The dynamic response of quorum sensing to density is robust to signal supplementation and individual signal synthase knockouts.

Quorum sensing (QS) is a widespread mechanism of environment sensing and behavioural coordination in bacteria. At its core, QS is based on the production, sensing and response to small signalling molecules. Previous work with Pseudomonas aeruginosa shows that QS can be used to achieve quantitative resolution and deliver a dosed response to the bacteria's density environment, implying a sophisticated mechanism of control. To shed light on how the mechanistic signal components contribute to graded responses to density, we assess the impact of genetic (AHL signal synthase deletion) and/or signal supplementation (exogenous AHL addition) perturbations on lasB reaction-norms to changes in density. Our approach condenses data from 2000 timeseries (over 74 000 individual observations) into a comprehensive view of QS-controlled gene expression across variation in genetic, environmental and signal determinants of lasB expression. We first confirm that deleting either (∆lasI, ∆rhlI) or both (∆lasIrhlI) AHL signal synthase gene attenuates QS response to density. In the ∆rhlI background we show persistent yet attenuated density-dependent lasB expression due to native 3-oxo-C12-HSL signalling. We then test if density-independent quantities of AHL signal (3-oxo-C12-HSL, C4-HSL) added to the WT either flatten or increase responsiveness to density and find that the WT response is robust to all tested concentrations of signal, alone or in combination. We then move to progressively supplementing the genetic knockouts and find that cognate signal supplementation of a single AHL signal (∆lasI +3-oxo-C12-HSL, ∆rhlI +C4HSL) is sufficient to restore the ability to respond in a density-dependent manner to increasing density. We also find that dual signal supplementation of the double AHL synthase knockout restores the ability to produce a graded response to increasing density, despite adding a density-independent amount of signal. Only the addition of high concentrations of both AHLs and PQS can force maximal lasB expression and ablate responsiveness to density. Our results show that density-dependent control of lasB expression is robust to multiple combinations of QS gene deletion and density-independent signal supplementation. Our work develops a modular approach to query the robustness and mechanistic bases of the central environmental sensing phenotype of quorum sensing.

Antibiotics Drive Expansion of Rare Pathogens in a Chronic Infection Microbiome Model.

Chronic (long-lasting) infections are globally a major and rising cause of morbidity and mortality. Unlike typical acute infections, chronic infections are ecologically diverse, characterized by the presence of a polymicrobial mix of opportunistic pathogens and human-associated commensals. To address the challenge of chronic infection microbiomes, we focus on a particularly well-characterized disease, cystic fibrosis (CF), where polymicrobial lung infections persist for decades despite frequent exposure to antibiotics. Epidemiological analyses point to conflicting results on the benefits of antibiotic treatment yet are confounded by the dependency of antibiotic exposures on prior pathogen presence, limiting their ability to draw causal inferences on the relationships between antibiotic exposure and pathogen dynamics. To address this limitation, we develop a synthetic infection microbiome model representing CF metacommunity diversity and benchmark on clinical data. We show that in the absence of antibiotics, replicate microbiome structures in a synthetic sputum medium are highly repeatable and dominated by oral commensals. In contrast, challenge with physiologically relevant antibiotic doses leads to substantial community perturbation characterized by multiple alternate pathogen-dominant states and enrichment of drug-resistant species. These results provide evidence that antibiotics can drive the expansion (via competitive release) of previously rare opportunistic pathogens and offer a path toward microbiome-informed conditional treatment strategies. IMPORTANCE We develop and clinically benchmark an experimental model of the cystic fibrosis (CF) lung infection microbiome to investigate the impacts of antibiotic exposures on chronic, polymicrobial infections. We show that a single experimental model defined by metacommunity data can partially recapitulate the diversity of individual microbiome states observed across a population of people with CF. In the absence of antibiotics, we see highly repeatable community structures, dominated by oral microbes. Under clinically relevant antibiotic exposures, we see diverse and frequently pathogen-dominated communities, and a nonevolutionary enrichment of antimicrobial resistance on the community scale, mediated by competitive release. The results highlight the potential importance of nonevolutionary (community-ecological) processes in driving the growing global crisis of increasing antibiotic resistance.

Bacterial Quorum Sensing Allows Graded and Bimodal Cellular Responses to Variations in Population Density.

Quorum sensing (QS) is a mechanism of cell-cell communication that connects gene expression to environmental conditions (e.g., cell density) in many bacterial species, mediated by diffusible signal molecules. Current functional studies focus on qualitatively distinct QS ON/OFF states. In the context of density sensing, this view led to the adoption of a "quorum" analogy in which populations sense when they are above a sufficient density (i.e., "quorate") to efficiently turn on cooperative behaviors. This framework overlooks the potential for intermediate, graded responses to shifts in the environment. In this study, we tracked QS-regulated protease (lasB) expression and showed that Pseudomonas aeruginosa can deliver a graded behavioral response to fine-scale variation in population density, on both the population and single-cell scales. On the population scale, we saw a graded response to variation in population density (controlled by culture carrying capacity). On the single-cell scale, we saw significant bimodality at higher densities, with separate OFF and ON subpopulations that responded differentially to changes in density: a static OFF population of cells and increasing intensity of expression among the ON population of cells. Together, these results indicate that QS can tune gene expression to graded environmental change, with no critical cell mass or "quorum" at which behavioral responses are activated on either the individual-cell or population scale. In an infection context, our results indicate there is not a hard threshold separating a quorate "attack" mode from a subquorate "stealth" mode. IMPORTANCE Bacteria can be highly social, controlling collective behaviors via cell-cell communication mechanisms known as quorum sensing (QS). QS is now a large research field, yet a basic question remains unanswered: what is the environmental resolution of QS? The notion of a threshold, or "quorum," separating coordinated ON and OFF states is a central dogma in QS, but recent studies have shown heterogeneous responses at a single cell scale. Using Pseudomonas aeruginosa, we showed that populations generate graded responses to environmental variation through shifts in the proportion of cells responding and the intensity of responses. In an infection context, our results indicate that there is not a hard threshold separating a quorate "attack" mode and a subquorate "stealth" mode.

Challenges and opportunities for cheat therapy in the control of bacterial infections.

Covering: 1999 to 2021Bacterial pathogens can be highly social, communicating and cooperating within multi-cellular groups to make us sick. The requirement for collective action in pathogens presents novel therapeutic avenues that seek to undermine cooperative behavior, what we call here 'cheat therapies'. We review two broad avenues of cheat therapy: first, the introduction of genetically engineered 'cheat' strains (bio-control cheats), and second the chemical induction of 'cheat' behavior in the infecting pathogens (chemical-control cheats). Both genetically engineered and chemically induced cheats can socially exploit the cooperative wildtype infection, reducing pathogen burden and the severity of disease. We review the costs and benefits of cheat therapies, highlighting advantages of evolutionary robustness and also the challenges of low to moderate efficacy, compared to conventional antibiotic treatments. We end with a summary of what we see as the most valuable next steps, focusing on adjuvant treatments and use as alternate therapies for mild, self-resolving infections - allowing the reservation of current and highly effective antibiotics for more critical patient needs.

Author Correction: In silico bacteria evolve robust cooperation via complex quorum-sensing strategies.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Combinatorial quorum sensing in Pseudomonas aeruginosa allows for novel cheating strategies.

In the opportunistic pathogen Pseudomonas aeruginosa, quorum sensing (QS) is a social trait that is exploitable by non-cooperating cheats. Previously it has been shown that by linking QS to the production of both public and private goods, cheats can be prevented from invading populations of cooperators and this was described by Dandekar et al. (Science 2012;338:264-266) as 'a metabolic incentive to cooperate'. We hypothesized that P. aeruginosa could evolve novel cheating strategies to circumvent private goods metabolism by rewiring its combinatorial response to two QS signals (3O-C12-HSL and C4-HSL). We performed a selection experiment that cycled P. aeruginosa between public and private goods growth media and evolved an isolate that rewired its control of cooperative protease expression from a synergistic (AND-gate) response to dual-signal input to a 3O-C12-HSL-only response. We show that this isolate circumvents metabolic incentives to cooperate and acts as a combinatorial signalling cheat, with higher fitness in competition with its ancestor. Our results show three important principles: first, combinatorial QS allows for diverse social strategies to emerge; second, restrictions levied by private goods are not sufficient to explain the maintenance of cooperation in natural populations; and third, modifying combinatorial QS responses could result in important physiological outcomes in bacterial populations.

In silico bacteria evolve robust cooperaion via complex quorum-sensing strategies.

Many species of bacteria collectively sense and respond to their social and physical environment via 'quorum sensing' (QS), a communication system controlling extracellular cooperative traits. Despite detailed understanding of the mechanisms of signal production and response, there remains considerable debate over the functional role(s) of QS: in short, what is it for? Experimental studies have found support for diverse functional roles: density sensing, mass-transfer sensing, genotype sensing, etc. While consistent with theory, these results cannot separate whether these functions were drivers of QS adaption, or simply artifacts or 'spandrels' of systems shaped by distinct ecological pressures. The challenge of separating spandrels from drivers of adaptation is particularly hard to address using extant bacterial species with poorly understood current ecologies (let alone their ecological histories). To understand the relationship between defined ecological challenges and trajectories of QS evolution, we used an agent-based simulation modeling approach. Given genetic mixing, our simulations produce behaviors that recapitulate features of diverse microbial QS systems, including coercive (high signal/low response) and generalized reciprocity (signal auto-regulation) strategists - that separately and in combination contribute to QS-dependent resilience of QS-controlled cooperation in the face of diverse cheats. We contrast our in silico results given defined ecological challenges with bacterial QS architectures that have evolved under largely unknown ecological contexts, highlighting the critical role of genetic constraints in shaping the shorter term (experimental evolution) dynamics of QS. More broadly, we see experimental evolution of digital organisms as a complementary tool in the search to understand the emergence of complex QS architectures and functions.

Patient-reported outcomes in sickle cell disease and association with clinical and psychosocial factors: Report from the sickle cell disease implementation consortium.

Understanding patient experiences, quality of life, and treatment needs in individuals with sickle cell disease (SCD) is essential in promoting health and well-being. We used measures from the Adult Sickle Cell Quality of Life Measurement Information System (ASCQ-Me), Patient Reported Outcomes Measurement Information System (PROMIS), and Quality of Life in Neurological Disorders (NeuroQol) to evaluate pain impact, sleep impact, social functioning, depressive symptoms, tiredness, and cognitive function (collectively, patient reported outcomes [PROs]) and to identify associated demographic and clinical characteristics. Participants (n = 2201) between 18 and 45 years were recruited through the eight Sickle Cell Disease Implementation Consortium (SCDIC) sites. In multivariate models, PROs were significantly associated with one another. Pain impact was associated with age, education, employment, time since last pain attack, hydroxyurea use, opioid use, sleep impact, social functioning, and cognitive function (F = 88.74, P < .0001). Sleep impact was associated with household income, opioid use, pain impact, social functioning, depressive symptoms, and tiredness (F = 101.40, P < .0001). Social functioning was associated with employment, pain attacks in the past year, autoimmune/inflammatory comorbidities, pain impact, sleep impact, depressive symptoms, tiredness, and cognitive function (F = 121.73, P < .0001). Depressive symptoms were associated with sex, sleep impact, social functioning, tiredness, and cognitive function (F = 239.51, P < .0001). Tiredness was associated with sex, education, sleep impact, social functioning, depressive symptoms, and cognitive function (F = 129.13, P < .0001). These findings reflect the baseline PRO assessments among SCDIC registry participants. Further research is needed to better understand these outcomes and new targets for interventions to improve quality of life and function in people with SCD.

Phage steering of antibiotic-resistance evolution in the bacterial pathogen, Pseudomonas aeruginosa.

BACKGROUND AND OBJECTIVES: Antimicrobial resistance is a growing global concern and has spurred increasing efforts to find alternative therapeutics. Bacteriophage therapy has seen near constant use in Eastern Europe since its discovery over a century ago. One promising approach is to use phages that not only reduce bacterial pathogen loads but also select for phage resistance mechanisms that trade-off with antibiotic resistance-so called 'phage steering'. METHODOLOGY: Recent work has shown that the phage OMKO1 can interact with efflux pumps and in so doing select for both phage resistance and antibiotic sensitivity of the pathogenic bacterium Pseudomonas aeruginosa. We tested the robustness of this approach to three different antibiotics in vitro (tetracycline, erythromycin and ciprofloxacin) and one in vivo (erythromycin). RESULTS: We show that in vitro OMKO1 can reduce antibiotic resistance of P. aeruginosa (Washington PAO1) even in the presence of antibiotics, an effect still detectable after ca.70 bacterial generations in continuous culture with phage. Our in vivo experiment showed that phage both increased the survival times of wax moth larvae (Galleria mellonella) and increased bacterial sensitivity to erythromycin. This increased antibiotic sensitivity occurred both in lines with and without the antibiotic. CONCLUSIONS AND IMPLICATIONS: Our study supports a trade-off between antibiotic resistance and phage sensitivity. This trade-off was maintained over co-evolutionary time scales even under combined phage and antibiotic pressure. Similarly, OMKO1 maintained this trade-off in vivo, again under dual phage/antibiotic pressure. Our findings have implications for the future clinical use of steering in phage therapies. Lay Summary: Given the rise of antibiotic-resistant bacterial infection, new approaches to treatment are urgently needed. Bacteriophages (phages) are bacterial viruses. The use of such viruses to treat infections has been in near-continuous use in several countries since the early 1900s. Recent developments have shown that these viruses are not only effective against routine infections but can also target antibiotic resistant bacteria in a novel, unexpected way. Similar to other lytic phages, these so-called 'steering phages' kill the majority of bacteria directly. However, steering phages also leave behind bacterial variants that resist the phages, but are now sensitive to antibiotics. Treatment combinations of these phages and antibiotics can now be used to greater effect than either one independently. We evaluated the impact of steering using phage OMKO1 and a panel of three antibiotics on Pseudomonas aeruginosa, an important pathogen in hospital settings and in people with cystic fibrosis. Our findings indicate that OMKO1, either alone or in combination with antibiotics, maintains antibiotic sensitivity both in vitro and in vivo, giving hope that phage steering will be an effective treatment option against antibiotic-resistant bacteria.

A meta-analysis of toxicities related to hydroxycarbamide dosing strategies.

Due to fear of short-term toxicities, there is nonconsensus of hydroxycarbamide dosing strategy (escalated vs fixed-dosing methods), which contributes to its suboptimal use. We performed a meta-analysis to summarize the incidence rates of toxicities associated with both dosing methods. Summarized incidence rates could not be statistically compared between dosing methods due to sparse data. Summarized neutropenia and thrombocytopenia incidence rates were slightly higher when using escalated dosing than with fixed. Summarized reticulocytopenia was comparable. Summarized hepatic and renal toxicities' incidence rates were slightly higher when using fixed doses than with escalated. We recommend diligent and transparent reporting of toxicities.

Evolving Antibiotics against Resistance: a Potential Platform for Natural Product Development?

To avoid an antibiotic resistance crisis, we need to develop antibiotics at a pace that matches the rate of evolution of resistance. However, the complex functions performed by antibiotics-combining, e.g., penetration of membranes, counteraction of resistance mechanisms, and interaction with molecular targets-have proven hard to achieve with current methods for drug development, including target-based screening and rational design. Here, we argue that we can meet the evolution of resistance in the clinic with evolution of antibiotics in the laboratory. On the basis of the results of experimental evolution studies of microbes in general and antibiotic production in Actinobacteria in particular, we propose methodology for evolving antibiotics to circumvent mechanisms of resistance. This exploits the ability of evolution to find solutions to complex problems without a need for design. We review evolutionary theory critical to this approach and argue that it is feasible and has important advantages over current methods for antibiotic discovery.

Height-corrected low bone density associates with severe outcomes in sickle cell disease: SCCRIP cohort study results.

Low bone mineral density (BMD) disproportionately affects people with sickle cell disease (SCD). Growth faltering is common in SCD, but most BMD studies in pediatric SCD cohorts fail to adjust for short stature. We examined low BMD prevalence in 6- to 18-year-olds enrolled in the Sickle Cell Clinical Research and Intervention Program (SCCRIP), an ongoing multicenter life span SCD cohort study initiated in 2014. We calculated areal BMD for chronological age and height-adjusted areal BMD (Ht-aBMD) z scores for the SCCRIP cohort, using reference data from healthy African American children and adolescents enrolled in the Bone Mineral Density in Childhood Study. We defined low BMD as Ht-aBMD z scores less than or equal to -2 and evaluated its associations with demographic and clinical characteristics by using logistic regression analyses. Of the 306 children and adolescents in our study cohort (mean age, 12.5 years; 50% female; 64% HbSS/Sß0-thalassemia genotype; 99% African American), 31% had low areal BMD for chronological age z scores and 18% had low Ht-aBMD z scores. In multivariate analyses, low Ht-aBMD z scores associated with adolescence (odds ratio [OR], 7.7; 95% confidence interval [CI], 1.94-30.20), hip osteonecrosis (OR, 4.0; 95% CI, 1.02-15.63), chronic pain (OR, 10.4; 95% CI, 1.51-71.24), and hemoglobin (OR, 0.74; 95% CI, 0.57-0.96). Despite adjusting for height, nearly 20% of this pediatric SCD cohort still had very low BMD. As the SCCRIP cohort matures, we plan to prospectively evaluate the longitudinal relationship between Ht-aBMD z scores and markers of SCD severity and morbidity.

Why put up with immunity when there is resistance: an excursion into the population and evolutionary dynamics of restriction-modification and CRISPR-Cas.

Bacteria can readily generate mutations that prevent bacteriophage (phage) adsorption and thus make bacteria resistant to infections with these viruses. Nevertheless, the majority of bacteria carry complex innate and/or adaptive immune systems: restriction-modification (RM) and CRISPR-Cas, respectively. Both RM and CRISPR-Cas are commonly assumed to have evolved and be maintained to protect bacteria from succumbing to infections with lytic phage. Using mathematical models and computer simulations, we explore the conditions under which selection mediated by lytic phage will favour such complex innate and adaptive immune systems, as opposed to simple envelope resistance. The results of our analysis suggest that when populations of bacteria are confronted with lytic phage: (i) In the absence of immunity, resistance to even multiple bacteriophage species with independent receptors can evolve readily. (ii) RM immunity can benefit bacteria by preventing phage from invading established bacterial populations and particularly so when there are multiple bacteriophage species adsorbing to different receptors. (iii) Whether CRISPR-Cas immunity will prevail over envelope resistance depends critically on the number of steps in the coevolutionary arms race between the bacteria-acquiring spacers and the phage-generating CRISPR-escape mutants. We discuss the implications of these results in the context of the evolution and maintenance of RM and CRISPR-Cas and highlight fundamental questions that remain unanswered. This article is part of a discussion meeting issue 'The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems'.

A Randomized Study of Exercise and Fitness Trackers in Obese Patients After Total Knee Arthroplasty.

Functional limitations persist in obese patients after total knee arthroplasty (TKA). This study assessed the effect of an exercise program (EP) and fitness trackers (FT) in obese patients with TKA. Sixty patients 1 year after orthopedic surgery were recruited and received a 16-week tailored EP; half were randomized to receive an FT. FT had no measurable effect compared with EP alone. EP improved knee range of motion, strength, and quality-of-life scores. This study provides preliminary evidence that a 16-week EP in obese individuals 1 year post TKA is feasible and effective in improving function and quality of life.

Transient negative effects of antibiotics on phages do not jeopardise the advantages of combination therapies.

Phages, the viruses of bacteria, have been proposed as antibacterial agents to complement or replace antibiotics due to the growing problem of resistance. In nature and in the clinic, antibiotics are ubiquitous and may affect phages indirectly via impacts on bacterial hosts. Even if the synergistic association of phages and antibiotics has been shown in several studies, the focus is often on bacteria with little known about the impact on phages. Evolutionary studies have demonstrated that time scale is an important factor in understanding the consequences of antimicrobial strategies, but this perspective is generally overlooked in phage-antibiotic combination studies. Here, we explore the effects of antibiotics on phages targeting the opportunistic pathogen Pseudomonas aeruginosa. We go beyond previous studies by testing the interaction between several types of antibiotics and phages, and evaluate the effects on several important phage parameters during 8 days of experimental co-evolution with bacteria. Our study reveals that antibiotics had a negative effect on phage density and efficacy early on, but not in the later stages of the experiment. The results indicate that antibiotics can affect phage adaptation, but that phages can nevertheless contribute to managing antibiotic resistance levels.

Sickle Cell Clinical Research and Intervention Program (SCCRIP): A lifespan cohort study for sickle cell disease progression from the pediatric stage into adulthood.

BACKGROUND: Previous natural history studies have advanced the understanding of sickle cell disease (SCD), but generally have not included sufficient lifespan data or investigation of the role of genetics in clinical outcomes, and have often occurred before the widespread use of disease-modifying therapies, such as hydroxyurea and chronic erythrocyte transfusions. To further advance knowledge of SCD, St. Jude Children's Research Hospital established the Sickle Cell Clinical Research and Intervention Program (SCCRIP), to conduct research in a clinically evaluated cohort of individuals with SCD across their lifetime. PROCEDURES: Initiated in 2014, the SCCRIP study prospectively recruits patients diagnosed with SCD and includes retrospective and longitudinal collection of clinical, neurocognitive, geospatial, psychosocial, and health outcomes data. Biological samples are banked for future genomics and proteomics studies. The organizational structure of SCCRIP is based upon organ/system-specific working groups and is opened to the research community for partnerships. RESULTS: As of August 2017, 1,044 (92.3% of eligible) patients with SCD have enrolled in the study (860 children and 184 adults), with 11,915 person-years of observation. Population demographics included mean age at last visit of 11.3 years (range 0.7-30.1), 49.8% females, 57.7% treated with hydroxyurea, 8.5% treated with monthly transfusions, and 62.9% hemoglobin (Hb) SS or HbSB0 -thalassemia, 25.7% HbSC, 8.4% HbsB+ -Thalassemia, 1.7% HbS/HPFH, and 1.2% other. CONCLUSIONS: The SCCRIP cohort will provide a rich resource for the conduct of high impact multidisciplinary research in SCD.

Behavioral symptoms and psychiatric disorders in child and adolescent long-term survivors of childhood acute lymphoblastic leukemia treated with chemotherapy only.

BACKGROUND: Prevalence of emotional, behavioral, and psychiatric outcomes in child and adolescent survivors of childhood acute lymphoblastic leukemia treated on a chemotherapy-only protocol were not well defined. METHODS: Self- and parent-reported emotional and behavioral symptoms were assessed for 161 survivors of childhood acute lymphoblastic leukemia (51.0% female; mean [SD] age 12.1[2.6] years; 7.5[1.6] years post-diagnosis). Age- and sex-adjusted scores were calculated for standardized measures and compared with 90th percentile of norms. Frequencies of survivor psychiatric disorders from structured diagnostic interviews with parents were compared with the general population. Parent emotional distress and post-traumatic stress symptoms were assessed. Associations between child symptoms/disorders and parent distress were examined with log-binomial models, adjusting for highest parent education. RESULTS: Compared with population expectations (10%), more survivors self-reported symptoms of inattention (27.9; 95% CI, 21.0%-35.7%), hyperactivity/impulsivity (26.0%; CI, 19.2%-33.6%), and oppositional-defiant behavior (20.1%; CI, 14.1%-27.3%). Parents reported survivors with more symptoms of inattention (23.6%; CI, 17.2%-31.0%), higher frequencies of obsessive-compulsive disorder (10.3% vs 2%) and oppositional defiant disorder (16.0% vs 9.5%), but not attention-deficit/hyperactivity disorder (7.1% vs 7.8%) or generalized anxiety disorder (3.2% vs 4.1%), compared with national norms. Parent-report of child anxiety disorders was associated with parent self-reported emotional distress but not survivor self-report of anxiety. CONCLUSION: A significant minority of survivors have long-term psychiatric morbidity, multi-informant assessment is important to understand these symptom profiles and to inform selection of appropriate interventions. Interventions targeting inattention and oppositional behavior in children and emotional distress in parents are warranted in families with survivors who display behavioral problems.

Evolution of neurocognitive function in long-term survivors of childhood acute lymphoblastic leukemia treated with chemotherapy only.

PURPOSE: The purpose of this study was to determine the evolution of neurocognitive problems from therapy completion to long-term follow-up in survivors of childhood acute lymphoblastic leukemia treated with chemotherapy only. METHODS: We evaluated whether attention problems observed at therapy completion evolve into long-term executive dysfunction in 158 survivors treated on a single institution protocol. Treatment data (high-dose intravenous methotrexate exposure [serum concentration] and triple intrathecal chemotherapy injections) were collected. Parent report of behavior and direct cognitive testing of survivors was conducted at end of therapy, and survivors completed neurocognitive testing when > 5 years post-diagnosis. RESULTS: At the end of chemotherapy, survivors (52% female; mean age 9.2 years) demonstrated higher frequency of impairment in sustained attention (38%) and parent-reported inattention (20%) compared to population expectations (10%). At long-term follow-up, survivors (mean age 13.7 years; 7.6 years post-diagnosis) demonstrated higher impairment in executive function (flexibility 24%, fluency 21%), sustained attention (15%), and processing speed (15%). Sustained attention improved from end of therapy to long-term follow-up (p < 0.001). Higher methotrexate AUC and greater number of intrathecal injections were associated with attention problems (p = 0.009, p = 0.002, respectively) at the end of chemotherapy and executive function (p < 0.001, p = 0.02, respectively) problems at long-term follow-up. Attention problems at the end of therapy were not associated with executive function problems at long-term follow-up (p's > 0.05). The direct effect of chemotherapy exposure predicted outcomes at both time points. CONCLUSIONS AND IMPLICATIONS FOR CANCER SURVIVORS: Survivors should be monitored for neurocognitive problems well into long-term survivorship, regardless of whether they show attention problems at the end of therapy. Treatment exposures are the best predictor of long-term complications.