The molecular landscape and associated clinical experience in infant medulloblastoma: prognostic significance of second-generation subtypes.

AIMS: Biomarker-driven therapies have not been developed for infant medulloblastoma (iMB). We sought to robustly sub-classify iMB, and proffer strategies for personalized, risk-adapted therapies. METHODS: We characterized the iMB molecular landscape, including second-generation subtyping, and the associated retrospective clinical experience, using large independent discovery/validation cohorts (n = 387). RESULTS: iMBGrp3 (42%) and iMBSHH (40%) subgroups predominated. iMBGrp3 harboured second-generation subtypes II/III/IV. Subtype II strongly associated with large-cell/anaplastic pathology (LCA; 23%) and MYC amplification (19%), defining a very-high-risk group (0% 10yr overall survival (OS)), which progressed rapidly on all therapies; novel approaches are urgently required. Subtype VII (predominant within iMBGrp4 ) and subtype IV tumours were standard risk (80% OS) using upfront CSI-based therapies; randomized-controlled trials of upfront radiation-sparing and/or second-line radiotherapy should be considered. Seventy-five per cent of iMBSHH showed DN/MBEN histopathology in discovery and validation cohorts (P < 0.0001); central pathology review determined diagnosis of histological variants to WHO standards. In multivariable models, non-DN/MBEN pathology was associated significantly with worse outcomes within iMBSHH . iMBSHH harboured two distinct subtypes (iMBSHH-I/II ). Within the discriminated favourable-risk iMBSHH DN/MBEN patient group, iMBSHH-II had significantly better progression-free survival than iMBSHH-I , offering opportunities for risk-adapted stratification of upfront therapies. Both iMBSHH-I and iMBSHH-II showed notable rescue rates (56% combined post-relapse survival), further supporting delay of irradiation. Survival models and risk factors described were reproducible in independent cohorts, strongly supporting their further investigation and development. CONCLUSIONS: Investigations of large, retrospective cohorts have enabled the comprehensive and robust characterization of molecular heterogeneity within iMB. Novel subtypes are clinically significant and subgroup-dependent survival models highlight opportunities for biomarker-directed therapies.

Minimal methylation classifier (MIMIC): A novel method for derivation and rapid diagnostic detection of disease-associated DNA methylation signatures.

Rapid and reliable detection of disease-associated DNA methylation patterns has major potential to advance molecular diagnostics and underpin research investigations. We describe the development and validation of minimal methylation classifier (MIMIC), combining CpG signature design from genome-wide datasets, multiplex-PCR and detection by single-base extension and MALDI-TOF mass spectrometry, in a novel method to assess multi-locus DNA methylation profiles within routine clinically-applicable assays. We illustrate the application of MIMIC to successfully identify the methylation-dependent diagnostic molecular subgroups of medulloblastoma (the most common malignant childhood brain tumour), using scant/low-quality samples remaining from the most recently completed pan-European medulloblastoma clinical trial, refractory to analysis by conventional genome-wide DNA methylation analysis. Using this approach, we identify critical DNA methylation patterns from previously inaccessible cohorts, and reveal novel survival differences between the medulloblastoma disease subgroups with significant potential for clinical exploitation.

Cross-species epigenetics identifies a critical role for VAV1 in SHH subgroup medulloblastoma maintenance.

The identification of key tumorigenic events in Sonic Hedgehog (SHH) subgroup medulloblastomas (MBSHH) will be essential for the development of individualized therapies and improved outcomes. However, beyond confirmation of characteristic SHH pathway mutations, recent genome-wide sequencing studies have not revealed commonly mutated genes with widespread relevance as potential therapeutic targets. We therefore examined any role for epigenetic DNA methylation events in MBSHH using a cross-species approach to candidate identification, prioritization and validation. MBSHH-associated DNA methylation events were first identified in 216 subgrouped human medulloblastomas (50 MBSHH, 28 Wnt/Wingless, 44 Group 3 and 94 Group 4) and their conservation then assessed in tumors arising from four independent murine models of Shh medulloblastoma, alongside any role in tumorigenesis using functional assessments in mouse and human models. This strategy identified widespread regional CpG hypo-methylation of VAV1, leading to its elevated expression, as a conserved aberrant epigenetic event, which characterizes the majority of MBSHH tumors in both species, and is associated with a poor outcome in MBSHH patients. Moreover, direct modulation of VAV1 in mouse and human models revealed a critical role in tumor maintenance, and its abrogation markedly reduced medulloblastoma growth. Further, Vav1 activity regulated granule neuron precursor germinal zone exit and migration initiation in an ex vivo model of early postnatal cerebellar development. These findings establish VAV1 as a critical epigenetically regulated oncogene with a key role in MBSHH maintenance, and highlight its potential as a validated therapeutic target and prognostic biomarker for the improved therapy of medulloblastoma.

Ethical tradeoffs in trial design: case study of an HPV vaccine trial in HIV-infected adolescent girls in lower income settings.

The Declaration of Helsinki and the Council of the International Organization of Medical Sciences provide guidance on standards of care and prevention in clinical trials. In the current and increasingly challenging research environment, the ethical status of a trial design depends not only on protection of participants, but also on social value, feasibility, and scientific validity. Using the example of a study assessing efficacy of a vaccine to prevent human papilloma virus in HIV-1 infected adolescent girls in low resource countries without access to the vaccine, we compare several trial designs which rank lower on some criteria and higher on others, giving rise to difficult trade-offs. This case demonstrates the need for developing more nuanced guidance documents to help researchers balance these often conflicting criteria.

Abacavir pharmacokinetics during chronic therapy in HIV-1-infected adolescents and young adults.

The pharmacokinetics of abacavir and its metabolites were investigated in 30 human immunodeficiency virus (HIV)-infected adolescents and young adults 13-25 years of age, equally divided into two groups: <18 years of age and >or=18 years of age. All the subjects received the recommended adult dose of 300 mg twice daily. The area under the plasma concentration-time curve (AUC) and half-life of abacavir did not differ significantly between the age groups or by gender or race, and there were only modest associations of age with apparent abacavir clearance and with volume of distribution. There were no significant correlations of carboxylate or glucuronide metabolite levels with age or gender, although glucuronide AUC was higher in Hispanic subjects than in African-American subjects. Zidovudine and lamivudine concentration profiles were also similar in the two age groups. A novel aspect of the study included an assessment of intracellular carbovir, zidovudine, and lamivudine triphosphate levels, and these were found to be similar in the two age-based groups. Overall, these findings suggest that current recommendations relating to adult dosages are appropriate for adolescents and young adults.

Alcohol disorders among Asian Americans: associations with unfair treatment, racial/ethnic discrimination, and ethnic identification (the national Latino and Asian Americans study, 2002-2003).

STUDY OBJECTIVE: To examine history of alcohol abuse/dependence disorder in relation to unfair treatment, racial/ethnic discrimination, and ethnic identification among Asian Americans. DESIGN: Weighted multivariate analyses of cross-sectional national survey data predicting lifetime history of alcohol abuse/dependence disorders. SETTING: USA, Asian Americans. PARTICIPANTS: 2007 Asian American adults recruited to the National Latino and Asian American Study (NLAAS; 2002-2003). RESULTS: Controlling for sociodemographic characteristics, Asian Americans who reported experiencing unfair treatment had higher odds of history of alcohol abuse/dependence disorder (OR 5.26, 95% CI 1.90 to 14.56). Participants who reported high levels of ethnic identification had lower odds of history of alcohol abuse/dependence disorders (OR 0.46, 95% CI 0.23 to 0.90). Ethnic identification moderated the influence of racial/ethnic discrimination (p = 0.097). Among participants with low levels of ethnic identification, racial/ethnic discrimination was associated with greater odds of having a history of alcohol disorder compared with those with high levels of ethnic identification. CONCLUSIONS: Social hazards such as unfair treatment and racial/ethnic discrimination should be considered in the development of programmes addressing alcohol disorders among Asian Americans. Interventions that promote ethnic identification in this population may be particularly relevant in mitigating the negative influence of racial/ethnic discrimination on alcohol disorders.

Epigenetic deregulation of multiple S100 gene family members by differential hypomethylation and hypermethylation events in medulloblastoma.

Deregulated expression of genes encoding members of the S100 family of calcium-binding proteins has been associated with the malignant progression of multiple tumour types. Using a pharmacological expression reactivation approach, we screened 16 S100 genes for evidence of epigenetic regulation in medulloblastoma, the most common malignant brain tumour of childhood. Four family members (S100A2, S100A4, S100A6 and S100A10) demonstrated evidence of upregulated expression in multiple medulloblastoma cell lines, following treatment with the DNA methyltransferase inhibitor, 5'-aza-2'-deoxycytidine. Subsequent analysis revealed methylation of critical CpG sites located within these four genes in an extended cell line panel. Assessment of these genes in the non-neoplastic cerebellum (from which medulloblastomas develop) revealed strong somatic methylation affecting S100A2 and S100A4, whereas S100A6 and S100A10 were unmethylated. Assessed against these normal tissue-specific methylation states, S100A6 and S100A10 demonstrated tumour-specific hypermethylation in medulloblastoma primary tumours (5 out of 40 and 4 out of 35, respectively, both 12%) and cell lines (both 7 out of 9, 78%), which was associated with their transcriptional silencing. Moreover, S100A6 hypermethylation was significantly associated with the aggressive large cell/anaplastic morphophenotype (P=0.026). In contrast, pro-metastatic S100A4 displayed evidence of hypomethylation relative to the normal cerebellum in a significant proportion primary tumours (7 out of 41, 17%) and cell lines (3 out of 9, 33%), which was associated with its elevated expression. In summary, these data characterise complex patterns of somatic methylation affecting S100 genes in the normal cerebellum and demonstrate their disruption causing epigenetic deregulation of multiple S100 family members in medulloblastoma development. Epigenetic events affecting S100 genes have potential clinical utility and merit further investigation as molecular biomarkers for this disease.

Impact of protease inhibitor-containing combination antiretroviral therapies on height and weight growth in HIV-infected children.

OBJECTIVE: To examine beneficial or detrimental effects of protease inhibitor (PI)-containing antiretroviral regimens on height and weight growth in children with human immunodeficiency virus (HIV) infection. METHODS: A prospective cohort study was conducted of 906 HIV-infected children, from pediatric research clinics in the United States, who were between 3 months and 18 years of age and who had height and weight assessed in 1995 (before introduction of PIs in this population) and at least once more through 1999. Changes in age- and gender-adjusted height and weight growth associated with PI use were assessed. RESULTS: Compared with a healthy reference population, children were more affected in height (mean z score: -0.90 [18th percentile]) than in weight (mean z score: -0.42 [34th percentile]) at baseline (1995). Two thirds of children received at least 1 PI during 1996 to 1999. In the multivariate mixed effects regression models adjusted for baseline log(10) CD4 cell count, baseline age, gender, and race/ethnicity, the use of PIs was associated with per-year gains of 0.13 z scores in height and 0.05 z scores in weight relative to the expected growth with non-PI-containing regimens (eg, after 1 year of PI use, a representative 6-year-old boy in our study would be approximately 0.7 cm taller and 0.1 kg heavier than if he had not received PIs). No significant differential effects of PIs on height or weight growth according to specific agents or children's sociodemographic or clinical characteristics were found. CONCLUSIONS: Although the use of PI-containing regimens was not associated with growth retardation, it was associated with only small annual increments in height and weight growth in HIV-infected children.

HIV-1 RNA levels and development of clinical disease in two different adolescent populations.

HIV infection rates in American youth continue to increase unabated. As adolescent-specific therapeutic interventions are planned, information on HIV infection's course and its predictors becomes critically important for valid and precise study design. We report on age-specific disease rates stratified by estimated time since infected and predictors of HIV disease progression through four clinical categories in two distinct adolescent populations. Adolescents with hemophilia infected through contaminated blood products showed disease progression rates of 18 to 23 events per 100 person-years (PYs) by age and years infected. Predictors of first progression included HIV-1 RNA >30,000 copies/ml (rate ratio [RR], 2.4; 95% confidence interval [CI], 1.5-3.9), antiretroviral monotherapy (RR, 2.4; 95% CI, 1.7-3.3); Latino/a ethnicity (RR, 2.2; 95% CI, 1.2-4.2) and initial intermediate clinical status (RR, 1.9; 95% CI, 1.3-2.9). Sexually-infected adolescents >18 years who had been infected >3 to 6 years had a disease progression rate of 16 events per 100 PY. For these youths, the sole predictor of first progression was viral load (VL) (RR for VL >30,000 copies per ml, 8.4; 95% CI, 2.8-25.1). This article examines the predictive capacity of viral load and evaluates other cofactors for disease progression in different adolescent populations. These data will be of value in clinical trial design.

Correlates of opportunistic infections in children infected with the human immunodeficiency virus managed before highly active antiretroviral therapy.

BACKGROUND: Opportunistic infections (OIs) are an important cause of morbidity and mortality in children infected with HIV. However, few data are available regarding the overall prevalence, incidence and immunologic correlates associated with these diseases in the pediatric HIV population. The Pediatric AIDS Clinical Trials Group (PACTG) has conducted multicenter studies in HIV-infected children since 1988 and through these studies has collected prospective data on the immunologic and virologic status of study participants and recorded complications, including infectious diseases, related to HIV infection and its treatments. Therefore data were analyzed from across 13 PACTG studies, performed before treatment with highly active antiretroviral therapy was given, to determine the rates of various infectious complications and the immunologic correlates, specifically CD4 cell counts, associated with these diseases. RESULTS: OIs were tabulated from 3331 HIV-infected children who participated in 13 clinic trials undertaken before highly effective antiretroviral therapy was available. Five OIs occurred at event rates of >1.0 per 100 patient years (person years): serious bacterial infections, 15.1; herpes zoster, 2.9; disseminated Mycobacterium avium complex (DMAC), 1.8; Pneumocystis carinii pneumonia, 1.3; and tracheobronchial and esophageal candidiasis, 1.2. Six other OIs evaluated, cytomegalovirus (CMV) disease, cryptosporidiosis, tuberculosis, systemic fungal infections, toxoplasmosis and progressive multifocal leukoencephalopathy, occurred at event rates of <1.0 per 100 person years. Pneumonia (11.1 per 100 person years) and bacteremia (3.3 per 100 person years) were the most common bacterial infections. An AIDS-defining OI before entry was a risk factor for the development of a new OI during a trial. Bacterial infections, herpes zoster and tuberculosis occurred frequently at all stages of HIV infection; whereas DMAC, P. carinii pneumonia, CMV and other OIs occurred primarily in children with severe immunosuppression. CONCLUSIONS: The frequency of OIs in HIV-infected children in the pre-highly active antiretroviral therapy era varies with age, pathogen, prior OI and immunologic status. Analysis of CD4 counts at the time of DMAC, CMV and PCP provide validation for current prophylaxis guidelines in children > or =2 years old. This information on infectious complications of pediatric HIV will be especially valuable for contemporary management of HIV infection that is poorly responsive to highly active antiretroviral therapy.

Paraplegin gene analysis in hereditary spastic paraparesis (HSP) pedigrees in northeast England.

OBJECTIVE: To identify the frequency and characterize the phenotype of paraplegin mutations in the hereditary spastic paraparesis (HSP) population in the northeast of England. BACKGROUND: HSP is a disorder that shows both clinical and genetic heterogeneity. To date, 13 loci have been associated with an HSP phenotype, with the causative gene having been identified in four of these. Two autosomal genes have been identified, paraplegin and spastin, and two X-linked genes have been identified, L1CAM (cell adhesion molecule) and proteolipid protein. METHODS: Thirty HSP pedigrees from the northeast of England were analyzed for mutation in each of the 17 exons of the paraplegin gene. RESULTS: A single family with a paraplegin mutation was identified in which the paraplegin mutation co-segregates with an HSP phenotype in an apparent dominant manner. The authors also describe frequent polymorphism in the paraplegin gene in both the HSP and control populations. CONCLUSION: Mutations in the paraplegin gene are not a common cause of HSP in the northeast of England. The phenotype of the paraplegin-related HSP family described had several striking features including amyotrophy, raised creatine kinase, sensorimotor peripheral neuropathy, and oxidative phosphorylation defect on muscle biopsy.

Serologic examination of hepatitis B infection and immunization in HIV-positive youth and associated risks. The Pediatric AIDS Clinical Trials Group Protocol 220 Team.

This seroprevalence report examines serologic evidence of hepatitis B immunization or infection and associated demographic/behavioral factors in adolescent (aged 12-20) subjects enrolled in a nontherapeutic clinical trial at 43 Pediatric AIDS Clinical Trials Group (PACTG) clinical centers. Subjects (n = 94) infected with the human immunodeficiency virus (HIV) through sexual activity were categorized as hepatitis B virus (HBV)-immunized, HBV-infected, or nonimmune by hepatitis B serology performed on specimens collected within the subject's first 48 weeks on study (1993-1995). Sixteen percent of the 94 serologically classified subjects were immunized; 19% HBV-infected; 65% nonimmune. Of the three risk factor scores examined (sociodemographic, sexual, and substance abuse), substance use alone demonstrated a significant difference among groups (despite virtually no reported injecting drug behavior), with the sexual risk score exhibiting marginally significant differences. Logistic regression analysis (restricted to nonimmunized subjects) showed that male-male sexual activity raised the odds of HBV infection by a factor of 5.14 (95% confidence interval [CI]: 1.45-18. 23) relative to heterosexual activity; and that for every one point increase on the substance abuse risk scale the odds of infection increased 5% (95% CI: 0.99-1.10). The HBV infection rate in PACTG 220 HIV-positive females is twice United States population-based rates; the rate in PACTG 220 HIV-positive males is nearly seven times higher. Past immunization efforts in this population appear to have been based on sexual activity volume without regard to injecting-drug use in sex partners.

Mutation analysis of the spastin gene (SPG4) in patients with hereditary spastic paraparesis.

BACKGROUND: Hereditary spastic paraparesis is a genetically heterogeneous condition. Recently, mutations in the spastin gene were reported in families linked to the common SPG4 locus on chromosome 2p21-22. OBJECTIVES: To study a population of patients with hereditary spastic paraparesis for mutations in the spastin gene (SPG4) on chromosome 2p21-22. METHODS: DNA from 32 patients (12 from families known to be linked to SPG4) was analysed for mutations in the spastin gene by single strand conformational polymorphism analysis and sequencing. All patients were also examined clinically. RESULTS: Thirteen SPG4 mutations were identified, 11 of which are novel. These mutations include missense, nonsense, frameshift, and splice site mutations, the majority of which affect the AAA cassette. We also describe a nucleotide substitution outside this conserved region which appears to behave as a recessive mutation. CONCLUSIONS: Recurrent mutations in the spastin gene are uncommon. This reduces the ease of mutation detection as a part of the diagnostic work up of patients with hereditary spastic paraparesis. Our findings have important implications for the presumed function of spastin and schemes for mutation detection in HSP patients.

Treatment-mediated changes in human immunodeficiency virus (HIV) type 1 RNA and CD4 cell counts as predictors of weight growth failure, cognitive decline, and survival in HIV-infected children.

This meta-analysis of 5 large studies of the Pediatric AIDS Clinical Trials Group was undertaken to evaluate the predictive value of antiretroviral treatment-mediated changes in 3 markers of human immunodeficiency virus (HIV) type 1 disease progression-HIV-1 RNA level, CD4 cell count, and CD4 percentage-for weight growth failure, cognitive decline, and survival in HIV-infected children. Proportional hazards models were used to assess the prognostic value of the markers at baseline and after 24 weeks of treatment, with data from 1089 children. Among children receiving nucleoside with or without nonnucleoside reverse-transcriptase inhibitors, higher immunologic and lower virologic markers at baseline and after 24 weeks were significant independent predictors of survival, whereas virologic markers were significant predictors of weight growth and cognitive failure in children >1 year old. The finding of differential age effects on pediatric-specific clinical outcomes emphasizes the need for continued investigation of treatment effects in children.

Methodological issues in analyzing psychological test scores in pediatric clinical trials.

Clinical trials to address drug dosing, safety, and efficacy issues in the pediatric population are becoming more common. In some studies, tests of mental ability are administered at regular intervals in drug trials for treatment of children with HIV, certain types of cancer, sickle cell anemia, and diabetes. The test scores are used to examine differences between treatments in efficacy and safety over time. In addition to the well-known problems of analyzing repeated measures with incomplete data profiles, the analyses of these data are complicated by a number of unique features, including that children can be so ill that their raw scores cannot be mapped to a normed scaled score, and that children may be in the studies long enough that they transition between the age-appropriate instruments. These issues are often ignored in data analyses and can potentially cause incorrect conclusions regarding treatment efficacy and safety. This article describes these issues and their possible consequences. A simple approach to determine their impact on the statistical analysis of a particular clinical trial is suggested. The approach is illustrated with data from a Phase III trial in HIV-infected children.

Combination therapy with stavudine (d4T) plus didanosine (ddI) in children with human immunodeficiency virus infection. The Pediatric AIDS Clinical Trials Group 327 Team.

OBJECTIVES: To evaluate the safety, tolerance, and antiviral activity of combination therapy with stavudine (d4T) plus didanosine (ddI) in symptomatic human immunodeficiency virus (HIV)-infected children. METHODS: The study enrolled HIV-infected children who successfully completed Pediatric AIDS Clinical Trials Group (PACTG) protocol 240 (d4T versus zidovudine [ZDV] monotherapy) without disease progression or who had received ZDV monotherapy by prescription for at least the preceding 6 months. Children who had received d4T monotherapy in PACTG 240 were assigned to treatment with d4T plus ddI (arm 1). Children who had received ZDV monotherapy in PACTG 240 or by prescription were randomized in a double-blind manner to treatment with either d4T alone (arm 2) or d4T plus ddI (arm 3). Patients were followed for 48 weeks each. RESULTS: A total of 108 children were enrolled. The mean age was 5.0 years (range, 1. 6 to 11.5 years), with mean baseline plasma HIV RNA concentration and CD4(+) lymphocyte count of 4.6 log10 copies/mL (range, 2.6 to 5. 9 log10 copies/mL) and 819 cells/microL (range, 8 to 3431 cells/microL), respectively. Both d4T monotherapy and d4T plus ddI combination therapy were well-tolerated, with 96 (89%) patients completing 48 weeks of study treatment. Plasma HIV RNA concentrations showed larger average declines in arm 3 compared with arm 2 at study week 12 (0.49 vs 0.18 log10 copies/mL, respectively); these average declines were maintained through week 48 (0.51 vs 0.17 log10 copies/mL, respectively). Fewer than 8% of the patients in any of the treatment arms had plasma HIV RNA concentrations below the limit of quantification (200 copies/mL) at any time point. CONCLUSIONS: Combination therapy with d4T plus ddI is safe and well-tolerated in HIV-infected children, producing durable, but incomplete, suppression of virus replication. This combination of nucleoside antiretroviral agents may provide a valuable backbone to protease inhibitor-containing treatment regimens for HIV-infected children.

Intrapolation and extrapolation of age-equivalent scores for the Bayley II: a comparison of two methods of estimation.

The Bayley Scales of Infant Development II (1) is a well established standardized test for assessing the mental ability of infants and young children. It provides an age-adjusted standard score as a summary measure, but for very low (or very high) functioning children the raw scores on this test may not allow the calculation of a standard score. The manual provides for the transformation of raw scores into age-equivalents but this translation is not unique and results in a step function. The availability of a unique and continuous transformation of raw scores to age-equivalents is critical when evaluating longitudinal mental development, particularly in the environment of controlled clinical trials or natural history studies. We compared two methods for deriving unique age equivalents: a local regression method, with estimates restricted to age-equivalents within the age range of the test, and a linear method, which also allows extrapolation outside the age range of the test. The linear method was found to be most useful and the values, which are provided in table format, can be used for assigning age equivalent scores to individual children. They are also useful in clinical trials which use the Bayley to assess the safety and efficacy of treatments with potential cognitive effects, when conducted in populations where the children are likely to record scaled scores below the limits of the test.

Local EM estimation of the hazard function for interval-censored data.

We propose a smooth hazard estimator for interval-censored survival data using the method of local likelihood. The model is fit using a local EM algorithm. The estimator is more descriptive than traditional empirical estimates in regions of concentrated information and takes on a parametric flavor in regions of sparse information. We derive two different standard error estimates for the smooth curve, one based on asymptotic theory and the other on the bootstrap. We illustrate the local EM method for times to breast cosmesis deterioration (Finkelstein, 1986, Biometrics 42, 845-854) and for times to HIV-1 infection for individuals with hemophilia (Kroner et al., 1994, Journal of AIDS 7, 279-286). Our hazard estimates for each of these data sets show interesting structures that would not be found using a standard parametric hazard model or empirical survivorship estimates.

Replication strand preference for deletions associated with DNA palindromes.

We have isolated and sequenced a set of deletions stimulated by DNA palindromes in Escherichia coli. All of the deletions are asymmetric with respect to the parental sequence and have occurred at short direct repeats. This is consistent with deletion by strand slippage during DNA replication. The orientation of the asymmetry in such deletion products is diagnostic of the direction of the strand slippage event. It is therefore also diagnostic of its occurrence on the leading or lagging strand of the replication fork when the direction of replication is known. In all cases in which the orientation of the asymmetry could be determined with respect to DNA replication, the products were consistent with a preference for deletion on the lagging strand of the fork. The data include replication slippage in three situations: on the chromosome of E. coli, in bacteriophage lambda and in high-copy-number pUC-based plasmids.

Tutorial in biostatistics methods for interval-censored data.

In standard time-to-event or survival analysis, occurrence times of the event of interest are observed exactly or are right-censored, meaning that it is only known that the event occurred after the last observation time. There are numerous methods available for estimating the survival curve and for testing and estimation of the effects of covariates in this context. In some situations, however, the times of the events of interest may only be known to have occurred within an interval of time. In clinical trials, for example, patients are often seen at pre-scheduled visits but the event of interest may occur in between visits. These data are interval-censored. Owing to the lack of well-known statistical methodology and available software, a common ad hoc approach is to assume that the event occurred at the end (or beginning or midpoint) of each interval, and then apply methods for standard time-to-event data. However, this approach can lead to invalid inferences, and in particular will tend to underestimate the standard errors of the estimated parameters. The purpose of this tutorial is to illustrate and compare available methods which correctly treat the data as being interval-censored. It is not meant to be a full review of all existing methods, but only those which are available in standard statistical software, or which can be easily programmed. All approaches will be illustrated on two data sets and compared with methods which ignore the interval-censored nature of the data. We hope this tutorial will allow those familiar with the application of standard survival analysis techniques the option of applying appropriate methods when presented with interval-censored data.