ProTarget: a Danish Nationwide Clinical Trial on Targeted Cancer Treatment based on genomic profiling - a national, phase 2, prospective, multi-drug, non-randomized, open-label basket trial.

BACKGROUND: An increasing number of trials indicate that treatment outcomes in cancer patients with metastatic disease are improved when targeted treatments are matched with druggable genomic alterations in individual patients (pts). An estimated 30-80% of advanced solid tumors harbor actionable genomic alterations. However, the efficacy of personalized cancer treatment is still scarcely investigated in larger, controlled trials due to the low frequency and heterogenous distribution of druggable alterations among different histologic tumor types. Therefore, the overall effect of targeted cancer treatment on clinical outcomes still needs investigation. STUDY DESIGN/METHODS: ProTarget is a national, non-randomized, multi-drug, open-label, pan-cancer phase 2 trial aiming to investigate the anti-tumor activity and toxicity of currently 13 commercially available, EMA-approved targeted therapies outside the labeled indication for treatment of advanced malignant diseases, harboring specific actionable genomic alterations. The trial involves the Danish National Molecular Tumor Board for confirmation of drug-variant matches. Key inclusion criteria include a) measurable disease (RECIST v.1.1), b) ECOG performance status 0-2, and c) an actionable genomic alteration matching one of the study drugs. Key exclusion criteria include a) cancer type within the EMA-approved label of the selected drug, and b) genomic alterations known to confer drug resistance. Initial drug dose, schedule and dose modifications are according to the EMA-approved label. The primary endpoint is objective response or stable disease at 16 weeks. Pts are assigned to cohorts defined by the selected drug, genomic alteration, and tumor histology type. Cohorts are monitored according to a Simon's two-stage-based design. Response is assessed every 8 weeks for the first 24 weeks, then every 12 weeks. The trial is designed similar to the Dutch DRUP and the ASCO TAPUR trials and is a partner in the Nordic Precision Cancer Medicine Trial Network. In ProTarget, serial fresh tumor and liquid biopsies are mandatory and collected for extensive translational research including whole genome sequencing, array analysis, and RNA sequencing. DISCUSSION: The ProTarget trial will identify new predictive biomarkers for targeted treatments and provide new data and essential insights in molecular pathways involved in e.g., resistance mechanisms and thereby potentially evolve and expand the personalized cancer treatment strategy. PROTOCOL VERSION: 16, 09-MAY-2022. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT04341181. Secondary Identifying No: ML41742. EudraCT No: 2019-004771-40.

Are cancer patients better off if they participate in clinical trials? A mixed methods study.

BACKGROUND: Research and cancer care are closely intertwined; however, it is not clear whether physicians and nurses believe that clinical trials offer the best treatment for patients and, if so, whether this belief is justified. The aim of this study was therefore: (i) to explore how physicians and nurses perceive the benefits of clinical trial participation compared with standard care and (ii) whether it is justified to claim that clinical trial participation improves outcomes for cancer patients. METHODS: A mixed methods approach was used employing semi-structured interviews with 57 physicians and nurses in oncology and haematology and a literature review of the evidence for trial superiority, i.e. the idea that receiving treatment in a clinical trial leads to a better outcome compared with standard care. Inductive thematic analysis was used to examine the interview data. A literature review comprising nine articles was conducted according to a conceptual framework developed by Peppercorn et al. and evaluated recent evidence on trial superiority. RESULTS: Our findings show that many physicians and nurses make claims supporting trial superiority, however very little evidence is available in the literature comparing outcomes for trial participants and non-participants that supports their assertions. CONCLUSIONS: Despite the recent rapid development and use of targeted therapy and immunotherapy, we find no support for trial participation to provide better outcomes for cancer patients than standard care. Hence, our present results are in line with previous results from Peppercorn et al. A weaker version of the superiority claim is that even if a trial does not bring about a direct positive effect, it brings about indirect positive effects. However, as the value of such indirect effects is dependent on the individual's specific circumstances and preferences, their existence cannot establish the general claim that treatment in trials is superior. Belief in trial superiority is therefore unfounded. Hence, if such beliefs are communicated to patients in a trial recruitment context, it would provide misleading information. Instead emphasis should be on patients volunteering to give an altruistic contribution to the furthering of knowledge and to the potential benefit of future patients.

Vitamin C - A new player in regulation of the cancer epigenome.

Over the past few years it has become clear that vitamin C, as a provider of reduced iron, is an essential factor for the function of epigenetic regulators that initiate the demethylation of DNA and histones. Vitamin C deficiency is rare in the general population, but is frequently observed in patients with cancer. Genes encoding epigenetic regulators are often mutated in cancer, underscoring their central roles in carcinogenesis. In hematological cancers, such as acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS), drugs that reverse epigenetic aberrations are now the standard of care. Recent in vitro studies suggest that vitamin C at physiological concentrations, combined with hypomethylating agents may act synergistically to cause DNA demethylation through active and passive mechanisms, respectively. Additionally, several recent studies have renewed interest in the use of pharmacological doses of vitamin C injected intravenously to selectively kill tumor cells. This review will focus on the potential of vitamin C to optimize the outcome of epigenetic therapy in cancer patients and alternatively to act as a therapeutic at high doses.

A study of associations between early DHA status and fatty acid desaturase (FADS) SNP and developmental outcomes in children of obese mothers.

DHA from diet or endogenous synthesis has been proposed to affect infant development, however, results are inconclusive. In this study, we aim to verify previously observed fatty acid desaturase gene cluster (FADS) SNP-specific associations with erythrocyte DHA status in 9-month-old children and sex-specific association with developmental outcomes. The study was performed in 166 children (55 % boys) of obese mothers. Erythrocyte fatty acid composition was analysed in blood-samples obtained at 9 months of age, and developmental outcomes assessed by the Ages and Stages Questionnaire at 3 years. Erythrocyte DHA level ranged from 4·4 to 9·9 % of fatty acids, but did not show any association with FADS SNP or other potential determinants. Regression analysis showed associations between erythrocyte DHA and scores for personal-social skills (ß 1·8 (95 % CI 0·3, 3·3), P=0·019) and problem solving (ß 3·4 (95 % CI 1·2, 5·6), P=0·003). A tendency was observed for an association in opposite direction between minor alleles (G-variant) of rs1535 and rs174575 and personal-social skills (P=0·062 and 0·068, respectively), which became significant when the SNP were combined based on their previously observed effect on erythrocyte DHA at 9 months of age (ß 2·6 (95 % CI 0·01, 5·1), P=0·011). Sex-SNP interaction was indicated for rs174575 genotype on fine motor scores (P=0·016), due to higher scores among minor allele carrying girls (P=0·043), whereas no effect was seen among boys. In conclusion, DHA-increasing FADS SNP and erythrocyte DHA status were consistently associated with improved personal-social skills in this small cohort of children of obese mothers irrespective of sex, but the sample was too small to verify potential sex-specific effects.

Fish oil supplementation from 9 to 18 months of age affects the insulin-like growth factor axis in a sex-specific manner in Danish infants.

Several studies have investigated the effects of fish oil (FO) on infant growth, but little is known about the effects of FO and sex on insulin-like growth factor-1 (IGF-1), the main regulator of growth in childhood. We explored whether FO v. sunflower oil (SO) supplementation from 9 to 18 months of age affected IGF-1 and its binding protein-3 (IGFBP-3) and whether the potential effects were sex specific. Danish infants (n 115) were randomly allocated to 5 ml/d FO (1·2 g/d n-3 long-chain PUFA (n-3 LCPUFA)) or SO. We measured growth, IGF-1, IGFBP-3 and erythrocyte EPA, a biomarker of n-3 LCPUFA intake and status, at 9 and 18 months. Erythrocyte EPA increased strongly with FO compared with SO (P<0·001). There were no effects of FO compared with SO on IGF-1 in the total population, but a sex × group interaction (P=0·02). Baseline-adjusted IGF-1 at 18 months was 11·1 µg/l (95% CI 0·4, 21·8; P=0·04) higher after FO compared with SO supplementation among boys only. The sex × group interaction was borderline significant in the model of IGFBP-3 (P=0·09), with lower IGFBP-3 with FO compared with SO among girls only (P=0·03). The results were supported by sex-specific dose-response associations between changes in erythrocyte EPA and changes in IGF-1 and IGFBP-3 (both P<0·03). Moreover, IGF-1 was sex specifically associated with BMI and length. In conclusion, FO compared with SO resulted in higher IGF-1 among boys and lower IGFBP-3 among girls. The potential long-term implications for growth and body composition should be investigated further.

Fish intake, erythrocyte n-3 fatty acid status and metabolic health in Danish adolescent girls and boys.

Marine n-3 long-chain PUFA (n-3 LCPUFA) may have a beneficial effect on several aspects of the metabolic syndrome (dyslipidaemia, insulin resistance, hypertension and abdominal obesity). The metabolic syndrome is increasing in prevalence during adolescence, but only few studies have investigated the effects of n-3 LCPUFA in adolescence. The present study examines associations between fish intake (assessed by a 7 d pre-coded food diary), erythrocyte (RBC) DHA status (analysed by GC) and metabolic syndrome measures (anthropometry, blood pressure and plasma lipids, insulin and glucose) in 109 17-year-old children from the Copenhagen Birth Cohort Study. Of the children, 8 % were overweight or obese and few showed signs of the metabolic syndrome, but all the metabolic syndrome variables were correlated. Median fish intake was 10·7 (interquartile range 3·6-21·2) g/d. Boys tended to have a higher fish intake (P = 0·052), but girls had significantly higher RBC levels of DHA (P = 0·001). Sex and fish intake explained 37 % of the variance in RBC-DHA (P < 0·001). After adjusting for confounders, high DHA status was found to be significantly correlated with higher systolic blood pressure (P = 0·014) and increased fasting insulin (P = 0·018), but no adverse association was observed with the mean metabolic syndrome z-score. Overall, the present study showed the expected association between fish intake and RBC-DHA, which in contrast to our expectations tended to be associated with a poorer metabolic profile. Whether these results reflect the physiological function of n-3 LCPUFA, lifestyle factors associated with fish intake in Denmark, or mere chance remains to be investigated.

Mendelian randomization shows sex-specific associations between long-chain PUFA-related genotypes and cognitive performance in Danish schoolchildren.

Background: Dietary and endogenously formed long-chain polyunsaturated fatty acids (LCPUFAs) are hypothesized to improve cognitive development, but results are inconclusive, with suggestions of sex specificity. One study suggested that single-nucleotide polymorphisms (SNPs) rs1535 and rs174448 in the fatty acid desaturase (FADS) gene cluster have opposite effects on erythrocyte LCPUFAs at 9 mo.Objective: To explore whether SNPs in FADS and elongase (ELOVL) genes were associated with school performance in a sex-specific manner, we performed a Mendelian randomization study using data from the Optimal well-being, development and health for Danish children through a healthy New Nordic Diet (OPUS) School Meal Study with 765 Danish schoolchildren 8-11 y old.Design: Associations between selected FADS1/2 SNPs (rs1535, rs174448, and rs174468) and ELOVL5 rs2397142, whole-blood fatty acid composition, and performance in the d2 Test of Attention and a reading test were analyzed in multiple regression models including all SNPs, SNP-sex interactions, and covariates related to testing conditions.Results:FADS, rs1535 minor allele carriage associated with lower whole-blood arachidonic acid (P ≤ 0.002), and minor alleles of rs174448 tended to associate with lower docosahexaenoic acid (DHA) (P = 0.052). We identified sex interactions in 50% of the SNP performance sets. Sex-dependent associations were observed for rs174448 and rs1535 on the d2 Test of Attention outcomes (P < 0.03) and for the associations between reading scores and rs174448 and rs2397142 (P < 0.01). All of the sex-specific analyses showed associations in opposite directions in girls and boys. The minor allele carriage of rs174448 was associated with lower d2 Test of Attention performance (P < 0.02) and reading scores (P < 0.001) in boys but with better reading scores in girls (P ≤ 0.002). The associations were consistently the opposite for rs1535 minor allele carriage (P < 0.05). Associations with rs2397142 also appeared to be opposite of those of rs174448, but only for reading and not significant after adjustment for parental educational level and whole-blood DHA.Conclusions: This study showed associations between rs1535 minor allele homozygosity and rs174448 major allele carriage and improved performance in 8- to 11-y-old boys but not in girls, thereby counteracting existing sex differences. This may be a consequence of increased endogenous DHA synthesis in infancy but not at school-age. This trial was registered at clinicaltrials.gov as NCT01457794.

DHA Effects in Brain Development and Function.

Docosahexaenoic acid (DHA) is a structural constituent of membranes specifically in the central nervous system. Its accumulation in the fetal brain takes place mainly during the last trimester of pregnancy and continues at very high rates up to the end of the second year of life. Since the endogenous formation of DHA seems to be relatively low, DHA intake may contribute to optimal conditions for brain development. We performed a narrative review on research on the associations between DHA levels and brain development and function throughout the lifespan. Data from cell and animal studies justify the indication of DHA in relation to brain function for neuronal cell growth and differentiation as well as in relation to neuronal signaling. Most data from human studies concern the contribution of DHA to optimal visual acuity development. Accumulating data indicate that DHA may have effects on the brain in infancy, and recent studies indicate that the effect of DHA may depend on gender and genotype of genes involved in the endogenous synthesis of DHA. While DHA levels may affect early development, potential effects are also increasingly recognized during childhood and adult life, suggesting a role of DHA in cognitive decline and in relation to major psychiatric disorders.

Aberrant methylation of cell-free circulating DNA in plasma predicts poor outcome in diffuse large B cell lymphoma.

BACKGROUND: The prognostic value of aberrant DNA methylation of cell-free circulating DNA in plasma has not previously been evaluated in diffuse large B cell lymphoma (DLBCL). The aim of this study was to investigate if aberrant promoter DNA methylation can be detected in plasma from DLBCL patients and to evaluate this as a prognostic marker. Furthermore, we wanted to follow possible changes in methylation levels during treatment. Seventy-four patients were enrolled in the study, of which 59 received rituximab and CHOP-like chemotherapy. Plasma samples were collected from all patients at the time of diagnosis and from 14 healthy individuals used as controls. In addition, plasma samples were collected during and after treatment for surviving patients. In total, 158 plasma samples were analyzed for DNA methylation in the promoter regions of DAPK (DAPK1), DBC1, MIR34A, and MIR34B/C using pyrosequencing. RESULTS: Aberrant methylation levels at the time of diagnosis were detected in 19, 16, 8, and 10 % of the DLBCL plasma samples for DAPK1, DBC1, MIR34A, and MIR34B/C, respectively. DAPK1 methylation levels were significantly correlated with DBC1 and MIR34B/C methylation levels (P < 0.001). For the entire cohort, 5-year overall survival (OS) rates were significantly lower in the groups carrying aberrant DAPK1 (P = 0.004) and DBC1 (P = 0.044) methylation, respectively. DAPK1 methylation status were significantly correlated with stage (P = 0.015), as all patients with aberrant DAPK1 methylation were stages III and IV. Multivariate analysis identified DAPK1 as an independent prognostic factor for OS with a hazard ratio of 8.9 (95 % CI 2.7-29.3, P < 0.0007). Patients with DAPK1 methylated cell-free circulating DNA at time of diagnosis, who became long-term survivors, lost the aberrant methylation after treatment initiation. Conversely, patients that maintained or regained aberrant DAPK1 methylation died soon thereafter. CONCLUSIONS: Aberrant promoter methylation of cell-free circulating DNA can be detected in plasma from DLBCL patients and hold promise as an easily accessible marker for evaluating response to treatment and for prognostication. In particular, aberrant DAPK1 methylation in plasma was an independent prognostic marker that may also be used to assess treatment response.

Reduced ex vivo stimulated IL-6 response in infants randomized to fish oil from 9 to 18 months, especially among PPARG2 and COX2 wild types.

We investigated whether n-3 LCPUFA affected immune function in late infancy and explored effect-modification by single nucleotide polymorphisms (SNPs) and links to intestinal microbiota. Infants (n=105) were randomized to fish oil (FO, 1.2g/d n-3 LCPUFA) or sunflower oil (SO)-supplements from age 9-18 months. Immune function was assessed by ex vivo cytokine production in stimulated blood and plasma immunoglobulin E (IgE). We genotyped functional SNPs in PPARG2 and COX2 and analyzed fecal microbiota by 16S-rRNA terminal restriction fragment length polymorphism. FO compared to SO reduced Lactobacillus paracasei-stimulated IL-6 at 18 months (P=0.03, n=104). This effect was most pronounced among infants wild-type for PPARG2-Pro12Ala and/or COX2-T8473C (P<0.05). Predominant bacterial fragments were associated with 18 months IgE in all infants (P=0.004) (bp100) and with IL-6 production among infants weaned before 9 months (P=0.047) (bp102). Thus, FO reduced IL-6 in a genotype-modified manner. The microbiota was partly linked to IL-6 and IgE, not directly to FO.

Effects on metabolic markers are modified by PPARG2 and COX2 polymorphisms in infants randomized to fish oil.

Long-chain n-3 fatty acids (n-3 LCPUFA) improve blood pressure (BP) and lipid profile in adults and improve insulin sensitivity in rodents. We have previously shown that n-3 LCPUFA reduces BP and plasma triacylglycerol (TAG) in infants. Few studies have found effects on glucose homeostasis in humans. We explored possible effect modification by FADS, PPARG2, and COX2 genotypes to support potential effects of n-3 LCPUFA on metabolic markers in infants. Danish infants (133) were randomly allocated to daily supplementation with a teaspoon (~5 mL/day) of fish oil (FO) or sunflower oil (SO) from 9 to 18 months of age. Before and after the intervention, we assessed BP, erythrocyte n-3 LCPUFA, plasma lipid profile, insulin, and glucose in addition to functional single nucleotide polymorphisms in FADS, PPARG2, and COX2. At 18 months, plasma TAG was lower in the FO compared with SO group (p = 0.014). This effect was modified by PPARG2-Pro12Ala, as TAG only decreased among heterozygotes. FO supplemented PPARG2 Pro12Ala heterozygotes also had decreased plasma glucose compared with the SO group (p = 0.043). The effect of FO on mean arterial BP at 18 months was gender dependent (p = 0.020) and reduced in boys only (p = 0.028). Diastolic BP was, however, lower among all FO supplemented homozygous COX2-T8473C variant allele carriers compared with the SO group (p = 0.001). In conclusion, our results confirm that FO supplementation in late infancy reduces TAG and BP and indicates that the effects are mediated via peroxisome proliferator-activated receptor-γ and cyclooxygenase-2. Furthermore, FO reduced plasma glucose only in PPARG2 heterozygotes.

FADS single-nucleotide polymorphisms are associated with behavioral outcomes in children, and the effect varies between sexes and is dependent on PPAR genotype.

BACKGROUND: Docosahexaenoic acid (DHA), supplied by the diet or endogenous biosynthesis from α-linolenic acid, accretes during the perinatal brain growth spurt. Results regarding a potential programming effect on cognitive function and behavior in humans are inconclusive. OBJECTIVE: Here we aimed to investigate whether behavioral outcomes in childhood were associated with FADS tag-single-nucleotide polymorphisms (SNPs) previously found to have opposing effects on infant erythrocyte DHA. DESIGN: At 36 mo, we assessed psychomotor development with the third edition of the Ages & Stages Questionnaire (n = 256) and physical activity by accelerometry (n = 231) in children from the SKOT [Småbørns Kost Og Trivsel (Diet and Thriving in Young Children)] cohort. Blood samples were taken to determine erythrocyte DHA (n = 200), FADS tag-SNPs (n = 255), and PPARG-Pro12Ala (n = 255). All outcomes were analyzed in models, including all 3 SNPs, SNP-sex interactions, erythrocyte DHA at 36 mo, and covariates. RESULTS: As previously shown, the minor allele carriers of the FADS SNP rs1535 had increased erythrocyte DHA at 9 mo, whereas DHA decreased in minor allele carriers of rs174448 and rs174575 (effect size around 0.5 percentage points per allele). No overall effects were observed for any of the FADS SNPs on the outcomes reported here, but FADS SNP-sex interactions were found for a number of DHA-increasing FADS alleles on both communication and problem solving (P = 0.005 and 0.013). DHA-increasing FADS alleles resulted in reduced scores in girls and improved abilities in boys, with an effect size of ∼1 score-point/allele. No associations were found between current erythrocyte DHA and any of the behavioral outcomes. The P value for the triple interaction between DHA-increasing FADS alleles, PPARG, and sex for communication was 0.051, and subsequent analyses showed the FADS-sex interaction only in PPARG minor allele carriers (n = 70). Furthermore, FADS-PPARG interactions were seen for problem solving in boys and for fine motor skills in girls. CONCLUSION: FADS SNPs seem to have a sex-specific, possibly peroxisome proliferator-activated receptor-mediated effect on behavior in children, indicating a programming effect of early DHA exposure.

FADS genotype and diet are important determinants of DHA status: a cross-sectional study in Danish infants.

BACKGROUND: Infant docosahexaenoic acid (DHA) status is supported by the DHA content of breast milk and thus can decrease once complementary feeding begins. Furthermore, it is unclear to what extent endogenous DHA synthesis contributes to status. OBJECTIVE: We investigated several determinants, including FADS genotypes on DHA status at 9 mo and 3 y. DESIGN: This was a cross-sectional study with Danish infants from 2 prospective studies [Essentielle Fedtsyrer i OvergangskosteN (EFiON) and the Småbørns Kost Og Trivsel (SKOT) cohort] in which we measured red blood cell (RBC) DHA status at 9 mo (n = 409) and 3 y (n = 176) and genotyped 4 FADS tag single nucleotide polymorphisms (SNPs): rs3834458, rs1535, rs174575, and rs174448 (n = 401). Information about breastfeeding was obtained by using questionnaires, and fish intake was assessed by using 7-d precoded food diaries. RESULTS: FADS genotype, breastfeeding, and fish intake explained 25% of the variation in infant RBC DHA status [mean ± SD: 6.6 ± 1.9% of fatty acids (FA%)]. Breastfeeding explained most of the variation (∼20%), and still being breastfed at 9 mo was associated with a 0.7 FA% higher DHA compared with no longer being breastfed (P < 0.001). The FADS SNPs rs1535 and rs3834458 were highly correlated (r = 0.98). Homozygous carriers of the minor allele of rs1535 had a DHA increase of 1.8 FA% (P = 0.001) relative to those with the wild-type allele, whereas minor allele carriers of rs174448 and rs174575 had a decrease of 1.1 FA% (P = 0.005) and 2.0 FA% (P = 0.001), respectively. Each 10-g increment in fish intake was associated with an increased DHA status of 0.3 FA%. At 3 y, fish intake was the only significant determinant of DHA status (0.2 FA%/10 g). CONCLUSION: Breastfeeding, FADS genotype, and fish intake are important determinants of DHA status in late infancy. The EFiON study was registered at clinicaltrials.gov as NCT 00631046.