What happens to cancer survivors attending a structured cancer survivorship clinic? Symptoms, quality of life and lifestyle changes over the first year at the Sydney Cancer Survivorship Centre clinic.

BACKGROUND: Sydney Cancer Survivorship Centre (SCSC) clinic provides multidisciplinary care after primary adjuvant treatment, with ~ 40% of attendees continuing follow-up with SCSC. METHODS: SCSC survivors completed measures of symptoms, quality-of-life and lifestyle factors at initial visit (T1), first follow-up (T2) and 1 year (T3). Analyses used mixed effect models, adjusted for age, sex and tumour type. RESULTS: Data from 206 survivors (2013-2019) were included: 51% male; median age 63 years; tumour types colorectal 68%, breast 12%, upper gastrointestinal 12%, other 8%. Mean time from: T1 to T2, 3.6 months; T1 to T3, 11.8 months. Mean weight remained stable, but 45% (35/77) of overweight/obese survivors lost weight from T1 to T3. Moderately-intense aerobic exercise increased by 63 mins/week at T2, and 68 mins/week T3. Proportion meeting aerobic exercise guidelines increased from 20 to 41%. Resistance exercise increased by 26 mins/week at T2. Global quality-of-life was unchanged from T1 to T2, improving slightly by T3 (3.7-point increase), mainly in males. Mean distress scores were stable, but at T3 the proportion scoring 4+/10 had declined from 41 to 33%. At T3, improvements were seen in pain, fatigue and energy, but > 20% reported moderate-severe fatigue, pain or sleep disturbance. Proportion reporting 5+ moderate-severe symptoms declined from 35% at T1 to 26% at T3, remaining higher in women. CONCLUSIONS: Survivors attending SCSC increased exercise by 3 months, and sustained it at 1 year. Most overweight/obese survivors avoided further weight gain. Survivors had relatively good quality-of-life, with improvement in many symptoms and lifestyle factors at 1 year.

Health concerns of cancer survivors after primary anti-cancer treatment.

PURPOSE: Cancer survivors experience significant health concerns compared to the general population. Sydney Survivorship Clinic (SSC) is a multi-disciplinary clinic aiming to help survivors treated with curative intent manage side effects, and establish a healthy lifestyle. Here, we determine the health concerns of survivors post-primary treatment. METHODS: Survivors completed questionnaires assessing symptoms, quality of life (QOL), distress, diet, and exercise before attending SSC, and a satisfaction survey after. Body mass index (BMI), clinical findings and recommendations were reviewed. Descriptive statistical methods were used. RESULTS: Overall, 410 new patients attended SSC between September 2013 and April 2018, with 385 survivors included in analysis: median age 57 years (range 18-86); 69% female; 43% breast, 31% colorectal and 19% haematological cancers. Median time from diagnosis, 12 months. Common symptoms of at least moderate severity: fatigue (45%), insomnia (37%), pain (34%), anxiety (31%) and with 56% having > 5 moderate-severe symptoms. Overall, 45% scored distress ≥ 4/10 and 62% were rated by clinical psychologist as having 'fear of cancer recurrence'. Compared to population mean of 50, mean global QOL T-score was 47.2, with physical and emotional well-being domains most affected. Average BMI was 28.2 kg/m2 (range 17.0-59.1); 61% overweight/obese. Only 31% met aerobic exercise guidelines. Overall, 98% 'agreed'/'completely agreed' attending the SSC was worthwhile, and 99% would recommend it to others. CONCLUSION: Distress, fear of cancer recurrence, fatigue, obesity and sedentary lifestyle are common in cancer survivors attending SSC and may best be addressed in a multi-disciplinary Survivorship Clinic to minimise longer-term effects. This model is well-rated by survivors.

Evaluation of the time resolved fluorescence immunoassay (TRFIA) for the detection of varicella zoster virus (VZV) antibodies following vaccination of healthcare workers.

Determination of varicella zoster virus (VZV) immunity in healthcare workers without a history of chickenpox is important for identifying those in need of vOka vaccination. Post immunisation, healthcare workers in the UK who work with high risk patients are tested for seroconversion. To assess the performance of the time-resolved fluorescence immunoassay (TRFIA) for the detection of antibody in vaccinated as well as unvaccinated individuals, a cut-off was first calculated. VZV-IgG specific avidity and titres six weeks after the first dose of vaccine were used to identify subjects with pre-existing immunity among a cohort of 110 healthcare workers. Those with high avidity (≥ 60%) were considered to have previous immunity to VZV and those with low or equivocal avidity (<60%) were considered naive. The former had antibody levels ≥ 400 mIU/mL and latter had levels < 400 mIU/mL. Comparison of the baseline values of the naive and immune groups allowed the estimation of a TRFIA cut-off value of > 130 mIU/mL which best discriminated between the two groups and this was confirmed by ROC analysis. Using this value, the sensitivity and specificity of TRFIA cut-off were 90% (95% CI 79-96), and 78% (95% CI 61-90) respectively in this population. A subset of samples tested by the gold standard Fluorescence Antibody to Membrane Antigen (FAMA) test showed 84% (54/64) agreement with TRFIA.

Persistence of varicella-zoster virus viraemia in patients with herpes zoster.

BACKGROUND: Herpes zoster is caused by the reactivation of varicella-zoster virus from sensory neurons. The commonest complication following zoster is chronic pain termed post herpetic neuralgia. OBJECTIVES: To investigate the dynamics of VZV viraemia and viral load following the resolution of zoster and its relationship to PHN development. STUDY DESIGN: Blood samples were collected at baseline, 1 month, 3 months and 6 month from a prospective study of 63 patients with active zoster. Quantification of VZV DNA in whole blood was performed using a real-time PCR assay. RESULTS: During acute zoster, all patients had detectable VZV DNA in their blood. VZV DNA remained detectable in the blood of 91% of patients at 6 months although levels declined significantly (p<0.0001). A history of prodromal symptoms (p=0.005) and severity of pain at baseline (p=0.038) as well as taking antivirals (p=0.046) and being immunocompromised (p=0.043) were associated, with longer time to recovery from PHN. Viral DNA loads were consistently higher in patients with risk factors for PHN and higher viral DNA loads over time were associated with longer time to recovery (p=0.058 overall and 0.038 in immunocompetent). CONCLUSIONS: Based on these observations we hypothesise that VZV replication persists following acute shingles and that higher viral DNA loads contribute to the risk factors for PHN.

Effect of viral load on the outcome of herpes zoster.

Varicella-zoster virus (VZV) is a member of the Herpesviridae family, primary infection with which causes varicella, more commonly known as chicken pox. Characteristic of members of the alphaherpesvirus subfamily, VZV is neurotropic and establishes latency in sensory neurons. Reactivation of VZV causes herpes zoster, also known as shingles. The most frequent complication following zoster is chronic and often debilitating pain called postherpetic neuralgia (PHN), which can last for months after the disappearance of a rash. During episodes of acute zoster, VZV viremia occurs in some, but not all, patients; however, the effect of the viral load on the disease outcome is not known. Here we describe the development of a highly specific, sensitive, and reproducible real-time PCR assay to investigate the factors that may contribute to the presence and levels of baseline viremia in patients with zoster and to determine the relationship between viremia and the development and persistence of PHN. VZV DNA was detected in the peripheral blood mononuclear cells (PBMCs) of 78% of patients with acute zoster and in 9% of healthy asymptomatic blood donors. The presence of VZV in the PBMCs of patients with acute zoster was independently associated with age and being on antivirals but not with gender, immune status, extent of rash, the age of the rash at the time of blood sampling, having a history of prodromal pain, or the extent of acute pain. Prodromal pain was significantly associated with higher baseline viral loads. Viral load levels were not associated with the development or persistence of PHN at 6, 12, or 26 weeks.

Population genetic models can be used to study the evolution of the interacting behaviours of parents and their progeny.

An example of the evolution of the interacting behaviours of parents and progeny is studied using iterative equations linking the frequencies of the gametes produced by the progeny to the frequencies of the gametes in the parental generation. This population genetics approach shows that a model in which both behaviours are determined by a single locus can lead to a stable equilibrium in which the two behaviours continue to segregate. A model in which the behaviours are determined by genes at two separate loci leads eventually to fixation of the alleles at both loci but this can take many generations of selection. Models of the type described in this paper will be needed to understand the evolution of complex behaviour when genomic or experimental information is available about the genetic determinants of behaviour and the selective values of different genomes.

DNA variation in a 13-Mb region including the F9 gene: inferring the genealogical history and causal role of a hemophilia B mutation (IVS 5+13 A-->G).

About 5.5% of all UK hemophilia B patients have the base substitution IVS 5+13 A-->G as the only change in their factor (F)IX gene (F9). This generates a novel donor splice site which fits the consensus better than the normal intron 5 donor splice. Use of the novel splice site should result in a missense mutation followed by the abnormal addition of four amino acids to the patients' FIX. In order to explain the prevalence of this mutation, its genealogical history is examined. Analysis of restriction fragment length polymorphism in the 21 reference UK individuals (from different families) with the above mutation showed identical haplotypes in 19 while two differed from the rest and from each other. In order to investigate the history of the mutation and to verify that it had occurred independently more than once, the sequence variation in 1.5-kb segments scattered over a 13-Mb region including F9 was examined in 18 patients and 15 controls. This variation was then analyzed with a recently developed Bayesian approach that reconstructs the genealogy of the gene investigated while providing evidence of independent mutations that contribute disconnected branches to the genealogical tree. The method also provides minimum estimates of the age of the mutation inherited by the members of coherent trees. This revealed that 17 or 18 mutant genes descend from a founder who probably lived 450 years ago, while one patient carries an independent mutation. The independent recurrence of the IVS5+13 A-->G mutation strongly supports the conclusion that it is the cause of these patients' mild hemophilia.

Short tandem repeat profiling provides an international reference standard for human cell lines.

Cross-contamination between cell lines is a longstanding and frequent cause of scientific misrepresentation. Estimates from national testing services indicate that up to 36% of cell lines are of a different origin or species to that claimed. To test a standard method of cell line authentication, 253 human cell lines from banks and research institutes worldwide were analyzed by short tandem repeat profiling. The short tandem repeat profile is a simple numerical code that is reproducible between laboratories, is inexpensive, and can provide an international reference standard for every cell line. If DNA profiling of cell lines is accepted and demanded internationally, scientific misrepresentation because of cross-contamination can be largely eliminated.

Measuring gametic disequilibrium from multilocus data.

We describe a Bayesian approach to analyzing multilocus genotype or haplotype data to assess departures from gametic (linkage) equilibrium. Our approach employs a Markov chain Monte Carlo (MCMC) algorithm to approximate the posterior probability distributions of disequilibrium parameters. The distributions are computed exactly in some simple settings. Among other advantages, posterior distributions can be presented visually, which allows the uncertainties in parameter estimates to be readily assessed. In addition, background knowledge can be incorporated, where available, to improve the precision of inferences. The method is illustrated by application to previously published datasets; implications for multilocus forensic match probabilities and for simple association-based gene mapping are also discussed.

Lipoprotein (a) as a predictor of myocardial infarction in middle-aged men.

PURPOSE: Whether serum lipoprotein (a) [Lp(a)] levels are an independent risk factor for coronary heart disease has been controversial. We have investigated its status in a prospective population survey, the Second Northwick Park Heart Study. METHODS: We recruited 2,616 men 50 to 61 years old from nine primary care practices in the United Kingdom. Baseline serum Lp(a) levels were measured by enzyme-linked immunosorbent assay (ELISA) and were analyzed in 3 groups (<25th percentile, 25th to 75th percentile, and >75th percentile) to overcome the problem of some measurements falling below the threshold of the assay. Coronary end points included sudden cardiac death, acute myocardial infarction, silent myocardial infarction on the electrocardiogram, and coronary artery bypass surgery. RESULTS: During a mean of 6 years of follow-up, 121 men had coronary events. In a multivariate analysis that also adjusted for fibrinogen, Apo-A1, Apo-B, and triglyceride levels, we identified several independent risk factors for coronary events, including cholesterol level (hazard ratio [HR] = 1.5 per SD 95% confidence interval [CI] 1.3 to 1.8), diabetes (HR = 4.1, 95% CI: 2. 0 to 8.4), current versus never smoking (HR = 2.5, 95% CI: 1.5 to 4.1), diastolic blood pressure (HR = 1.4 per SD, 95% CI: 1.1 to 1.7), Apo-A1 (HR = 0.8 per SD, 95% CI: 0.6 to 0.9), age (HR = 1.3 per SD, 95% CI: 1.1 to 1.6), and Lp(a) (>26.3 mg/dL [75th percentile] versus <2.9 mg/dL [25th percentile], HR = 1.9, 95% CI: 1.1 to 3.3]. There was a statistically significant (P = 0.01) difference in risk between the three levels of Lp(a). CONCLUSIONS: We found that a high Lp(a) level was an independent predictor of the development of coronary heart disease in middle-aged men.

Analysis of disputed single-parent/child and sibling relationships using 16 STR loci.

This study describes the validation of short tandem repeat (STR) systems for the resolution of cases of disputed parentage where only a single parent is available for testing or where the claimed relationship of both parents is in doubt and also cases where sibship must be tested. Three separate multiplex systems the Second Generation Multiplex, Powerplex 1.2 and FFFL have been employed, giving a total of 16 STR loci. Both empirical and theoretical approaches to the validation have been adopted. Appropriate equations have been derived to calculate likelihood ratios for different relationships, incorporating a correction for subpopulation effects. An F(ST) point estimate of 1% has been applied throughout. Empirically, 101 cases of alleged father, alleged mother and child where analysed using six SLP systems and also using the three multiplex STR systems. Of the 202 relationships tested, 197 were independently resolved by both systems, providing either clear evidence of non-parentage or strong support for the relationship.

Relatedness testing in subdivided populations.

Assigning probabilities to alleged relationships, given DNA profiles, requires, among other things, calculation of a likelihood ratio (LR). Such calculations usually assume independence of genes: this assumption is not appropriate when the tested individuals share recent ancestry due to population substructure. Adjusted LR formulae, incorporating the coancestry coefficient F(ST), are presented here for various two-person relationships, and the issue of mutations in parentage testing is also addressed.

A two-locus forensic match probability for subdivided populations.

A two-locus match probability is presented that incorporates the effects of within-subpopulation inbreeding (consanguinity) in addition to population subdivision. The usual practice of calculating multi-locus match probabilities as the product of single-locus probabilities assumes independence between loci. There are a number of population genetics phenomena that can violate this assumption: in addition to consanguinity, which increases homozygosity at all loci simultaneously, gametic disequilibrium will introduce dependence into DNA profiles. However, in forensics the latter problem is usually addressed in part by the careful choice of unlinked loci. Hence, as is conventional, we assume gametic equilibrium here, and focus instead on between-locus dependence due to consanguinity. The resulting match probability formulae are an extension of existing methods in the literature, and are shown to be more conservative than these methods in the case of double homozygote matches. For two-locus profiles involving one or more heterozygous genotypes, results are similar to, or smaller than, the existing approaches.

Validation of short tandem repeat analysis for the investigation of cases of disputed paternity.

This study details validation of two separate multiplex STR systems for use in paternity investigations. These are the Second Generation Multiplex (SGM) developed by the UK Forensic Science Service and the PowerPlex 1 multiplex commercially available from Promega Inc. (Madison, WI, USA). These multiplexes contain 12 different STR systems (two are duplicated in the two systems). Population databases from Caucasian, Asian and Afro-Caribbean populations have been compiled for all loci. In all but two of the 36 STR/ethnic group combinations, no evidence was obtained to indicate inconsistency with Hardy-Weinberg (HW) proportions. Empirical and theoretical approaches have been taken to validate these systems for paternity testing. Samples from 121 cases of disputed paternity were analysed using established Single Locus Probe (SLP) tests currently in use, and also using the two multiplex STR systems. Results of all three test systems were compared and no non-conformities in the conclusions were observed, although four examples of apparent germ line mutations in the STR systems were identified. The data was analysed to give information on expected paternity indices and exclusion rates for these STR systems. The 12 systems combined comprise a highly discriminating test suitable for paternity testing. 99.96% of non-fathers are excluded from paternity on two or more STR systems. Where no exclusion is found, Paternity Index (PI) values of > 10,000 are expected in > 96% of cases.

Measuring departures from Hardy-Weinberg: a Markov chain Monte Carlo method for estimating the inbreeding coefficient.

Many well-established statistical methods in genetics were developed in a climate of severe constraints on computational power. Recent advances in simulation methodology now bring modern, flexible statistical methods within the reach of scientists having access to a desktop workstation. We illustrate the potential advantages now available by considering the problem of assessing departures from Hardy-Weinberg (HW) equilibrium. Several hypothesis tests of HW have been established, as well as a variety of point estimation methods for the parameter which measures departures from HW under the inbreeding model. We propose a computational, Bayesian method for assessing departures from HW, which has a number of important advantages over existing approaches. The method incorporates the effects-of uncertainty about the nuisance parameters--the allele frequencies--as well as the boundary constraints on f (which are functions of the nuisance parameters). Results are naturally presented visually, exploiting the graphics capabilities of modern computer environments to allow straightforward interpretation. Perhaps most importantly, the method is founded on a flexible, likelihood-based modelling framework, which can incorporate the inbreeding model if appropriate, but also allows the assumptions of the model to he investigated and, if necessary, relaxed. Under appropriate conditions, information can be shared across loci and, possibly, across populations, leading to more precise estimation. The advantages of the method are illustrated by application both to simulated data and to data analysed by alternative methods in the recent literature.

Synthesis and antiviral evaluation of certain disubstituted benzimidazole ribonucleosides.

Ribosylation of 2-chloro-5(6)-nitrobenzimidazole (3) gave 2-chloro-5-nitro-1-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)benzimidazol e (4a) and 2-chloro-6-nitro-1-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)benzimidazol e (4b) as a mixture of positional isomers. Subsequent hydrogenation of this mixture over Raney Nickel afforded the corresponding 5-amino and 6-amino derivatives 5 and 6. At this stage the products were readily resolved via silica column chromatography into pure isomeric forms, and the pure isomers 5 and 6 were diazotized with tert-butyl nitrite and cupric chloride to furnish the isomerically pure 5-chloro derivative 2a and 6-chloro derivative 2b. Deprotection of 5, 6, 2a, and 2b with methanolic ammonia yielded the free nucleosides 5-amino-2-chloro-1-(beta-D-ribofuranosyl)benzimidazole (7), 6-amino-2-chloro-1-(beta-D-ribofuranosyl)-benzimidazole (8), 2,5-dichloro-1-(beta-D-ribofuranosyl)benzimidazole (9), and 2,6-dichloro-1-(beta-D-ribofuranosyl)benzimidazole (10), respectively. Treatment of 10 with thiourea afforded 6-chloro-1-(beta-D-ribofuranosyl)benzimidazole-2-thione (14). Alkylation of 14 with methyl iodide and benzyl bromide gave good yields of the corresponding 2-methylthio (12) and the 2-benzylthio (13) analogs. The synthesis of 6-chloro-2-methoxy-1-(beta-D-ribofuranosyl)benzimidazole (11) was accomplished by the treatment of 2b with sodium methoxide in methanol. A difference NOE spectroscopic experiment was conducted to allow unequivocal assignment of regiochemistry to the positional isomers 5 and 6. Evaluation of compounds for activity against human cytomegalovirus (HCMV) and herpes simplex virus type 1 revealed that the heterocycle 3 was active against both viruses but also was cytotoxic. Only the dichloro compounds 9 and 10 were weakly active against HCMV and noncytotoxic in their antiviral dose range. These data further substantiate the conclusion that activity against HCMV at noncytotoxic concentrations by benzimidazole ribonucleosides requires a halogen not only at the 2-position, but also more than one halogen on the benzene moiety.

Developmental regulation of DNase I-hypersensitive sites in Dictyostelium discoideum.

We have studied two regions of Dictyostelium discoideum chromatin and identified several DNase I-hypersensitive sites in these regions. One of these sites is located about 300 to 500 bases upstream of the transcriptional start site of a gene that is expressed at all stages of development. This site is present in both vegetative cells and postaggregation cells. Another hypersensitive site is associated with a gene that is expressed only after the multicellular stage. This site is located about 400 bases upstream of the start site, and it is present only in postaggregation cells. Thus, much like higher eucaryotes, D. discoideum contains DNase I-hypersensitive sites that may be involved in the regulation of the genes with which they are associated.

Cellular aspects of DNA repair.

DNA repair reactions are under cellular control. In bacteria, the reactions removing 0(6)-methylguanine and 3-methyladenine are inducible. It is not clear whether similar inducibility occurs in human lymphoblastoid cells. Nonetheless, the ability to manufacture the 0(6)-methylguanine acceptor protein does seem to be controlled by some chromosomal mechanism which is superimposed on the structural gene. This control system may affect reactions other than the removal of 0(6)-methylguanine. Insofar as this is so, transformed human lymphoblastoid cells have a system reminiscent of that found in bacteria.

Regulation of the capacity for O6-methylguanine removal from DNA in human lymphoblastoid cells studied by cell hybridization.

Hybrids were made between a ouabain-resistant, thioguanine-resistant human lymphoma line able to remove O6-methylguanine from its DNA (Mex+) and human lymphoblastoid lines deficient in this capability (Mex-). The formation of hybrids was confirmed by chromosomal analysis. Hybrid cells had an O6-methylguanine removal capacity per mole of guanine about one third to one half that of the Mex+ parents, i.e., about the same per cell. Cell hybrids removed the same amount of the alkylation adduct 3-methyladenine as did their parents per mole of guanine, i.e., about twice as much per cell. Although the cell hybrids had intermediate resistance to the cytotoxic action of N-methyl-N'-nitro-N-nitrosoguanidine used to induce O6-methylguanine and 3-methyladenine, there is evidence that the ability to remove O6-methylguanine and resistance to the cytotoxic effect of N-methyl-N'-nitro-N-nitrosoguanidine are dissociable characteristics.

High cloning efficiency of human lymphoid cells in agarose without feeder layers.

A protocol has been developed that consistently gives high cloning efficiencies for human lymphoid cell lines (lymphoblastoid and lymphoma) in agarose without the use of feeder layers. This procedure utilizes a cloning medium that contains horse serum, alpha-ketoglutarate or oxalacetate, and high levels of glutamine.