Delineating functional and molecular impact of ex vivo sample handling in precision medicine.

Consistent handling of samples is crucial for achieving reproducible molecular and functional testing results in translational research. Here, we used 229 acute myeloid leukemia (AML) patient samples to assess the impact of sample handling on high-throughput functional drug testing, mass spectrometry-based proteomics, and flow cytometry. Our data revealed novel and previously described changes in cell phenotype and drug response dependent on sample biobanking. Specifically, myeloid cells with a CD117 (c-KIT) positive phenotype decreased after biobanking, potentially distorting cell population representations and affecting drugs targeting these cells. Additionally, highly granular AML cell numbers decreased after freezing. Secondly, protein expression levels, as well as sensitivity to drugs targeting cell proliferation, metabolism, tyrosine kinases (e.g., JAK, KIT, FLT3), and BH3 mimetics were notably affected by biobanking. Moreover, drug response profiles of paired fresh and frozen samples showed that freezing samples can lead to systematic errors in drug sensitivity scores. While a high correlation between fresh and frozen for the entire drug library was observed, freezing cells had a considerable impact at an individual level, which could influence outcomes in translational studies. Our study highlights conditions where standardization is needed to improve reproducibility, and where validation of data generated from biobanked cohorts may be particularly important.

Prediction model for drug response of acute myeloid leukemia patients.

Despite some encouraging successes, predicting the therapy response of acute myeloid leukemia (AML) patients remains highly challenging due to tumor heterogeneity. Here we aim to develop and validate MDREAM, a robust ensemble-based prediction model for drug response in AML based on an integration of omics data, including mutations and gene expression, and large-scale drug testing. Briefly, MDREAM is first trained in the BeatAML cohort (n = 278), and then validated in the BeatAML (n = 183) and two external cohorts, including a Swedish AML cohort (n = 45) and a relapsed/refractory acute leukemia cohort (n = 12). The final prediction is based on 122 ensemble models, each corresponding to a drug. A confidence score metric is used to convey the uncertainty of predictions; among predictions with a confidence score >0.75, the validated proportion of good responders is 77%. The Spearman correlations between the predicted and the observed drug response are 0.68 (95% CI: [0.64, 0.68]) in the BeatAML validation set, -0.49 (95% CI: [-0.53, -0.44]) in the Swedish cohort and 0.59 (95% CI: [0.51, 0.67]) in the relapsed/refractory cohort. A web-based implementation of MDREAM is publicly available at https://www.meb.ki.se/shiny/truvu/MDREAM/ .

Serum amyloid A as a marker to detect sepsis and predict outcome in hospitalized neonatal foals.

BACKGROUND: Serum amyloid A (SAA) has been reported to hold promise as diagnostic and prognostic marker in foals. This has not been investigated thoroughly. OBJECTIVES: Evaluate admission SAA concentrations as predictor of sepsis and outcome. ANIMALS: Five hundred and ninety hospitalized foals <14 days old. METHODS: Retrospective multicenter study. Foals were scored with sepsis and survival scores, grouped according to health category (septic, sick but nonseptic, uncertain sepsis status) and outcome; septic foals were further categorized according to severity (normal sepsis, severe sepsis, and septic shock). SAA was compared between groups using Mann-Whitney test and Kruskal-Wallis test. Receiver operating characteristic curves identified optimal SAA cut off values for detecting sepsis and predicting outcome. RESULTS: Admission SAA concentrations differed significantly between sick nonseptic foals (312.1 ± 685.4 mg/L) and septic foals (1079.7 ± 1254.5 mg/L) and increased with increasing sepsis score. SAA did not differ between sepsis severity groups. The optimal cut off for sepsis detection was 1050 mg/L (sensitivity 30.2%, specificity 90.7%). Admission SAA concentrations were lower in surviving (435.0 ± 723.6 mg/L) compared to nonsurviving foals (1062.7 ± 1440.1 mg/L) and decreased with increasing survival score. The optimal cut off for nonsurvival prediction was 1250 mg/L (sensitivity 22.1%, specificity 90.8%). CONCLUSIONS AND CLINICAL IMPORTANCE: SAA concentration was higher in septic foals and nonsurviving foals. Even though optimal cut offs for SAA to detect sepsis and predict outcome had low sensitivity, they had good specificity. SAA can therefore be used as a marker to rule out sepsis and nonsurvival.

Targeting SAMHD1 with hydroxyurea in first-line cytarabine-based therapy of newly diagnosed acute myeloid leukaemia: Results from the HEAT-AML trial.

BACKGROUND: Treatment of newly diagnosed acute myeloid leukaemia (AML) is based on combination chemotherapy with cytarabine (ara-C) and anthracyclines. Five-year overall survival is below 30%, which has partly been attributed to cytarabine resistance. Preclinical data suggest that the addition of hydroxyurea potentiates cytarabine efficacy by increasing ara-C triphosphate (ara-CTP) levels through targeted inhibition of SAMHD1. OBJECTIVES: In this phase 1 trial, we evaluated the feasibility, safety and efficacy of the addition of hydroxyurea to standard chemotherapy with cytarabine/daunorubicin in newly diagnosed AML patients. METHODS: Nine patients were enrolled and received at least two courses of ara-C (1 g/m2 /2 h b.i.d. d1-5, i.e., a total of 10 g/m2 per course), hydroxyurea (1-2 g d1-5) and daunorubicin (60 mg/m2 d1-3). The primary endpoint was safety; secondary endpoints were complete remission rate and measurable residual disease (MRD). Additionally, pharmacokinetic studies of ara-CTP and ex vivo drug sensitivity assays were performed. RESULTS: The most common grade 3-4 toxicity was febrile neutropenia (100%). No unexpected toxicities were observed. Pharmacokinetic analyses showed a significant increase in median ara-CTP levels (1.5-fold; p = 0.04) in patients receiving doses of 1 g hydroxyurea. Ex vivo, diagnostic leukaemic bone marrow blasts from study patients were significantly sensitised to ara-C by a median factor of 2.1 (p = 0.0047). All nine patients (100%) achieved complete remission, and all eight (100%) with validated MRD measurements (flow cytometry or real-time quantitative polymerase chain reaction [RT-qPCR]) had an MRD level <0.1% after two cycles of chemotherapy. Treatment was well-tolerated, and median time to neutrophil recovery >1.0 × 109 /L and to platelet recovery >50 × 109 /L after the start of cycle 1 was 19 days and 22 days, respectively. Six of nine patients underwent allogeneic haematopoietic stem-cell transplantation (allo-HSCT). With a median follow-up of 18.0 (range 14.9-20.5) months, one patient with adverse risk not fit for HSCT experienced a relapse after 11.9 months but is now in second complete remission. CONCLUSION: Targeted inhibition of SAMHD1 by the addition of hydroxyurea to conventional AML therapy is safe and appears efficacious within the limitations of the small phase 1 patient cohort. These results need to be corroborated in a larger study.

Adapting pediatric obesity care to better suit adolescent patients: Design of a treatment platform and results compared with standard care in the national patient quality register.

BACKGROUND: Obesity constitutes a critical risk for adolescent health. This study aimed at identifying youth-friendly components of obesity treatment. METHODS: In this feasibility study, an adolescent obesity treatment platform was implemented at two Pediatric outpatient clinics in Sweden. Body mass index (BMI), BMI z-score, and the category of obesity (International Obesity Task Force) were compared before and after the intervention and with data on standard care from the Swedish Childhood Obesity Treatment Register. RESULTS: The study included 99 participants (49 females) aged 13-18 years from 1 September 2014, to 31 December 2016. A pediatric nurse met the participants on average 6.5 times in the average inclusion period of 15 months. Physical activity sessions attracted 63 participants. Acceptance Commitment Therapy and In Real Life groups attracted 24 participants. At inclusion, 62 participants had obesity and 37 severe obesity, and 71/99 (72%) remained in the same category. The mean BMI increased from 32.0 to 33.4 kg/m2 (p < 0.01), but 56/94 (60%) participants lowered their BMI or increased less than 1 kg/m2 and 73% stayed to the end of the study. Participants who were new to treatment and participants coming for more than eight visits to the nurse did not increase in BMI. BMI did not change for the 221 out of 641 register patients who had two recordings of BMI in the study period. CONCLUSIONS: The platform was successful in increasing retention, and 60% of participants lowered or maintained their BMI. Still, seven out of ten adolescents with obesity or severe obesity remained in the same weight category.

The transcriptome-wide landscape of molecular subtype-specific mRNA expression profiles in acute myeloid leukemia.

Molecular classification of acute myeloid leukemia (AML) aids prognostic stratification and clinical management. Our aim in this study is to identify transcriptome-wide mRNAs that are specific to each of the molecular subtypes of AML. We analyzed RNA-sequencing data of 955 AML samples from three cohorts, including the BeatAML project, the Cancer Genome Atlas, and a cohort of Swedish patients to provide a comprehensive transcriptome-wide view of subtype-specific mRNA expression. We identified 729 subtype-specific mRNAs, discovered in the BeatAML project and validated in the other two cohorts. Using unique proteomics data, we also validated the presence of subtype-specific mRNAs at the protein level, yielding a rich collection of potential protein-based biomarkers for the AML community. To enable the exploration of subtype-specific mRNA expression by the broader scientific community, we provide an interactive resource to the public.

Promising results from an implemented treatment model for paediatric obesity.

AIM: To investigate the implementation of a plan of action for treatment of childhood obesity, and the effect after 2 years of treatment. METHODS: Children aged 6-12.9 years who started obesity treatment between 2008 and 2015 in a paediatric clinic in Stockholm County were included. The treatment model included staff education and support and group activities for parents and children separately followed by individual sessions to a multidisciplinary team. The main outcome was change in body mass index standard deviation score (BMI SDS), in comparison to a matched control group. RESULTS: In the intervention group, 1334 children (52% boys) with an average age of 9.3 years and BMI SDS of 2.7 and 3012 children in the control group were included. The intervention group decreased their BMI SDS more after two years compared with the control group, (-0.31 vs -0.23, P < .001). Younger age and higher BMI SDS at treatment initiation and families that completed the group sessions (all P < .001) had greater decreases in BMI SDS after 2 years. Sex did not affect the outcome. CONCLUSION: Even though the treatment in the control group was effective, the implementation of the action plan yielded a better treatment response compared with the control group.

Evaluation of the foal survival score in a Danish-Swedish population of neonatal foals upon hospital admission.

BACKGROUND: It is highly desirable to assess the probability of survival in sick neonatal foals upon admission. The foal survival score (FSS) is a published scoring system used to estimate the probability of survival in hospitalized neonatal foals <4 days old. HYPOTHESIS/OBJECTIVES: To evaluate the ability of the FSS to predict survival in older foals from a geographically different area compared to the original study. ANIMALS: Five-hundred ninety hospitalized neonatal foals ≤14 days of age. METHODS: Retrospective Danish-Swedish multicenter study that included details of signalment, history, clinical examination, laboratory results, necropsy findings, and outcome. Scores and score variables were compared between survivors and nonsurvivors using logistic regression. The optimal cutoff and its test parameters were calculated using a receiver operator characteristic curve. RESULTS: Prematurity, cold extremities, ≥2 infectious or inflammatory sites, blood glucose concentration, and total white blood cell counts were significantly associated with nonsurvival (P ≤ .02). The optimal cutoff to predict survival was ≥6, resulting in sensitivity 78%, specificity 58%, 92% positive predictive value, and 31% negative predictive value. The test performed equally well in foals <4 days old compared to those 4-14 days old. CONCLUSIONS AND CLINICAL IMPORTANCE: Using the suggested optimal cutoff of ≥6, the FSS performed moderately well and may aid in early determination of prognosis for survival. However, the FSS did perform differently in another population and therefore should be assessed under local conditions so that its diagnostic potential is not overestimated.

Lawsonia intracellularis associated equine proliferative enteropathy in Danish weanling foals.

BACKGROUND: Lawsonia intracellularis, an obligate intracellular bacterium, causes equine proliferative enteropathy, mainly in horses around weaning. This disease is rarely reported in the Scandinavian countries. RESULTS: Five cases of equine proliferative enteropathy were diagnosed between 2008-2016 at the University of Copenhagen Large Animal Teaching Hospital. Cases were Danish Warmbloods and a Friesian horse, aged 6-7 months, presenting with typical clinical signs of lethargy, poor body condition, pyrexia and diarrhea. Clinical pathology was consistent with previous reports of severe hypoalbuminemia and leukocytosis. Diagnosis was confirmed by fecal polymerase chain reaction, serum immunomonolayer peroxidase assay and/or immunofluorescence and fluorescence in situ hybridization performed on formalin-fixed ileum samples. Concurrent intestinal parasitism was present in all five cases. Treatment consisted of antimicrobial therapy, anti-inflammatories, intravenous crystalloids and plasma. Three foals were euthanised due to deterioration and poor response to treatment, one with complications of septic arthritis and Strongylus vulgaris associated intestinal infarct. The other two foals survived and were reported by the owners to be healthy on long-term follow-up. CONCLUSIONS: Equine proliferative enteropathy is a disease to consider in young horses presenting with diarrhea and hypoproteinemia in Denmark.

Five-year outpatient programme that provided children with continuous behavioural obesity treatment enjoyed high success rate.

AIM: Results from long-time follow-up of obesity treatment in early childhood are lacking. We investigate long-term continuous behavioural childhood obesity treatment and factors of importance for treatment effect. METHOD: A five-year longitudinal retrospective controlled study of children aged five to 13 years in obesity treatment, divided into three groups depending on age at start of treatment. Outcome is presented as change in degree of obesity, body mass index standard deviation score (BMI SDS), change in weight status and decrease of ≥0.5 BMI SDS units, in relation to a age-matched obese comparison group. RESULTS: In total, 220 children (46% females) were included. After five years of treatment, the decrease in BMI SDS was significant in all age groups with the largest effect in age group 4-6 years. Compared to the comparison group (n = 369), the decline in BMI SDS was greater (p = 0.001). After five years of treatment, 48% of the patients were cured from their obesity and 72% reached a decline of 0.5 BMI SDS units. Age at start of treatment was the only factor affecting treatment efficacy. CONCLUSION: The ability to reach a significant weight loss in a paediatric outpatient clinic is promising through a long-term behavioural obesity treatment.

Dissecting antibodies with regards to linear and conformational epitopes.

An important issue for the performance and specificity of an antibody is the nature of the binding to its protein target, including if the recognition involves linear or conformational epitopes. Here, we dissect polyclonal sera by creating epitope-specific antibody fractions using a combination of epitope mapping and an affinity capture approach involving both synthesized peptides and recombinant protein fragments. This allowed us to study the relative amounts of antibodies to linear and conformational epitopes in the polyclonal sera as well as the ability of each antibody-fraction to detect its target protein in Western blot assays. The majority of the analyzed polyclonal sera were found to have most of the target-specific antibodies directed towards linear epitopes and these were in many cases giving Western blot bands of correct molecular weight. In contrast, many of the antibodies towards conformational epitopes did not bind their target proteins in the Western blot assays. The results from this work have given us insights regarding the nature of the antibody response generated by immunization with recombinant protein fragments and has demonstrated the advantage of using antibodies recognizing linear epitopes for immunoassay involving wholly or partially denatured protein targets.

Perceived gender inequality in the couple relationship and musculoskeletal pain in middle-aged women and men.

AIMS: Musculoskeletal pain is a major health problem, especially in women, and is partially determined by psychosocial factors. The aim of the present study was to investigate whether gender inequality in the couple relationship was related to musculoskeletal pain. METHODS: Participants (n=721; 364 women and 357 men) were all individuals living in a couple relationship in the Northern Swedish Cohort, a 26-year Swedish cohort study. Self-administered questionnaire data at age 42 years comprised perceived gender inequality in the couple relationship and musculoskeletal pain (in three locations, summarised into one score and median-split), concurrent demographic factors, psychological distress, and previous musculoskeletal pain at age 30 years. Associations were examined using logistic regression. RESULTS: Gender inequality was positively associated with symptoms of musculoskeletal pain in the total sample, remaining significant after addition of possible confounders and of previous musculoskeletal pain. Separate adjustment for concurrent psychological distress attenuated the association but not below significance. The association was present and of comparable strength in both women and men. CONCLUSIONS: Gender inequality in the couple relationship might contribute to the experience of musculoskeletal pain in both women and men. The results highlight the potential adverse bodily consequences of living in unequal relationships.