Cell-free DNA analysis in current cancer clinical trials: a review.

Cell-free DNA (cfDNA) analysis represents a promising method for the diagnosis, treatment selection and clinical follow-up of cancer patients. Although its general methodological feasibility and usefulness has been demonstrated, several issues related to standardisation and technical validation must be addressed for its routine clinical application in cancer. In this regard, most cfDNA clinical applications are still limited to clinical trials, proving its value in several settings. In this paper, we review the current clinical trials involving cfDNA/ctDNA analysis and highlight those where it has been useful for patient stratification, treatment follow-up or development of novel approaches for early diagnosis. Our query included clinical trials, including the terms 'cfDNA', 'ctDNA', 'liquid biopsy' AND 'cancer OR neoplasm' in the FDA and EMA public databases. We identified 1370 clinical trials (FDA = 1129, EMA = 241) involving liquid-biopsy analysis in cancer. These clinical trials show promising results for the early detection of cancer and confirm cfDNA as a tool for real-time monitoring of acquired therapy resistance, accurate disease-progression surveillance and improvement of treatment, situations that result in a better quality of life and extended overall survival for cancer patients.

Telomere shortening accompanies increased cell cycle activity during serial transplantation of hematopoietic stem cells.

Reactivation of telomerase and maintenance of telomere length can lead to the prevention of replicative senescence in some human somatic cells grown in vitro. To investigate whether telomere shortening might also play a role in the limitation of hematopoietic stem cell (HSC) division capacity in vivo, we analyzed telomere length during serial transplantation of murine HSCs. Southern blot analysis of telomere length in donor bone marrow cells revealed extensive shortening ( approximately 7 kb) after just two rounds of HSC transplantation. The number of cycling HSCs increased after transplantation and remained elevated for at least 4 mo, while the frequency of HSCs in the bone marrow was completely regenerated by 2 mo after transplantation. Direct analysis of telomeres in HSCs by fluorescent in situ hybridization during serial transplantation also revealed a reduction in telomere size. Together, these data show that telomeres shorten during division of HSCs in vivo, and are consistent with the hypothesis that telomere shortening may limit the replicative capacity of HSCs.

Death-associated protein kinase (DAPK1) in cerebral cortex of late-onset Alzheimer's disease patients and aged controls.

AIMS: Here our objective was to detect the pro-apoptotic serine/threonine kinase death-associated protein kinase (DAPK1) in aged human cerebral cortex and to test the hypothesis that DAPK1 abundance is associated with late-onset Alzheimer's disease (AD). METHODS: Using Western analysis and immunohistochemistry we evaluated post mortem frontal cerebral cortex from patients with severe AD (mean age 76 years, range 66-91, n = 11, all male), and from control cases without serious central nervous system illness (mean age 77 years, range 61-95, n = 12, all male). We also examined brains of Tg2576 transgenic mice (males, aged 16-21 months), a model for chronic amyloid-induced brain injury. RESULTS: Immunohistochemical labelling showed DAPK1 expression in cortical neurones of human cortex and axonal tracts within subcortical white matter, both in AD and in control brains. Western analysis confirmed DAPK1 expression in all samples, although expression was very low in some control cases. DAPK1 abundance in the AD group was not significantly different from that in controls (P = 0.07, Mann-Whitney test). In brains of Tg2576 mice DAPK1 abundance was very similar to that in wild-type littermates (P = 0.96, Mann-Whitney test). CONCLUSION: We found that DAPK1 was expressed in neurones of aged human frontal cortex, both in AD and in control cases.

Quality of life and health-related utility analysis of adults with moderate and severe atopic dermatitis treated with tacrolimus ointment vs. topical corticosteroids.

BACKGROUND: The purpose of this study was to measure change in quality of life (QoL) and estimate health-related utility in adults with moderate and severe atopic dermatitis (AD) following the use of either tacrolimus ointment or topical corticosteroids. METHODS: Data were analysed from a double-blind, randomized controlled trial comparing the treatment of adults with moderate and severe AD with either tacrolimus ointment or a standard corticosteroid regimen. Following randomisation, patients applied their medication twice-daily for 6 months. Monthly assessments determined response and QoL. Health-related utility (EQ5Dindex) was estimated by Monte Carlo simulation from SF-12 responses via a published mapping algorithm. RESULTS: At baseline, estimated utility data were available for 926 (95%) of the intention-to-treat patients, 57% of whom had AD of moderate severity (43% severe). The mean age at baseline was 32.5 years (SD +/- 11.8), 46.2% were male, with a mean EQ5Dindex for moderate cases of 0.770 (SD +/- 0.157), and 0.665 (SD +/- 0.225) for those with severe disease (P < 0.001). Patients treated with tacrolimus ointment showed significantly greater improvement in all but one domain of the SF-36. At baseline, there was no difference in estimated utility between the two groups; however, a difference in utility in favour of tacrolimus ointment emerged after 1 month's treatment (0.849 vs. 0.820; P = 0.004). Over the 6-month study period, the mean, marginal utility difference between the study arms was 0.032 U (utility) in favour of tacrolimus (P < 0.001). CONCLUSION: Treatment with 0.1% tacrolimus ointment rather than a standard topical corticosteroid ointment regimen was associated with clinically significant, incremental improvement in QoL, sustained over a 6-month period. A within-trial cost-utility estimate based on study medication cost alone suggests that tacrolimus ointment is highly cost-effective given existing willingness-to-pay thresholds.

In situ analysis of changes in telomere size during replicative aging and cell transformation.

Telomeres have been shown to gradually shorten during replicative aging in human somatic cells by Southern analysis. This study examines telomere shortening at the single cell level by fluorescence in situ hybridization (FISH). FISH and confocal microscopy of interphase human diploid fibroblasts (HDFs) demonstrate that telomeres are distributed throughout the nucleus with an interchromosomal heterogeneity in size. Analysis of HDFs at increasing population doubling levels shows a gradual decrease in spot size, intensity, and detectability of telomeric signal. FISH of metaphase chromosomes prepared from young and old HDFs shows a heterogeneity in detection frequency for telomeres on chromosomes 1, 9, 15, and Y. The interchromosomal distribution of detection frequencies was similar for cells at early and late passage. The telomeric detection frequency for metaphase chromosomes also decreased with age. These observations suggest that telomeres shorten at similar rates in normal human somatic cels. T-antigen transformed HDFs near crisis contained telomere signals that were low compared to nontransformed HDFs. A large intracellular heterogeneity in telomere lengths was detected in two telomerase-negative cell lines compared to normal somatic cells and the telomerase-positive 293 cell line. Many telomerase-negative immortal cells had telomeric signals stronger than those in young HDFs, suggesting a different mechanism for telomere length regulation in telomerase-negative immortal cells. These studies provide an in situ demonstration of interchromosomal heterogeneity in telomere lengths. Furthermore, FISH is a reliable and sensitive method for detecting changes in telomere size at the single cell level.

The RNA component of human telomerase.

Eukaryotic chromosomes are capped with repetitive telomere sequences that protect the ends from damage and rearrangements. Telomere repeats are synthesized by telomerase, a ribonucleic acid (RNA)-protein complex. Here, the cloning of the RNA component of human telomerase, termed hTR, is described. The template region of hTR encompasses 11 nucleotides (5'-CUAACCCUAAC) complementary to the human telomere sequence (TTAGGG)n. Germline tissues and tumor cell lines expressed more hTR than normal somatic cells and tissues, which have no detectable telomerase activity. Human cell lines that expressed hTR mutated in the template region generated the predicted mutant telomerase activity. HeLa cells transfected with an antisense hTR lost telomeric DNA and began to die after 23 to 26 doublings. Thus, human telomerase is a critical enzyme for the long-term proliferation of immortal tumor cells.

Long telomeres in the mature human placenta.

OBJECTIVE: To investigate whether telomere shortening may play a role in senescence of the placenta. STUDY DESIGN: Villous tissue was collected from single, random sites of full-term placentas (39-41 weeks of gestation; n=10) as well as multiple, specific sites of the same placenta (39-41 weeks of gestation; n=5). For the latter group of placentas, samples were taken near the umbilical cord and at the periphery on both the maternal and fetal sides (a total of 4 samples per placenta). Cord blood samples were also obtained from all placental donors. Telomerase activity was assessed by the TRAP assay, and telomere length measured by Southern analysis of mean terminal restriction fragment (TRF) length. RESULTS: We show for the first time that telomeres are longer ( approximately 25% longer; P<0.001) in placenta tissue than in cord blood from the same donor. CONCLUSION: Telomere shortening is unlikely to have a significant role in senescence or terminal maturation of the placenta.

Expression of mouse telomerase reverse transcriptase during development, differentiation and proliferation.

We have identified the mouse telomerase reverse transcriptase component (mTERT) and demonstrate both substantial sequence homology to the human ortholog (hTERT), and the presence of reverse transcriptase and telomerase specific motifs. Furthermore, we show functional interchangeability with hTERT in in vitro telomerase reconstitution experiments, as mTERT produces strong telomerase activity in combination with the human telomerase RNA component hTR. The mouse TERT is widely expressed at low levels in adult tissues, with greatest abundance during embryogenesis and in adult thymus and intestine. The mTERT component mRNA levels were regulated during both differentiation and proliferation, while mTR levels remained constant throughout both processes. Comparison of mTERT and mTR levels to telomerase activity indicates that mTERT expression is more tightly linked to the regulation of telomerase activity during these processes than is mTR. In contrast to the situation in human cell cultures, mTERT transcript levels are present at readily detectable levels in primary cultured cells and are not upregulated following crisis. The widespread expression of mTERT in primary cells and mouse tissues could explain the increased frequency of spontaneous immortalization of mouse cells in culture and tumorigenesis in vivo.

Telomere end-replication problem and cell aging.

Since DNA polymerase requires a labile primer to initiate unidirectional 5'-3' synthesis, some bases at the 3' end of each template strand are not copied unless special mechanisms bypass this "end-replication" problem. Immortal eukaryotic cells, including transformed human cells, apparently use telomerase, an enzyme that elongates telomeres, to overcome incomplete end-replication. However, telomerase has not been detected in normal somatic cells, and these cells lose telomeres with age. Therefore, to better understand the consequences of incomplete replication, we modeled this process for a population of dividing cells. The analysis suggests four things. First, if single-stranded overhangs generated by incomplete replication are not degraded, then mean telomere length decreases by 0.25 of a deletion event per generation. If overhangs are degraded, the rate doubles. Data showing a decrease of about 50 base-pairs per generation in fibroblasts suggest that a full deletion event is 100 to 200 base-pairs. Second, if cells senesce after 80 doublings in vitro, mean telomere length decreases about 4000 base-pairs, but one or more telomeres in each cell will lose significantly more telomeric DNA. A checkpoint for regulation of cell growth may be signalled at that point. Third, variation in telomere length predicted by the model is consistent with the abrupt decline in dividing cells at senescence. Finally, variation in length of terminal restriction fragments is not fully explained by incomplete replication, suggesting significant interchromosomal variation in the length of telomeric or subtelomeric repeats. This analysis, together with assumptions allowing dominance of telomerase inactivation, suggests that telomere loss could explain cell cycle exit in human fibroblasts.

Shortened telomeres in the expanded CD28-CD8+ cell subset in HIV disease implicate replicative senescence in HIV pathogenesis.

OBJECTIVE: To test the hypothesis that the expanded population of non-proliferative CD28-CD8+ T cells in HIV disease have shortened telomeres, thereby providing evidence that increased rounds of CD8+ cell division occur during HIV disease, possibly leading to replicative senescence and exhaustion of CD8+ T-cell responses. DESIGN: CD8+ cells play a central role in control of HIV infection. In late HIV disease, an expanded population of CD28-CD8+ cells with reduced proliferative potential has been documented. A similar population of CD28-CD8+ cells has been identified in ageing humans, where telomere length measurements have suggested that these cells have reached the irreversible state of replicative senescence. METHODS: CD8+ cells from HIV-infected and control subjects were sorted by flow cytometry into CD28+ and CD28- fractions. Telomere lengths were determined as mean terminal restriction fragment (TRF) lengths by Southern hybridization. RESULTS: The TRF lengths of sorted CD28-CD8+ cells in HIV-infected subjects ranged between 5 and 7 kilobases (kb) and were significantly shorter than TRF lengths of CD28-CD8+ cells in uninfected subjects (P = 0.003). The TRF length in CD28-CD8+ cells from HIV-infected subjects was the same as that observed for centenarian peripheral blood mononuclear cells and is compatible with a state of replicative senescence. CONCLUSIONS: The shortened telomeres in the CD28-CD8+ cells in HIV-infected subjects and the poor proliferative potential of these cells identifies CD8+ cell replicative senescence as a newly described feature of HIV disease. Our results provide a mechanism for the loss of CD8+ cell control of viral replication that accompanies advanced HIV disease. Replicative senescence may contribute to exhaustion of the T-cell response as a result of chronic HIV disease. Whether this phenomenon occurs in other chronic viral infections is unknown.

Options for vector control against trypanosomiasis in Africa.

Tsetse control has long been an important option for reducing the impact of African trypanosomiasis but, although many effective methods have been used, the results have seldom proved sustainable. Developments to reduce cost and environmental impact increasingly limit the choices available for control and the scale of operations has declined. Conversely, human trypanosomiasis has reached epidemic proportions in some countries. Here, Reg Allsopp argues that those tasked with managing trypanosomiasis or committed to poverty alleviation in Africa should consider large-scale, area-wide tsetse control involving all proven methods, including aerial spraying and the sterile insect technique.

Models of initiation of replicative senescence by loss of telomeric DNA.

Situated at the ends of all eukaryotic chromosomes are telomeres, genetic elements that are essential for genomic stability. It has recently been established that telomere length shortens during replicative aging of normal human somatic cells. Although the cause of replicative senescence of somatic cells is still debated, we believe that telomere shortening plays a causal role in this process. In support of this hypothesis, mutant strains of yeast and ciliates that are incapable of maintaining telomere length during cell division eventually acquire a senescent-like phenotype wherein the cells become sickly, stop growing and die. Also, replicative capacity of cultured human skin fibroblast strains shows a strong positive correlation with telomere length. Several theories explaining how telomere shortening could lead to the induction of replicative senescence are now presented. We favor a model in which replicative senescence is caused by the shortening of telomeres below a length that is critical for the maintenance of proper telomere structure and function.

The effects of ambient temperature on life span, lipid peroxidation, superoxide dismutase, and phospholipase A2 activity in Drosophila melanogaster.

Aging changes were examined in Drosophila melanogaster. Lifespan was determined in two strains of male and female Drosophila raised at 19 degrees, 24 degrees, and 29 degrees C. The results show an inverse relationship between lifespan and temperature. In addition, lipid peroxidation rates and superoxide dismutase activity were measured in homogenates and phospholipase A2 activity was determined in crude membrane samples prepared from this species. Temperature was found to be directly correlated with the rate of lipid peroxidation in each group. The longest-lived group, wild-type females, exhibited the lowest rate of lipid peroxidation at each temperature; whereas the shortest-lived group, vestigial wing males, displayed the highest rates of lipid peroxidation. Older (40-53 day) vestigial wing males also exhibited significantly higher superoxide dismutase activity than younger vestigial wing males (0-5 day) and higher phospholipase A2 activity than wild-type females of the same age. These results indicate that there is an association between lipid peroxidation rates and lifespan in Drosophila, and that aging changes may include an increase in superoxide dismutase and phospholipase A2 activity. These findings agree with the hypothesis that free radicals are involved in the aging process in Drosophila.

Cross-hybridization of equid herpesvirus-2 (EHV-2) and herpes simplex virus-1 (HSV-1) genes to equid herpesvirus-1 (EHV-1).

In contrast to previous findings, the Ab4 isolate of equid herpesvirus-1 (EHV-1) was shown to share homology with the G9 isolate of equid herpesvirus-2 (EHV-2). Using Southern blotting and stringent hybridization conditions, a significant proportion of this cross-hybridization was identified by the immediate-early gene-3 (IE-3) probe from herpes simplex virus-1 (HSV-1). The HSV-1 UL48 gene probe (encoding the IE gene transactivating protein VmW65, which is also known as alpha-TIF or VP16) was used to identify and isolate its counterpart in EHV-1. The relevance of shared homology to transactivation is being investigated.

Food insufficiency in Queensland.

To investigate the prevalence of food insufficiency and factors associated with it, two questions assessing household and individual food insufficiency were included in 13 regional health surveys conducted in Queensland in 1993. The surveys used computer-assisted telephone interviewing methodology. Of the 10,451 people interviewed, 9.7 per cent and 6.4 per cent reported household and individual food insufficiency, respectively, and 11.3 per cent reported at least one type. Prevalence was significantly higher in women than men and in urban than rural residents, and decreased monotonically with increasing age from 16.6 per cent in 18- to 30-year-olds to 1.7 per cent in over-70-year-olds. Higher prevalence also was associated with lower income, unemployment, single or separated, divorced or widowed status versus married (or de facto), one-adult households, and shared accommodation. Lower prevalence was associated with more education in those aged 50 and under but not in those over 50 years. Using logistic regression to control simultaneously for important sociodemographic factors, we found that risk of food insufficiency was most highly associated with age and income (threefold risk), unemployment and shared accommodation (twofold risk) and one-adult households, and being single versus separated, widowed or divorced (one-and-a-half-fold risk). Some differences in risks existed between men and women and between rural and urban residents, although none excluded the role of chance. Association of the items with lower reported fruit, vegetable and meat intake, poorer health status, and greater underweight supports their validity.

Molecular basis of selective antagonism of the P2X1 receptor for ATP by NF449 and suramin: contribution of basic amino acids in the cysteine-rich loop.

BACKGROUND AND PURPOSE: The cysteine-rich head region, which is adjacent to the proposed ATP-binding pocket in the extracellular ligand-binding loop of P2X receptors for ATP, is absent in the antagonist-insensitive Dictyostelium receptors. In this study we have determined the contribution of the head region to the antagonist action of NF449 and suramin at the human P2X1 receptor. EXPERIMENTAL APPROACH: Chimeras and point mutations in the cysteine-rich head region were made between human P2X1 and P2X2 receptors. Mutant receptors were expressed in Xenopus oocytes and P2X receptor currents characterized using two-electrode voltage clamp. KEY RESULTS: The chimera replacing the region between the third and fourth conserved cysteine residues of the P2X1 receptor with the corresponding part of P2X2 reduced NF449 sensitivity a thousand fold from an IC(50) of ∼1 nM at the P2X1 receptor to that of the P2X2 receptor (IC(50) ∼1 µM). A similar decrease in sensitivity resulted from mutation of four positively charged P2X1 receptor residues in this region that are absent from the P2X2 receptor. These chimeras and mutations were also involved in determining sensitivity to the antagonist suramin. Reciprocal chimeras and mutations in the P2X2 receptor produced modest increases in antagonist sensitivity. CONCLUSIONS AND IMPLICATIONS: These data indicate that a cluster of positively charged residues at the base of the cysteine-rich head region can account for the highly selective antagonism of the P2X1 receptor by the suramin derivative NF449.

Fetal growth restriction is associated with accelerated telomere shortening and increased expression of cell senescence markers in the placenta.

A hallmark of fetal growth restriction (FGR) is restricted placental development and insufficient nutrient supply to the fetus. It has previously been shown that activity levels of telomerase, the enzyme responsible for completing replication of telomeric DNA during cell division, is suppressed in FGR placenta samples as compared to control placenta samples from donors of the same gestational age. Here we examine whether telomere length maintenance is also compromised in FGR placenta samples. Southern analysis of telomere length for placenta and cord blood samples from 32 FGR and 36 control donors, ranging in gestational age from 37 to 40 weeks, revealed significantly shorter telomeres (P

The role of game animals in the maintenance of endemic and enzootic trypanosomiases in the Lambwe Valley, South Nyanza District, Kenya.

Rhodesian sleeping sickness and bovine trypanosomiasis were endemic in the Lambwe Valley of western Kenya, where the vector of both diseases was a tsetse fly Glossina pallidipes. Since a large resident population of game animals also inhabited the valley, a programme was designed to assess the relationships between game animals, tsetse, and trypanosome populations and the results were collated with some aspects of local human ecology and the general epidemiological situation in the valley. Of 134 game animals examined for trypanosome infections, 16.4% were found to be positive. This overall infection rate was similar to that found in cattle in the same area. T. brucei was the commonest trypanosome found in game animals, but the isolation of T. rhodesiense from a reedbuck (Redunca redunca) was of greater interest. Altogether, 90% of the bushbuck (Tragelaphus scriptus) examined were infected with animal trypanosomes; this is particularly significant since bushbuck was the preferred host of G. pallidipes and greatly influenced the distribution pattern and behaviour of the tsetse. It was concluded that the association existing between bushbuck and G. pallidipes was extremely important in creating disease foci and that the spread of infections to man was largely a result of poaching activities.

Immobilization of game animals in trypanosomiasis research.

The proximity of human populations to communities of large animals in Africa creates suitable conditions for the transmission of zoonotic diseases. Since the indiscriminate destruction of these communities to control zoonoses is highly undesirable, the epidemiological role of the animals must be properly assessed in order that alternative methods of control can be developed. For this purpose, techniques should be available to permit large animals to be examined alive. Immobilization techniques using various drugs were tried but only limited success was achieved with the species most likely to be involved in the transmission cycle of trypanosomiasis. In the study reported here, xylazine was the drug selected, one reason being that an antagonist was not required. The drug was administered from a distance by means of a projectile syringe shot from a special rifle. In seven attempts, two waterbuck (Kobus defassa) and one reedbuck (Redunca redunca) were sufficiently immobilized to be handled. The reactions of all seven animals, whether successfully immobilized or not, are discussed.