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.

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.

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.

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.

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

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.

Genetic differentiation of Glossina morsitans centralis populations.

Variation at mitochondrial and microsatellite loci was used to study the breeding and dispersal structure of Glossina morsitans centralis, in six natural populations from Botswana, the Caprivi Strip (Namibia), Zambia, and in a laboratory culture derived from Singida, Tanzania. Only seven mitochondrial haplotypes were found. Mean diversity averaged over the six natural populations was 0.216 +/- 0.085. The fixation index FST = 0.866 indicated a high degree of genetic differentiation among populations. Fifty-three alleles were detected among six microsatellite loci and six natural populations. Mean microsatellite diversity was 0.702 +/- 0.091. Depending on the estimating model used, fixation indices varied from 0.15 to 0.225 confirming that G. m. centralis populations are strongly subdivided. For all FST estimates, positive correlations were detected between pair-wise genetic distance measures and geographical distances. The difference in fixation indices estimated from mitochondrial or nuclear loci was explained by the greater sensitivity of mitochondrial genomes to genetic drift. Population differentiation can be explained by genetic drift and the subsequent recovery of extant populations from small, discontinuous populations. These data confirm genetically the collapse and retreat of G. m. centralis populations caused by the rinderpest epizootic of the late 19th and early 20th centuries.

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.

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.

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.

ATM-dependent telomere loss in aging human diploid fibroblasts and DNA damage lead to the post-translational activation of p53 protein involving poly(ADP-ribose) polymerase.

Telomere loss has been proposed as a mechanism for counting cell divisions during aging in normal somatic cells. How such a mitotic clock initiates the intracellular signalling events that culminate in G1 cell cycle arrest and senescence to restrict the lifespan of normal human cells is not known. We investigated the possibility that critically short telomere length activates a DNA damage response pathway involving p53 and p21(WAF1) in aging cells. We show that the DNA binding and transcriptional activity of p53 protein increases with cell age in the absence of any marked increase in the level of p53 protein, and that p21(WAF1) promoter activity in senescent cells is dependent on both p53 and the transcriptional co-activator p300. Moreover, we detected increased specific activity of p53 protein in AT fibroblasts, which exhibit accelerated telomere loss and undergo premature senescence, compared with normal fibroblasts. We investigated the possibility that poly(ADP-ribose) polymerase is involved in the post-translational activation of p53 protein in aging cells. We show that p53 protein can associate with PARP and inhibition of PARP activity leads to abrogation of p21 and mdm2 expression in response to DNA damage. Moreover, inhibition of PARP activity leads to extension of cellular lifespan. In contrast, hyperoxia, an activator of PARP, is associated with accelerated telomere loss, activation of p53 and premature senescence. We propose that p53 is post-translationally activated not only in response to DNA damage but also in response to the critical shortening of telomeres that occurs during cellular aging.

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.

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.

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.

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 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.

Telomere shortening is associated with cell division in vitro and in vivo.

In humans, the amount of terminal (TTAGGG)n, telomeric DNA decreases during aging of various somatic cell types in vitro and in vivo. While the factors accounting for telomere shortening have not been thoroughly established, the inability of the DNA replication machinery to completely copy chromosomal termini (the "end replication problem") and the absence in somatic cells of telomerase, the enzyme that synthesizes telomeric DNA de novo, is a likely mechanism. One prediction of this hypothesis is that telomere shortening should be dependent on cell division. Thus we analyzed telomere length in actively dividing and quiescent cells in vitro and in vivo. In circular outgrowths of cultured human diploid fibroblasts (HDF), cells at the outer periphery had a significantly lower mean terminal restriction fragment (TRF) length (P = 0.011) and telomeric signal intensity (P = 0.024) than cells at the center. Also, the rate of telomere shortening over time for HDFs held quiescent was not statistically significant (m = -12 bp/day, P = 0.16) while that for serially passaged cells was significant (m = -34 bp/day, P = 0.017). To examine the rate of telomere shortening for quiescent cells in vivo, we measured mean TRF length in brain tissue from adult donors ranging in age from 32-75 years. No significant decrease was observed as a function of donor age (P = 0.087), in contrast to the shortening of telomere length that occurs during in vivo aging of mitotically active cells (P = 0.0001). These observations show that telomere shortening is largely, if not entirely, dependent on cell division and support the end replication problem as a mechanism for this process and the use of telomere length as a biomarker for replicative capacity.

Evidence for a critical telomere length in senescent human fibroblasts.

Telomeres, the G/C-rich DNA sequences capping the ends of all eukaryotic chromosomes, have been shown to shorten during replicative aging of normal cells in vitro and in vivo. Moreover, variation in the initial length of terminal restriction fragments (TRF) accounts for much of the variation in replicative capacity of fibroblast cultures from different donors. Since replicative capacity also varies significantly between clones in a mass culture of fibroblasts from a single donor, we wished to further test the hypothesis that the shortening of telomeres to a critical or threshold length acts as a signal for cell senescence. Thus, we measured TRF length and total telomeric signal intensity for 35 clonal fibroblast populations at early passage and at senescence. Replicative capacity was found to be directly proportional to mean TRF length (m = 7.2 population doublings/kbp, r = 0.65, P = 0.0004) and total signal intensity (m = 25.0 population doublings/unit, r = 0.63, P < 0.003) at early passage. More importantly, the variability in both mean TRF length and signal intensity (F = 2.0 and 2.9; P = 0.02 and 0.03, respectively) at senescence was markedly less than that at early passage. Although initial telomere length cannot account for all of the interclonal variability in replicative capacity, our observations support the existence of a critical telomere length in senescing cells and a causal role of telomere shortening in cell senescence.

Evidence for a mitotic clock in human hematopoietic stem cells: loss of telomeric DNA with age.

The proliferative life-span of the stem cells that sustain hematopoiesis throughout life is not known. It has been proposed that the sequential loss of telomeric DNA from the ends of human chromosomes with each somatic cell division eventually reaches a critical point that triggers cellular senescence. We now show that candidate human stem cells with a CD34+CD38lo phenotype that were purified from adult bone marrow have shorter telomeres than cells from fetal liver or umbilical cord blood. We also found that cells produced in cytokine-supplemented cultures of purified precursor cells show a proliferation-associated loss of telomeric DNA. These findings strongly suggest that the proliferative potential of most, if not all, hematopoietic stem cells is limited and decreases with age, a concept that has widespread implications for models of normal and abnormal hematopoiesis as well as gene therapy.

Equid herpesviruses 1 and 4 encode functional homologs of the herpes simplex virus type 1 virion transactivator protein, VP16.

The herpes simplex virus type 1 (HSV-1) tegument protein VP16 is a potent transcriptional inducer of immediate-early gene expression, comprising an N-terminal domain involved in binding DNA linked to an acidic transactivating C-terminal domain. The gene encoding the counterpart of this protein in equid herpesvirus 4 (EHV-4) was sequenced. Comparisons with VP16 and the homologous proteins of equine herpesvirus 1 (EHV-1) and varicella-zoster virus (VZV) showed that a region in the N-terminal domain involved in formation of a complex with cellular proteins is partially conserved in all four proteins. In contrast, the C-terminal regions of the EHV proteins, like that of VZV, are not particularly acidic and are not significantly conserved with respect to the C-terminal region of VP16. Nevertheless, transient expression experiments indicated that the EHV-1 and EHV-4 proteins are able to transactivate HSV-1 and EHV-1 immediate-early promoters in a dose-dependent manner, which suggests that this activity is not dependent on an acidic C-terminal domain.