting of mutant and wild-type colonies. The variation among identically treated cultures is here modeled as arising from 3 sources: (1) the number of mutant cells surviving treatment, (2) the number of mutant cells sampled in steps of sampling and growth required in assays involving phenotypic lag, and (3) the number of mutant and nonmutant colonies actually observed. The arithmetical statements describing the expectation of variance from each step are presented and used to provide means to calculate an expected overall variance for typical experiments. The model is then tested by comparing its predictions with the observed mutant fractions in human lymphoblastoid cells at the loci coding for 6-thioguanine, ouabain, podophyllotoxin, and 5,6-dichlororibofuranosyl benzimidazole resistances. The model is found to have excellent predictive qualities and should be useful in experimental design of studies involving induction of rare variants in single-cell systems.

A statistical model to estimate variance in long term-low dose mutation assays: testing of the model in a human lymphoblastoid mutation assay.

Long term-low dose mutation assays offer a means to study the genetic effects of environmental mutagens at concentrations relevant to human exposure. These assays involve continuous induction of mutants, serial dilution of cultures and sampling to determine the mutant fraction as a function of time and mutagen concentration. An arithmetic model for the expected variance among identically treated cultures is presented. This model provides means to calculate a predicted variance of the mutant fractions and mutation rates in typical long term-low dose experiments. We have calculated the expected variances of the mutant fraction with this model and compared them to the observed variances among 4 independent experiments in which human lymphoblastoid cells were treated for 5, 10, 15 and 20 days with a non-toxic concentration of the mutagen 4-aminobiphenyl. Mutations at the HPRT locus were measured by determining the 6-thioguanine-resistant mutant fraction. The expected and observed variances of the mutant fractions are in close agreement. This model is adequate to predict the variance of the mutant fraction and should be useful in experimental design and objective evaluation of long term-low dose mutation assays.

Population risk and physiological rate parameters for colon cancer. The union of an explicit model for carcinogenesis with the public health records of the United States.

The relationship between the molecular mechanisms of mutagenesis and the actual processes by which most people get cancer is still poorly understood. One missing link is a physiologically based but quantitative model uniting the processes of mutation, cell growth and turnover. Any useful model must also account for human heterogeneity for inherited traits and environmental experiences. Such a coherent algebraic model for the age-specific incidence of cancer has been developing over the past 50 years. This development has been spurred primarily by the efforts of Nordling [N.O. Nordling, A new theory on the cancer-inducing mechanism, Br. J. Cancer 7 (1953) 68-72], Armitage and Doll [P. Armitage, R. Doll, The age distribution of cancer and a multi-stage theory of carcinogenesis, Br. J. Cancer 8 (1) (1954) 1-12; P. Armitage, R. Doll, A two-stage theory of carcinogenesis in relation to the age distribution of human cancer, Br. J. Cancer 9 (2) (1957) 161-169], and Moolgavkar and Knudson [S.H. Moolgavkar, A.G. Knudson Jr., Mutation and cancer: a model for human carcinogenesis. JNCI 66 (6) (1981) 1037-1052], whose work defined two rate-limiting stages identified with initiation and promotion stages in experimental carcinogenesis. Unfinished in these efforts was an accounting of population heterogeneity and a complete description of growth and genetic change during the growth of adenomas. In an attempt to complete a unified model, we present herein the first means to explicitly compute the essential parameters of the two-stage initiation-promotion model using colon cancer as an example. With public records from the 1930s to the present day, we first calculate the fraction at primary risk for each birth year cohort and note historical changes. We then calculate the product of rates for n initiation-mutations, the product of rates for m promotion-mutations and the average growth rate of the intermediate adenomatous colonies from which colon carcinomas arise. We find that the population fraction at primary risk for colon cancer risk was historically invariant at about 42% for the birth year cohorts from 1860 through 1930. This was true for each of the four cohorts we examined (European- and African-Americans of each gender). Additionally, the data indicate an historical increase in the initiation-mutation rates for the male cohorts and the promotion-mutation rates for the female cohorts. Interestingly, the calculated rates for initiation-mutations are in accord with mutation rates derived from observations of mutations in peripheral blood cells drawn from persons of different ages. Adenoma growth rates differed significantly between genders but were essentially historically invariant. In its present form, the model has also allowed us to calculate the rate of loss of heterozygosity (LOH) or loss of genomic imprinting (LOI) in adenomas to result in the high LOH/LOI fractions in tumors. But it has not allowed us to specify the number of events m required during promotion.

Mutation, cell kinetics, and subpopulations at risk for colon cancer in the United States.

We have extended the algebraic models for cancer initiation and progression developed by Nordling, Armitage-Doll and Knudson-Moolgavkar to include the effect of cell turnover rate in normal tissue, stochastic growth of preneoplastic adenomas, and the general case wherein a subfraction of the population is at risk. We have also gathered the mortality data available for the United States from 1900 to 1991 and categorically organized them by birth year cohorts and age specific death rates for ages 0 to 104 in 5-year groupings. Using these data, we first explored the quantitative nature of the biases of underreporting or misdiagnosis as historical age-dependent functions. Then we used the extended algebraic model to calculate the parameters of subpopulation fraction at risk, mutation rates and adenoma growth rates. We observe that death rates for all cancers are low in childhood and early adulthood, rise in middle age in an approximately linear manner, reach a maximum in old age, and even after correction for reporting bias, decrease markedly in extreme old age. We represent this behavior as the natural result of a continuous process of cell division, death and mutation within a subpopulation at risk. This population at risk within any birth cohort is defined by the product of a constant inherited risk factor multiplied by a historically valuable environmental risk factor. Our formulation permits explicit calculation of the fraction at risk of death from any cancer as a historical function. With regard to the algebraic description of the process of carcinogenesis, we use Nordling's concept that n genetic events in a cell population of constant cell number are required to initiate a colony capable of net cell growth or 'adenoma.' We adopt and extend Moolgavkar's use of the 'Gambler's Ruin' stochastic process to describe the probability of adenoma survival and the canonical expectation that a surviving adenoma will soon contain many initiated cells by virtue of stochastic distribution of surviving cells. We consider that within the growing adenoma, it is necessary for a cell to acquire m additional mutations in order to attain the carcinoma phenotype of cell growth rapid enough to kill in a short time. This would be irrespective of the need for any additional genetic events that may define the subsequent phenotypes of large lethal tumors, as these would be automatically acquired and be physiologically selected in any rapidly growing cell mass. It is evident that the steps of initiation and progression are dependent on both the rates of genetic change per cell division and the cell kinetic rates of division and death. We have chosen to first examine colon cancer because the rates of cell division in normal colonic epithelium, dysplastic adenomas and small carcinomas have been directly observed as reported herein. For colon cancer, we calculate that about 65% of the US population is at risk for both males and females, and that this fraction has been constant for the earliest recorded birth cohorts of the mid-19th century to the beginning of the 20th century. The changes that have been observed in colon cancer mortality rates appear to arise from historical changes in death rates by unknown 'other causes of death', which share both genetic and environmental risk factors with colon cancer and explicitly include undiagnosed deaths by colon cancer. Considering all possible values of n and m, we find the case of n=2 and m=1 to give the best concordance with present knowledge of mutations in the colon by the loss of two alleles of the APC gene and the observation that for m=1, a rate of genetic change approximately equal to that calculated for initiation mutation rates is obtained. Our estimates for the rate of initiation and progression mutation rates show no significant historical shifts and are approximately 1-2x10-7 events per cell division. (ABSTRACT TRUNCATED)

Random walk and gap plots of DNA sequences.

Genomic sequence analysis is usually performed with the help of specialized software packages written for molecular biologists. The scope of such pre-programmed techniques is quite limited. Because DNA sequences contain a large amount of information, analysis of such sequences without underlying assumptions may provide additional insights. The present article proposes two new graphical representations as examples of such methods. The random walk plot is designed to show the base composition in a compact form, whereas the gap plot visualizes positional correlations. The random walk plot represents the DNA sequence as a curve, a random walk, in a plane. The four possible moves, left/right and up/down, are used to encode the four possible bases. Gap plots provide a tool to exhibit various features in a sequence. They visualize the periodic patterns within a sequence, both with regard to a single type of base or between two types of bases.

Combined therapy with saquinavir, ritonavir and stavudine in moderately to severely immunosuppressed HIV-infected protease inhibitor-naive patients.

OBJECTIVE: To assess the short-term and long-term effect of a combination of saquinavir, ritonavir and stavudine in moderately to severely immunosuppressed protease inhibitor-naive patients. DESIGN: Prospective open-label multicentre study. PATIENTS AND METHODS: A total of 64 protease inhibitor-naive and stavudine-naive HIV-infected patients with a CD4 count of < 250 cells/microL and > 10 000 HIV-1 RNA copies/mL received saquinavir hard-gelatin capsules, ritonavir and stavudine. Full (drop in viraemia of > 2 log10 and/or < 500 copies/mL) and partial responders (drop to between 500 and 5000 viraemia copies/mL) at week 9 (end of phase I) entered the second phase (additional 12-month period). RESULTS: Fifty-six patients completed phase I, 45 (70%) full responders and nine (14%) partial responders by intent-to-treat analysis. Thirty-nine patients completed phase II, 33 (52%) full responders and two (3%) partial responders. Six patients had < 50 HIV-1 RNA copies/mL at week 9, and 20 (31%) patients at month 12 of phase II. Mean CD4 cell counts increased significantly in the 56 patients from 89 to 184 cells/microL after 9 weeks and from 100 to 292 cells/microL in the 39 patients treated for another 12 months. Higher baseline viraemia and lower baseline CD4 cell counts were not associated with an unfavourable virological response at week 9 and month 12 of phase II. HIV DNA in peripheral blood monocytes decreased substantially (- 1.5 log10) but was detectable in all except one patient at the end of phase II. CONCLUSION: In protease- and stavudine-naive HIV-infected patients with moderate to severe immunosuppression, saquinavir in combination with ritonavir and stavudine caused a substantial long-term decrease in plasma viral load in approximately half the participants and a substantial increase in CD4 cell counts.