A nationwide, population-based study of school grades, delayed graduation, and qualification for school years 10-12, in children with brain tumors in Sweden.

BACKGROUND: As many as 95.7% of children diagnosed with a brain tumor will experience persistent late effects as adults. These include difficulties with general executive functions, lower IQ, and mental fatigue, which may negatively affect school performance. METHODS: Through the Swedish Childhood Cancer Registry, we identified 475 children born between 1988 and 1996, diagnosed with a brain tumor before their 15th birthday. School grades in "Swedish," "mathematics," and "English," if their graduation was delayed, and qualification for school years 10-12 were compared with 2197 matched controls. Furthermore, we checked for interaction effects between sex and age at diagnosis, and possible effects of tumor grade (high or low) as well as parents' education. RESULTS: Children treated for a brain tumor performed worse in the subjects compared to controls and also had delayed graduation to a greater extent. Fewer children treated for a brain tumor than controls qualified for school years 10-12. Children treated at a young age, especially females, and children whose parents have low education seem to be at particular risk. Unexpectedly, there were no differences in outcomes between survivors with high- and low-grade tumors. CONCLUSIONS: It is important that schools provide regular pedagogical assessment and individualized support to meet the different needs of children treated for a brain tumor. Children treated for low-grade tumors do not perform better than children treated for high-grade tumors, despite the lighter treatment, and hence require the same attention and support.

A gene transfer agent and a dynamic repertoire of secretion systems hold the keys to the explosive radiation of the emerging pathogen Bartonella.

Gene transfer agents (GTAs) randomly transfer short fragments of a bacterial genome. A novel putative GTA was recently discovered in the mouse-infecting bacterium Bartonella grahamii. Although GTAs are widespread in phylogenetically diverse bacteria, their role in evolution is largely unknown. Here, we present a comparative analysis of 16 Bartonella genomes ranging from 1.4 to 2.6 Mb in size, including six novel genomes from Bartonella isolated from a cow, two moose, two dogs, and a kangaroo. A phylogenetic tree inferred from 428 orthologous core genes indicates that the deadly human pathogen B. bacilliformis is related to the ruminant-adapted clade, rather than being the earliest diverging species in the genus as previously thought. A gene flux analysis identified 12 genes for a GTA and a phage-derived origin of replication as the most conserved innovations. These are located in a region of a few hundred kb that also contains 8 insertions of gene clusters for type III, IV, and V secretion systems, and genes for putatively secreted molecules such as cholera-like toxins. The phylogenies indicate a recent transfer of seven genes in the virB gene cluster for a type IV secretion system from a cat-adapted B. henselae to a dog-adapted B. vinsonii strain. We show that the B. henselae GTA is functional and can transfer genes in vitro. We suggest that the maintenance of the GTA is driven by selection to increase the likelihood of horizontal gene transfer and argue that this process is beneficial at the population level, by facilitating adaptive evolution of the host-adaptation systems and thereby expansion of the host range size. The process counters gene loss and forces all cells to contribute to the production of the GTA and the secreted molecules. The results advance our understanding of the role that GTAs play for the evolution of bacterial genomes.

The mutational landscape in pediatric acute lymphoblastic leukemia deciphered by whole genome sequencing.

Genomic characterization of pediatric acute lymphoblastic leukemia (ALL) has identified distinct patterns of genes and pathways altered in patients with well-defined genetic aberrations. To extend the spectrum of known somatic variants in ALL, we performed whole genome and transcriptome sequencing of three B-cell precursor patients, of which one carried the t(12;21)ETV6-RUNX1 translocation and two lacked a known primary genetic aberration, and one T-ALL patient. We found that each patient had a unique genome, with a combination of well-known and previously undetected genomic aberrations. By targeted sequencing in 168 patients, we identified KMT2D and KIF1B as novel putative driver genes. We also identified a putative regulatory non-coding variant that coincided with overexpression of the growth factor MDK. Our results contribute to an increased understanding of the biological mechanisms that lead to ALL and suggest that regulatory variants may be more important for cancer development than recognized to date. The heterogeneity of the genetic aberrations in ALL renders whole genome sequencing particularly well suited for analysis of somatic variants in both research and diagnostic applications.

Hormone Replacement Therapy Associated White Blood Cell DNA Methylation and Gene Expression are Associated With Within-Pair Differences of Body Adiposity and Bone Mass.

The loss of estrogen during menopause causes changes in the female body, with wide-ranging effects on health. Estrogen-containing hormone replacement therapy (HRT) leads to a relief of typical menopausal symptoms, benefits bone and muscle health, and is associated with tissue-specific gene expression profiles. As gene expression is controlled by epigenetic factors (including DNA methylation), many of which are environmentally sensitive, it is plausible that at least part of the HRT-associated gene expression is due to changes in DNA methylation profile. We investigated genome-wide DNA methylation and gene expression patterns of white blood cells (WBCs) and their associations with body composition, including muscle and bone measures of monozygotic (MZ) female twin pairs discordant for HRT. We identified 7,855 nominally significant differentially methylated regions (DMRs) associated with 4,044 genes. Of the genes with DMRs, five (ACBA1, CCL5, FASLG, PPP2R2B, and UHRF1) were also differentially expressed. All have been previously associated with HRT or estrogenic regulation, but not with HRT-associated DNA methylation. All five genes were associated with bone mineral content (BMC), and ABCA1, FASLG, and UHRF1 were also associated with body adiposity. Our study is the first to show that HRT associates with genome-wide DNA methylation alterations in WBCs. Moreover, we show that five differentially expressed genes with DMRs associate with clinical measures, including body fat percentage, lean body mass, bone mass, and blood lipids. Our results indicate that at least part of the known beneficial HRT effects on body composition and bone mass may be regulated by DNA methylation associated alterations in gene expression in circulating WBCs.

Accurate detection of subclonal single nucleotide variants in whole genome amplified and pooled cancer samples using HaloPlex target enrichment.

BACKGROUND: Target enrichment and resequencing is a widely used approach for identification of cancer genes and genetic variants associated with diseases. Although cost effective compared to whole genome sequencing, analysis of many samples constitutes a significant cost, which could be reduced by pooling samples before capture. Another limitation to the number of cancer samples that can be analyzed is often the amount of available tumor DNA. We evaluated the performance of whole genome amplified DNA and the power to detect subclonal somatic single nucleotide variants in non-indexed pools of cancer samples using the HaloPlex technology for target enrichment and next generation sequencing. RESULTS: We captured a set of 1528 putative somatic single nucleotide variants and germline SNPs, which were identified by whole genome sequencing, with the HaloPlex technology and sequenced to a depth of 792-1752. We found that the allele fractions of the analyzed variants are well preserved during whole genome amplification and that capture specificity or variant calling is not affected. We detected a large majority of the known single nucleotide variants present uniquely in one sample with allele fractions as low as 0.1 in non-indexed pools of up to ten samples. We also identified and experimentally validated six novel variants in the samples included in the pools. CONCLUSION: Our work demonstrates that whole genome amplified DNA can be used for target enrichment equally well as genomic DNA and that accurate variant detection is possible in non-indexed pools of cancer samples. These findings show that analysis of a large number of samples is feasible at low cost, even when only small amounts of DNA is available, and thereby significantly increases the chances of indentifying recurrent mutations in cancer samples.

Occupational outcomes after high-grade or low-grade brain tumors in childhood: A Swedish, nationwide, registry-based study.

BACKGROUND: Survivors of pediatric brain tumors are at high risk of late complications that may affect their daily life in both short- and long-term perspectives. METHODS: In this nationwide registry-based study we explored the occupational outcomes, including employment, sickness or activity compensation and parental leave, in 452 individuals in Sweden, born 1988-1996, and diagnosed with a brain tumor before their 15th birthday. Their results were compared with 2188 matched controls. RESULTS: There were significant differences between cases and controls for all assessed variables. The cases had benefitted from sickness or activity compensation 11 times more often than controls (CI 7.90-15.83; p < 0.001) between 2005 and 2016. Controls were almost three times more likely to have an employment (OR 0.36; CI 0.28-0.47; p < 0.001) and nearly twice as likely to have been on parental leave (OR 0.56; CI 0.39-0.80; p = 0.002). Although cases treated for high-grade tumors typically fared worse than those treated for low-grade tumors, significant differences for all assessed variables were also observed for cases treated for a low-grade tumor compared with controls. CONCLUSIONS: Our findings emphasize the need for follow-up programs for all brain tumor diagnoses, not only those known to be at most risk. This is evident, for example, from the high number of cases who received sickness or activity compensation.

A genome-wide study of recombination rate variation in Bartonella henselae.

BACKGROUND: Rates of recombination vary by three orders of magnitude in bacteria but the reasons for this variation is unclear. We performed a genome-wide study of recombination rate variation among genes in the intracellular bacterium Bartonella henselae, which has among the lowest estimated ratio of recombination relative to mutation in prokaryotes. RESULTS: The 1.9 Mb genomes of B. henselae strains IC11, UGA10 and Houston-1 genomes showed only minor gene content variation. Nucleotide sequence divergence levels were less than 1% and the relative rate of recombination to mutation was estimated to 1.1 for the genome overall. Four to eight segments per genome presented significantly enhanced divergences, the most pronounced of which were the virB and trw gene clusters for type IV secretion systems that play essential roles in the infection process. Consistently, multiple recombination events were identified inside these gene clusters. High recombination frequencies were also observed for a gene putatively involved in iron metabolism. A phylogenetic study of this gene in 80 strains of Bartonella quintana, B. henselae and B. grahamii indicated different population structures for each species and revealed horizontal gene transfers across Bartonella species with different host preferences. CONCLUSIONS: Our analysis has shown little novel gene acquisition in B. henselae, indicative of a closed pan-genome, but higher recombination frequencies within the population than previously estimated. We propose that the dramatically increased fixation rate for recombination events at gene clusters for type IV secretion systems is driven by selection for sequence variability.

Run-off replication of host-adaptability genes is associated with gene transfer agents in the genome of mouse-infecting Bartonella grahamii.

The genus Bartonella comprises facultative intracellular bacteria adapted to mammals, including previously recognized and emerging human pathogens. We report the 2,341,328 bp genome sequence of Bartonella grahamii, one of the most prevalent Bartonella species in wild rodents. Comparative genomics revealed that rodent-associated Bartonella species have higher copy numbers of genes for putative host-adaptability factors than the related human-specific pathogens. Many of these gene clusters are located in a highly dynamic region of 461 kb. Using hybridization to a microarray designed for the B. grahamii genome, we observed a massive, putatively phage-derived run-off replication of this region. We also identified a novel gene transfer agent, which packages the bacterial genome, with an over-representation of the amplified DNA, in 14 kb pieces. This is the first observation associating the products of run-off replication with a gene transfer agent. Because of the high concentration of gene clusters for host-adaptation proteins in the amplified region, and since the genes encoding the gene transfer agent and the phage origin are well conserved in Bartonella, we hypothesize that these systems are driven by selection. We propose that the coupling of run-off replication with gene transfer agents promotes diversification and rapid spread of host-adaptability factors, facilitating host shifts in Bartonella.

Utility of a physiologically-based pharmacokinetic (PBPK) modeling approach to quantitatively predict a complex drug-drug-disease interaction scenario for rivaroxaban during the drug review process: implications for clinical practice.

BACKGROUND: Rivaroxaban is an oral Factor Xa inhibitor. The primary objective of this communication was to quantitatively predict changes in rivaroxaban exposure when individuals with varying degrees of renal impairment are co-administered with another drug that is both a P-gp and a moderate CYP3A4 inhibitor. METHODS: A physiologically based pharmacokinetic (PBPK) model was developed to simulate rivaroxaban pharmacokinetics in young (20-45 years) or older (55-65 years) subjects with normal renal function, mild, moderate and severe renal impairment, with or without concomitant use of the combined P-gp and moderate CYP3A4 inhibitor, erythromycin. RESULTS: The simulations indicate that combined factors (i.e., renal impairment and the use of erythromycin) have a greater impact on rivaroxaban exposure than expected when the impact of these factors are considered individually. Compared with normal young subjects taking rivaroxaban, concurrent mild, moderate or severe renal impairment plus erythromycin resulted in 1.9-, 2.4- or 2.6-fold increase in exposure, respectively in young subjects; and 2.5-, 2.9- or 3.0-fold increase in exposure in older subjects. CONCLUSIONS: These simulations suggest that a drug-drug-disease interaction is possible, which may significantly increase rivaroxaban exposure and increase bleeding risk. These simulations render more mechanistic insights as to the possible outcomes and allow one to reach a decision to add cautionary language to the approved product labeling for rivaroxaban.

Post-Compulsory Education in Teenagers and Young Adults Treated for Brain Tumors in Childhood: A Swedish Nationwide Registry-Based Study.

The risk of late complications after a brain tumor in childhood is high. Both the tumor itself and the treatments give rise to sequelae that affect daily life activities. In this registry study, we explored post-compulsory education, i.e., further education following the nine compulsory years in school, in 452 cases born 1988-1996 and diagnosed with a brain tumor before their fifteenth birthday. They were compared with 2188 individual controls who were not treated for cancer. Significantly fewer teenagers and young adults treated for brain tumors in childhood attended high school or university compared with controls, especially individuals treated for embryonal tumors or optic pathway gliomas. A significantly larger proportion of subjects treated for embryonal tumors and craniopharyngiomas attended folk high schools, a type of post-compulsory school with a more accessible learning environment. For both cases and controls, we observed a positive correlation between parental education levels and attendance in high school and university. In our previous studies we have shown that children treated for brain tumors, as a group, tend to perform worse during their last year of compulsory school compared with their peers, and the current study confirms that these differences remain over time.

A Study Protocol for Validation and Implementation of Whole-Genome and -Transcriptome Sequencing as a Comprehensive Precision Diagnostic Test in Acute Leukemias.

Background: Whole-genome sequencing (WGS) and whole-transcriptome sequencing (WTS), with the ability to provide comprehensive genomic information, have become the focal point of research interest as novel techniques that can support precision diagnostics in routine clinical care of patients with various cancer types, including hematological malignancies. This national multi-center study, led by Genomic Medicine Sweden, aims to evaluate whether combined application of WGS and WTS (WGTS) is technically feasible and can be implemented as an efficient diagnostic tool in patients with acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). In addition to clinical impact assessment, a health-economic evaluation of such strategy will be performed. Methods and Analysis: The study comprises four phases (i.e., retrospective, prospective, real-time validation, and follow-up) including approximately 700 adult and pediatric Swedish AML and ALL patients. Results of WGS for tumor (90×) and normal/germline (30×) samples as well as WTS for tumors only will be compared to current standard of care diagnostics. Primary study endpoints are diagnostic efficiency and improved diagnostic yield. Secondary endpoints are technical and clinical feasibility for routine implementation, clinical utility, and health-economic impact. Discussion: Data from this national multi-center study will be used to evaluate clinical performance of the integrated WGTS diagnostic workflow compared with standard of care. The study will also elucidate clinical and health-economic impacts of a combined WGTS strategy when implemented in routine clinical care. Clinical Trial Registration: [https://doi.org/10.1186/ISRCTN66987142], identifier [ISRCTN66987142].

Can National Tests from the Last Year of Compulsory School Be Used to Obtain More Detailed Information about Academic Performance in Children Treated for Brain Tumours? A Nationwide, Population-Based Study from Sweden.

Children treated for brain tumours often have late-appearing complications that may affect their school performance. Uneven skill profiles may help reveal late complications that can be compensated for but otherwise remain undetected. We investigated Swedish national school tests of oral, reading and writing skills in the first foreign language (English), the mother tongue (Swedish) and mathematics. Data were obtained from The Swedish Childhood Cancer Registry and Statistics Sweden. The results from 475 children diagnosed with a brain tumour before their 15th birthday and 2197 matched controls showed that children treated for brain tumours evinced more difficulties with national tests than controls in almost all subtests, especially in the subject English, and that they may perform better on oral than written tasks. There were larger differences between female cases and controls than between male cases and controls; age at diagnosis played a significant role for some subtests, whereas tumour grade did not. Missing information from national tests proved to be a strong predictor of poor academic performance. Our results show that regular educational follow-ups, as a complement to neuropsychological follow-ups, are important for all children treated for brain tumours, regardless of sex, age at diagnosis or tumour grade.

A best practice framework for applying physiologically-based pharmacokinetic modeling to pediatric drug development.

Pediatric physiologically-based pharmacokinetic (PBPK) models have broad application in the drug development process and are being used not only to project doses for clinical trials but increasingly to replace clinical studies. However, the approach has yet to become fully integrated in regulatory submissions. Emerging data support an expanded integration of the PBPK model informed approach in regulatory guidance on pediatrics. Best practice standards are presented for further development through interaction among regulators, industry, and model providers.

Mutational patterns and clonal evolution from diagnosis to relapse in pediatric acute lymphoblastic leukemia.

The mechanisms driving clonal heterogeneity and evolution in relapsed pediatric acute lymphoblastic leukemia (ALL) are not fully understood. We performed whole genome sequencing of samples collected at diagnosis, relapse(s) and remission from 29 Nordic patients. Somatic point mutations and large-scale structural variants were called using individually matched remission samples as controls, and allelic expression of the mutations was assessed in ALL cells using RNA-sequencing. We observed an increased burden of somatic mutations at relapse, compared to diagnosis, and at second relapse compared to first relapse. In addition to 29 known ALL driver genes, of which nine genes carried recurrent protein-coding mutations in our sample set, we identified putative non-protein coding mutations in regulatory regions of seven additional genes that have not previously been described in ALL. Cluster analysis of hundreds of somatic mutations per sample revealed three distinct evolutionary trajectories during ALL progression from diagnosis to relapse. The evolutionary trajectories provide insight into the mutational mechanisms leading relapse in ALL and could offer biomarkers for improved risk prediction in individual patients.

Genome dynamics of Bartonella grahamii in micro-populations of woodland rodents.

BACKGROUND: Rodents represent a high-risk reservoir for the emergence of new human pathogens. The recent completion of the 2.3 Mb genome of Bartonella grahamii, one of the most prevalent blood-borne bacteria in wild rodents, revealed a higher abundance of genes for host-cell interaction systems than in the genomes of closely related human pathogens. The sequence variability within the global B. grahamii population was recently investigated by multi locus sequence typing, but no study on the variability of putative host-cell interaction systems has been performed. RESULTS: To study the population dynamics of B. grahamii, we analyzed the genomic diversity on a whole-genome scale of 27 B. grahamii strains isolated from four different species of wild rodents in three geographic locations separated by less than 30 km. Even using highly variable spacer regions, only 3 sequence types were identified. This low sequence diversity contrasted with a high variability in genome content. Microarray comparative genome hybridizations identified genes for outer surface proteins, including a repeated region containing the fha gene for filamentous hemaggluttinin and a plasmid that encodes a type IV secretion system, as the most variable. The estimated generation times in liquid culture medium for a subset of strains ranged from 5 to 22 hours, but did not correlate with sequence type or presence/absence patterns of the fha gene or the plasmid. CONCLUSION: Our study has revealed a geographic microstructure of B. grahamii in wild rodents. Despite near-identity in nucleotide sequence, major differences were observed in gene presence/absence patterns that did not segregate with host species. This suggests that genetically similar strains can infect a range of different hosts.

Rapid diversification by recombination in Bartonella grahamii from wild rodents in Asia contrasts with low levels of genomic divergence in Northern Europe and America.

Bartonella is a genus of vector-borne bacteria that infect the red blood cells of mammals, and includes several human-specific and zoonotic pathogens. Bartonella grahamii has a wide host range and is one of the most prevalent Bartonella species in wild rodents. We studied the population structure, genome content and genome plasticity of a collection of 26 B. grahamii isolates from 11 species of wild rodents in seven countries. We found strong geographic patterns, high recombination frequencies and large variations in genome size in B. grahamii compared with previously analysed cat- and human-associated Bartonella species. The extent of sequence divergence in B. grahamii populations was markedly lower in Europe and North America than in Asia, and several recombination events were predicted between the Asian strains. We discuss environmental and demographic factors that may underlie the observed differences.

Nationwide, population-based study of school grades in practical and aesthetic subjects of children treated for brain tumour.

BACKGROUND: Children treated for brain tumour (hereafter termed paediatric brain tumour survivors (PBTS)) often need extra support in school because of late-appearing side effects after their treatment. We explored how this group of children perform in the five practical and aesthetic (PRAEST) subjects: home and consumer studies, physical education and health, art, crafts and music. METHODS: In this nationwide population-based study of data from the Swedish Childhood Cancer Registry and Statistics Sweden, we included 475 children born between 1988 and 1996, diagnosed with a brain tumour before their 15th birthday. We compared their grades in PRAEST subjects with those of 2197 matched controls. We also investigated if there were any differences between girls and boys, children diagnosed at different ages, and children with high-grade or low-grade tumours. RESULTS: The odds for failing a subject were two to three times higher for girls treated for a brain tumour compared with their controls in all five PRAEST subjects, whereas there were no significant differences between the boys and their controls in any subject. PBTS had lower average grades from year 9 in all PRAEST subjects, and girls differed from their controls in all five subjects, while boys differed in physical education and health and music. PBTS treated for high-grade tumours neither did have significantly different average grades nor did they fail a subject to a significantly higher extent than PBTS treated for low-grade tumours. CONCLUSIONS: Children treated for a brain tumour, especially girls, are at risk of lower average grades or failing PRAEST subjects. All children treated for brain tumour may need extra support as these subjects are important for their well-being and future skills.

Inhibitors of Organic Anion-Transporting Polypeptides 1B1 and 1B3: Clinical Relevance and Regulatory Perspective.

Organic anion-transporting polypeptides (OATPs) 1B1 and 1B3 are the primary hepatic transporters responsible for uptake of drugs into the liver and, as such, an area of growing research focus. Currently, evaluation of these transporters as potential mediators of drug-drug interactions (DDIs) is recommended by regulatory agencies worldwide during the drug development process. Despite the growing focus on OATP1B1/1B3 as mediators of DDIs, only 2 drugs are recommended as index inhibitors for use in clinical studies, single-dose rifampin and cyclosporine, each with limitations for the utility of the resulting data. In this study a thorough analysis of the available in vitro and clinical data was conducted to identify drugs that are clinically relevant inhibitors of OATP1B1/1B3 and, from those, to select any novel index inhibitors. A total of 13 drugs and 16 combination products were identified as clinical inhibitors of OATP1B1/1B3, showing significant changes in exposure for sensitive substrates of the transporters, with strong supporting in vitro evidence. Although none of the identified inhibitors qualified as index inhibitors, this study confirmed the utility of cyclosporine and single-dose rifampin as index inhibitors to evaluate the effect of broad, multiple-pathway inhibition and more selective OATP1B1/1B3 inhibition, respectively.

Artificial Intelligence and Machine Learning Applied at the Point of Care.

INTRODUCTION: The increasing availability of healthcare data and rapid development of big data analytic methods has opened new avenues for use of Artificial Intelligence (AI)- and Machine Learning (ML)-based technology in medical practice. However, applications at the point of care are still scarce. OBJECTIVE: Review and discuss case studies to understand current capabilities for applying AI/ML in the healthcare setting, and regulatory requirements in the US, Europe and China. METHODS: A targeted narrative literature review of AI/ML based digital tools was performed. Scientific publications (identified in PubMed) and grey literature (identified on the websites of regulatory agencies) were reviewed and analyzed. RESULTS: From the regulatory perspective, AI/ML-based solutions can be considered medical devices (i.e., Software as Medical Device, SaMD). A case series of SaMD is presented. First, tools for monitoring and remote management of chronic diseases are presented. Second, imaging applications for diagnostic support are discussed. Finally, clinical decision support tools to facilitate the choice of treatment and precision dosing are reviewed. While tested and validated algorithms for precision dosing exist, their implementation at the point of care is limited, and their regulatory and commercialization pathway is not clear. Regulatory requirements depend on the level of risk associated with the use of the device in medical practice, and can be classified into administrative (manufacturing and quality control), software-related (design, specification, hazard analysis, architecture, traceability, software risk analysis, cybersecurity, etc.), clinical evidence (including patient perspectives in some cases), non-clinical evidence (dosing validation and biocompatibility/toxicology) and other, such as e.g. benefit-to-risk determination, risk assessment and mitigation. There generally is an alignment between the US and Europe. China additionally requires that the clinical evidence is applicable to the Chinese population and recommends that a third-party central laboratory evaluates the clinical trial results. CONCLUSIONS: The number of promising AI/ML-based technologies is increasing, but few have been implemented widely at the point of care. The need for external validation, implementation logistics, and data exchange and privacy remain the main obstacles.

Regulatory aspects of pharmacokinetic profiling in special populations: a European perspective.

The clinical efficacy and safety profile of a new medicinal product is established in phase III studies, which are usually restricted to a well defined patient population. This population may not fully represent the population in which the product will be used once it is on the market. Pharmacokinetic studies in special populations are performed to estimate drug exposure in subpopulations of patients with characteristics that may affect drug exposure. The clinical consequences of altered exposure are then assessed, taking pharmacokinetic/pharmacodynamic relationships into consideration. If needed, specific treatment recommendations should be developed.Recommendations regarding pharmacokinetic characterization in special populations are given in a number of European guidelines. The pharmacokinetic characteristics, therapeutic window and intended use of the medicinal product influence the need for pharmacokinetic studies of a new medicinal product. There are a number of methodological issues to be considered when designing pharmacokinetic studies in special populations: the study design, study population and control group, the dosing regimen to be used, the analytes to be measured, and the distribution and range of the factor to be studied. The data should be presented in sufficient detail to enable assessment by regulatory authorities of the conducted analysis and conclusions drawn. Assessment of the data should include an evaluation of how and to what extent the pharmacokinetics in specific subpopulations deviate from the exposure at the therapeutic dose in the clinical efficacy and safety studies, and if there is a need for specific treatment recommendations. Based on the available information on the pharmacokinetic/pharmacodynamic relationships for efficacy and safety and/or the exposure at the therapeutic dose in the phase III population where efficacy and safety have been demonstrated, target criteria (a target exposure range) should be defined. Within the target exposure range, there should be no clinically relevant difference in efficacy and safety. Should the exposure in a specific group fall outside the defined target criteria, appropriate treatment recommendations need to be developed. The aim should be to develop dosing recommendations that will allow the majority of the patients to obtain exposure within the defined target range. If it is not possible to develop suitable dosing recommendations in a subgroup of patients, there may be a need for specific warnings or wordings regarding monitoring of the patients. It may also be an option to exclude that patient group from the indication. The resulting treatment recommendations should ensure safe and effective use of the drug in the entire population for which it has been approved.