Liquid biopsies are providing new opportunities for detection of residual disease in cell-free DNA (cfDNA) after surgery but may be confounded through identification of alterations arising from clonal hematopoiesis. Here, we identify circulating tumor-derived DNA (ctDNA) alterations through ultrasensitive targeted sequencing analyses of matched cfDNA and white blood cells from the same patient. We apply this approach to analyze samples from patients in the CRITICS trial, a phase III randomized controlled study of perioperative treatment in patients with operable gastric cancer. After filtering alterations from matched white blood cells, the presence of ctDNA predicts recurrence when analyzed within nine weeks after preoperative treatment and after surgery in patients eligible for multimodal treatment. These analyses provide a facile method for distinguishing ctDNA from other cfDNA alterations and highlight the utility of ctDNA as a predictive biomarker of patient outcome to perioperative cancer therapy and surgical resection in patients with gastric cancer. Identifying patients that will respond to a particular therapy remains a key challenge in precision oncology. Here, in gastric cancer, the authors show that circulating tumour DNA can predict recurrence, provided that the signal from white blood cells is filtered out. Currently available imaging techniques and traditional blood biomarkers to capture minimal residual disease (MRD) state after surgery have poor sensitivity and do not play a role in clinical practice5. Histopathological assessment of the effects of neoadjuvant chemotherapy on resection specimens has become an important tool to provide prognostic information8. However, microscopic residual tumor, lymph node infiltration, and poor histopathological response do not measure the real-time presence of residual disease. More recent approaches such as detection of circulating tumor-derived DNA (ctDNA) through liquid biopsies may provide new opportunities for identifying patients that would benefit from adjuvant treatment options and further follow-up12.A major challenge after multimodal curative treatment for resectable gastric cancer is identifying patients with microscopic residual disease at high risk of recurrence after surgery14. Cell-free ctDNA is released from tumor cells into the circulation and has been detected in patients with early- and late-stage cancers19. A key challenge of liquid biopsy approaches has been developing methods to detect and characterize small amounts of ctDNA in large populations of cell-free DNA (cfDNA). A variety of studies have focused on changes in ctDNA during the course of therapy, but mostly in the setting of metastatic disease and largely centered on the analysis of a limited number of genomic positions that may only represent a subset of clones of the tumor28. More recent studies have used panels of commonly mutated driver genes to allow detection of multiple driver clones, typically at the time of diagnosis, after surgery, or at disease progression34. However, blood-based deep sequencing approaches have raised concerns about detection and misclassification of white blood cell (WBC)-derived variants in cfDNA associated with clonal hematopoiesis of indeterminate potential37. Such studies have typically assessed WBC DNA and cfDNA in patients with cancer at a single timepoint40 or in healthy individuals41, and did not attempt to evaluate these during the course of therapy to predict clinical outcome.Theoretically, the ability to non-invasively detect tumor-specific alterations in the circulation after neoadjuvant chemotherapy and surgery has the potential to rapidly and dynamically inform the presence of MRD or preclinical metastasesIn the present study, we apply a matched cfDNA and WBC sequencing approach to accurately detect ctDNA alterations after preoperative chemotherapy and after surgery in patients with resectable gastric cancer. We hypothesize that ctDNA detection after completion of preoperative treatment as well as MRD detection after surgery can predict recurrence and survival in patients with resectable gastric cancer treated with multimodal therapeutic regimens. Overall, these analyses evaluate a strategy to distinguish ctDNA alterations from cfDNA variants related to clonal hematopoiesis and investigate whether ctDNA elimination before or after surgery can serve as a predictive biomarker of patient outcome to perioperative treatment.NCT00407186), an investigator-initiated, open-label, multi-center, phase III randomized controlled trial of perioperative chemotherapy (chemotherapy group) vs. preoperative chemotherapy with postoperative chemoradiotherapy (chemoradiotherapy group) for patients with resectable gastric cancer42. Between 11 January 2007 and 17 April 2015, a total of 788 patients from 56 hospitals in the Netherlands, Sweden, and Denmark were randomized upfront to receive three preoperative 21-day cycles of intravenous epirubicin, cisplatin or oxaliplatin, and oral capecitabine followed by three postoperative cycles of intravenous epirubicin, cisplatin or oxaliplatin, and oral capecitabine (chemotherapy group) or to receive the same preoperative regimen followed by radiation combined with daily capecitabine and weekly cisplatin (chemoradiotherapy group).The current study was an exploratory analysis of the predictive value of ctDNA assessment in a subset of patients from the CRITICS (ChemoRadiotherapy after Induction chemoTherapy In Cancer of the Stomach) study  were enrolled in one of the two arms of the study and received three cycles of preoperative chemotherapy; (ii) had plasma available from at least two consecutive timepoints processed within 24 h after blood collection for cfDNA analyses; (iii) had buffy coat or whole blood at baseline for WBC analysis; and (iv) had clinical follow-up longer than 18 months if they were free of disease after therapy  of DNA fragments to identify single base substitutions and small insertions or deletions in cfDNA across 80,930 bp of coding gene regions while distinguishing these from PCR amplification and sequencing artifacts18. For alterations detected in cfDNA that were not identified by matched WBC sequencing, we determined the probability that such an alteration was tumor derived from a Bayesian statistical model using the frequency of altered alleles and total coverage of cfDNA and WBC sequences (see Methods).For each patient, plasma and buffy coat were collected at the time of trial enrollment (baseline timepoint), after patients received three cycles of preoperative chemotherapy (preoperative timepoint), and after surgery but before the initiation of the adjuvant treatment (postoperative timepoint) Fig.  and 2. W43 with alterations in one or more of the 58 analyzed genes. These analyses showed that our targeted panel would have a sensitivity of ~88% as 384 of 436 gastric cancer cases had at least one alteration in these genes   , MYC (15%), PIK3CA (15%), KRAS (11%), HRAS (11%), BRAF (11%), ALK (11%), ATM (11%), KIT (11%), and CDH1 (7%)  of PIK3CA mutations in ctDNA of patients with Epstein–Barr virus (EBV)-positive (n = 3) or MSI-high tumors (n = 2) compared to the frequency (3%) in EBV-negative (n = 36) and MSS tumors (n = 37).We evaluated cfDNA in all 50 patients at baseline and after three cycles of preoperative chemotherapy. At baseline, we detected sequence alterations in cfDNA from 40 patients 80%) Fig.  and in W0% Fig.  7%) Fig. . In accoDNMT3A (45%), TP53 (29%), EGFR (10%), APC (6%), AR (6%), ATM (6%), and MLH1 (6%)    Fig. . Functio%), EGFR %, APC (6st) Fig. . We obsest) Fig. . As expeon) Fig. . InteresTP53 in cfDNA, including 17 missense mutations, two nonsense mutations, one in-frame deletion, and one splice site mutation  as well as four missense mutations . We further evaluated fragment length distributions of the 21 TP53 alterations detected in cfDNA. We observed that fragments harboring tumor-specific TP53 mutations in the circulation were significantly shorter than fragments harboring TP53 variants associated with clonal hematopoiesis , as well as fragments harboring wild-type TP53 coding regions    in the specimen obtained at the time of surgery   and four achieved a major pathological response, exhibiting fibrotic surgical specimens with scattered tumor cells (TRG 2). All seven responders (100%) had no ctDNA detected at the preoperative timepoint Fig. . Of the st) Fig. .p = 0.03, Fisher’s exact test)    = 3.0; 95% confidence interval (CI) = 1.3–6.9)   Fig. , at leasst) Fig. , and detst) Fig. . TRG scost) Fig. . It is nst) Fig.  or deathst) Fig. . However.9) Fig.  as well BRAF G469A and KRAS G13R, respectively    Fig. . Disease.1) Fig. .4. Despite the value of multimodal curative treatment approaches, a significant fraction of patients will eventually perish as a consequence of locoregional relapse, peritoneal recurrence, or distant metastases3. Current methods to estimate the risk of disease recurrence after surgery mostly rely on the assessment of pathological staging and microscopic residual disease score systems8. However, there are several limitations with these approaches, especially with tumor regression grading scales, which make their implementation difficult in daily clinical practice8. Furthermore, the poor sensitivity of currently available imaging methods and blood protein biomarkers to detect remaining disease after curative surgery has provided an opportunity for ctDNA analyses for MRD assessment in gastric cancer. There is an urgent clinical need to select patients who need adjuvant treatment because of the presence of MRD. Here, we have developed a tissue-independent sequencing approach using ultrasensitive sequencing of matched cfDNA and WBCs to detect tumor-specific mutations in cfDNA after completion of preoperative chemotherapy as well as after surgery in patients with resectable gastric cancer.High mortality rates associated with gastric cancer reflect the prevalence of advanced disease at presentation, when treatment options are limited48, these efforts have used a tumor-guided approach to identify potential alterations. An advantage of our approach is the identification of ctDNA without requiring tumor tissue, which may be unobtainable or available to a limited extent and where sequencing analyses may be hampered by intra-tumoral heterogeneity.In this study, we investigated the value of parallel deep sequencing of cfDNA and WBCs to detect cfDNA alterations associated with clonal hematopoiesis and used this approach to longitudinally infer bona fide tumor-specific alterations independent of matched tumor analyses. Although ctDNA analyses for detection of MRD have been utilized for a variety of solid tumors49, a major challenge for the development of MRD assays using noninvasive liquid biopsies is distinguishing tumor-specific mutations from background changes associated with biological variation. WBC-derived alterations that arise as a consequence of clonal hematopoiesis may confound liquid biopsy analyses that are based on characterization of cfDNA as these may occur in common cancer driver genes41. As we have shown here, cfDNA analyses without parallel analyses of WBC DNA would have been unable to appropriately identify patients that benefit from perioperative treatment in terms of event-free and overall survival.As the majority of cfDNA typically arises from hematopoietic cells51 and we did not assess ctDNA levels after adjuvant therapy. Future studies will address the value of post-adjuvant ctDNA status as a surrogate marker for adjuvant treatment efficacy. Tumor-guided personalized approaches may improve the accuracy of such methods32, but have the disadvantage of requiring tumor tissue and are challenging to implement as widely available standardized tests. Although our approach can be improved in the future with targeted panels specifically designed for gastric cancer and incorporating fragmentation analyses of cfDNA19, our efforts provide a proof of principle of the utility of ctDNA analyses in patients receiving perioperative treatment and deserves further validation in randomized interventional trials.However, there are some limitations when using these approaches to noninvasively detect gastric cancer, especially in patients with localized disease. In this study, although we analyzed a relatively small number of patients with available plasma samples, the extensive follow-up time of the CRITICS trial allowed for accurate determination of clinical recurrences. Despite the fact that the panel we utilized was not developed for gastric cancer we detected the majority of patients at the baseline timepoint. Of the patients not detected at baseline, 61% had diffuse subtype, highlighting potential differences in DNA shedding associated with this histological feature. Some patients did not recur despite positive ctDNA results after surgery, but this observation is consistent with the fact that a fraction of patients receiving adjuvant therapy would be expected to be cured of diseaseNCT00407186) who had plasma samples available and suitable for genomic analyses from at least two timepoints  or to receive the same preoperative regimen followed by postoperative radiotherapy combined with daily capecitabine and weekly cisplatin (chemoradiotherapy group)  Fig. . Tumor-s52, as assessed by esophagogastroduodenoscopy and CT of the chest, abdomen, and pelvis. Patients with tumors of the gastroesophageal junction were permitted to enroll when the bulk of the tumor was predominantly located in the stomach and could therefore consist of Siewert types II  and III  tumors. Patients with Siewert type I  tumors were not eligible. An exploratory laparoscopy was indicated when the preoperative CT scan suggested peritoneal carcinomatosis. Patient enrollment and genomic studies were conducted in accordance with the Declaration of Helsinki. The study was approved by the medical ethical committee of the Netherlands Cancer Institute and by the review boards of all participating centers. All patients provided oral and written informed consent for sample acquisition for research purposes.Patients were eligible for the study if they had histologically proven gastric adenocarcinoma , stage IB–IVA53. Histopathological regression was determined by NCTvG according to Mandard’s TRG system: (i) TRG 1, no residual tumor left ; (ii) TRG 2, scattered tumor cells left; (iii) TRG 3, fibrosis outgrows tumor; (iv) TRG4, tumor outgrows fibrosis; and (v) TRG 5, no histological signs of regression  according to the manufacturer’s instructions. PCR products were separated by capillary electrophoresis using an ABI 3500 Genetic Analyzer , and analyzed using the GeneMapper software . An internal lane size standard was added to the PCR samples for accurate sizing of alleles and to adjust for run-to run variations. When all markers were stable, the tumor was interpreted as microsatellite stable (MSS). The tumor was interpreted as MSI-low (MSI-L) if one marker was unstable and MSI-high (MSI-H) if two or more markers showed instability. MSI-L tumors were included in the MSS category  consisting of five nearly monomorphic mononucleotide markers  and eluted in LoBind tubes (Eppendorf AG). High-molecular weight DNA from WBCs was extracted using the Qiagen DNA Blood Mini Kit (Qiagen GmbH), followed by shearing using a focused-ultrasonicator (Covaris). Concentration and quality of cfDNA was assessed using the Bioanalyzer 2100 (Agilent Technologies). cfDNA samples with high-molecular weight DNA were excluded from the study.Whole blood was collected in K18. Briefly, the NEBNext DNA Library Prep Kit for Illumina [New England Biolabs (NEB)] was used with four main modifications to the manufacturer’s guidelines: (i) the library purification steps utilized the on-bead Ampure XP approach, (ii) reagent volumes were adjusted accordingly to accommodate the on-bead strategy, (iii) a pool of eight unique Illumina dual index adapters with 8 bp barcodes were used in the ligation reaction, and (iv) cfDNA libraries were amplified with HotStart Phusion Polymerase. Genomic library preparation was performed as previously described18. Concentration and quality of cfDNA genomic libraries were assessed using the Bioanalyzer 2100 (Agilent Technologies).Next-generation sequencing libraries from cfDNA and sheared high-molecular weight DNA from WBCs were prepared from 8.4 to 250 ng  software (version 1.8) was used for demultiplexing and masking of dual index adapter sequences. Sequence reads were aligned against the human reference genome (hg19) using NovoAlign with additional realignment of select regions using the Needleman–Wunsch method55.Primary processing of next-generation sequence data for analyses of sequence alterations in cfDNA and WBC samples were performed as previously described55  across the targeted regions of interest as previously described18. Briefly, an alteration was considered a candidate somatic mutation only when: (i) three distinct paired reads contained the mutation in the cfDNA and the number of distinct paired reads containing a particular mutation in the plasma was at least 0.05% of the total distinct read pairs; or (ii) one distinct paired read contained the mutation in the cfDNA and the mutation had also been detected in at least one additional timepoint at the level specified in (i); (iii) the mismatched base or small indel was not identified in matched WBC sequencing data of samples collected at baseline at the level of one distinct read  was the same as the Bayes factor and we obtained the probability that the mutation was tumor derived by PO/(1 + PO). We performed this analysis for each mutation that was identified only by cfDNA sequencing.For mutations identified by cfDNA sequencing but not identified by WBC sequencing, we computed the probability that the mutation was tumor derived relative to the probability that the mutation was hematopoietic. The sampling distribution of the observed number of reads with an altered mutation in cfDNA and WBC sequencing is a binomial parameterized by the total coverage at that mutation and unknown probability χ2 tests. Visualizations of groups of mutations were carried out in R using the package maftools56. Pearson’s correlation coefficients were determined for the association between WBC variants and their correspondent alterations identified in cfDNA, as well as for the association between the number of WBC variants and age. Differences in survival between two groups were evaluated using log-rank tests and implemented in R using the survival package. All hypothesis tests were two sided.Statistical significance was determined using a variety of methods. Wilcoxon’s rank-sum test or Kruskal–Wallis test were performed for two- and three-way comparisons of continuous variables. Independence of categorical variables was evaluated by Fisher’s exact test and Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Supplementary Data 7Supplementary Data 8Supplementary Data 9Supplementary Data 10Reporting Summary


OXTR and AVPR1A, respectively. To explore this hypothesis we examined the expression of both genes in peripheral blood mononuclear cells (PBMC) from ASD children, their non-ASD siblings, and age-matched neurotypical children aged 3 to 16 years of age as well as datamined published ASD datasets. Both genes were found to have large inter-individual variations. Higher OXTR and AVPR1A expression was associated with lower Aberrant Behavior Checklist (ABC) scores. OXTR expression was associated with less severe behavior and higher adaptive behavior on additional standardized measures. Combining the sum expression levels OXTR, AVPR1A, and IGF1 yielded the strongest correlation with ABC scores. We propose that future clinical trials in ASD children with oxytocin, oxytocin mimetics and additional tentative therapeutics should assess the prognostic value of their PBMC mRNA expression of OXTR, AVPR1A, and IGF1.The peptide hormone oxytocin is an established regulator of social function in mammals, and dysregulated oxytocin signaling is implicated in autism spectrum disorder (ASD). Several clinical trials examining the effects of intranasal oxytocin for improving social and behavioral function in ASD have had mixed or inclusive outcomes. The heterogeneity in clinical trials outcomes may reflect large inter-individual expression variations of the oxytocin and/or vasopressin receptor genes  Intranasal oxytocin was shown to improve social function and empathy in healthy individuals6, and is thus a candidate therapeutic for children with ASD.The peptide hormone oxytocin is strongly implicated in social behavior and is conserved in mammals. Oxytocin has been extensively studied in the context of social function in animals and humans, in particular in children and adults with autism spectrum disorder (ASD)OXTR alleles, promoter methylation, and/or expression levels in brain areas implicated in social function may contribute to the variable response observed in clinical trials with intranasal oxytocin in ASD8. In addition, differences between and within animal species in sociality may in part be explained by substantial differences in OXTR expression levels within the social brain network, such as the amygdala, the anterior cingulate cortex, the prefrontal cortex and the nucleus accumbens9. In primates, OXTR is also expressed in the superior colliculus, the pulvinar, and the primary visual cortex, where oxytocin functions as a modulator of visual processing and allocation of attention10. Thus, variations in OXTR expression seem critical for the diversity of social behaviors across and within mammal and in particular primate species, including humans. Indeed, inter-individual variations in OXTR expression levels were shown to be associated with resilience to the effects of neonatal isolation on adult social attachment in female prairie voles11.It has been suggested that genetic variations of the 22. A follow-up study23 of one of these published clinical trials12 reported correlations of treatment efficacy with the OXTR single nucleotide polymorphisms (SNPs) rs53576 and rs2254298; another oxytocin trial reported efficacy correlations with OXTR rs679161913. The large variation in these clinical trials could in-part reflect genetic or epigenetic variations in the expression levels of the oxytocin receptor gene, OXTR. In addition to oxytocin, the closely similar peptide hormone arginine-vasopressin is also implicated in social function via its V1AR receptor, encoded by AVPR1A27. Another peptide hormone studied as a tentative ASD therapeutic is insulin-like growth factor 1 (IGF-1), encoded in humans by IGF1. A single small study with recombinant human IGF-1 reported favorable response in ASD children28 and another study (NCT01970345) is ongoing.Several clinical trials have studied intranasal oxytocin as a tentative ASD therapeutic for improving social and behavioral function in children, adolescents, or adults diagnosed with ASD, albeit with mixed outcomesOXTR, AVPR1A and IGF1 genes could explain the variation in response to these therapeutic agents in clinical trials. To explore this hypothesis we examined OXTR, AVPR1A and IGF1 expression levels in peripheral blood mononuclear cells (PBMC) samples from 63 ASD children, their non-ASD siblings, and matched neurotypical children aged 3–16 years  datasets for exploring OXTR and AVPR1A expression variations in blood and postmortem brain samples from ASD and matched controls, respectively. Our findings suggest that PBMC expression levels of OXTR, AVPR1A and IGF1 correlate with several standardized measures of behavior and development, including the Aberrant Behavior Checklist (ABC), Vineland Adaptive Behavior Scale (VABS), Social Responsiveness Scale (SRS), and Childhood Behavior Checklist (CBCL). Based on our findings we suggest that the PBMC expression levels of these genes should be evaluated as tentative prognostic biomarkers by clinical trials with oxytocin or other tentative ASD therapeutics.We hypothesize that variations in 25, we searched for expression differences of the genes which code for their receptors, OXTR and AVPR1A, in PBMCs collected from the study participants. No statistically significant differences were found for the PBMC mRNA expression levels of OXTR or AVPR1A between ASD children, their neurotypical siblings, and/or neurotypical age-matched children . We found that higher OXTR expression levels correlated with i) better development as measured by the VABS Adaptive behavioral composite, ii) less severe social impairment as measured by the SRS total t-score and iii) less severe behavior problems as measured by the lower total raw ABC scores and lower total CBCL t-scores .Next, we searched for correlations between PBMC expression levels of res Fig. . The PBMres Fig. . Notablyr 1 Fig. .Figure 236 and as the ABC scores of the ASD children correlated with the OXTR, AVPR1A, and IGF1 expression levels, we determined whether combining the expression of these genes affords a better correlation with ABC scores as compared to correlation with individual gene expression. As shown in Fig. OXTR, AVPR1A, and IGF1 expression levels with ABC scores, we randomly selected 30 individuals from our dataset. For each of 500 random selections we determined the Pearson’s correlation coefficient and p-value of the correlation between ABC score and the summed gene expression. We found that 410 (82%) of these random subsets yielded a significant correlation  between ABC score and summed gene expression (mean Spearman’s correlation coefficient: 0.47). This is compared to 25 (5%) significant subsets expected by chance. This result demonstrates that the observed correlation is robust and is not underlined by specific outlier individuals.Since combining the expression levels of several genes may afford more robust diagnostic or prognostic power38, and oxytocin receptor knockout mice display behavioral deficits resembling autism-related behaviors39, such mouse models typically reflect a single mutation or deletion, and do not reflect the real-world state and wide spectrum of ASD individuals. Our study aimed to explore correlations between development and behavior in children with ASD and PBMC expression levels of the oxytocin and vasopressin receptor genes OXTR or AVPR1A, respectively.Dysfunctional signaling by the peptide hormones oxytocin and vasopressin were suggested as contributing to ASD. While the role of these hormones has been extensively studied in several mouse models of ASDOXTR , as well as with AVPR1A and IGF1 (ABC score). The PBMC expression of all three genes therefore seem to be associated with the severity of aberrant behavior in children with ASD.Our findings indicate that the severity of developmental and behavioral deficits in children with ASD aged 3 to 16 years is associated with their PBMC mRNA expression levels of 22 (Table Our literature search (PubMed search of May 2019) identified 11 completed and published clinical trials  of intranasal oxytocin prescribed for at least 5 days and including at least 10 ASD participants who received oxytocin22 examined the OXTR expression levels, and reported lack of correlation of its expression with the effect of intranasal oxytocin on behavioral scores of children diagnosed with ASD. However, this trial measured OXTR expression in whole blood and compared it with the participants Social Responsiveness Scale (SRS) Total Raw Score (while we studied PBMC mRNA and report correlations with ABC scores).Of the 11 trials listed in Table OXTR SNPs on the efficacy of intranasal oxytocin13; both studies applied a placebo-controlled crossover design in adolescents and adults with ASD. Watanabe et al.12 reported that intranasal oxytocin has a smaller effect for participants carrying OXTR rs53576 or rs2254298; while Kosaka et al.13 reported lower efficacy for those carrying OXTR rs6791619. Of note, all three of these OXTR SNPs are intronic. We searched for the effects of these OXTR SNPs on its expression in datasets derived from studies with postmortem brain tissues  deposited in the UK Brain Expression Consortium  by the MRC Sudden Death Brain and Tissue Bank40. Our datamining showed that postmortem brain tissues from individuals carrying the minor (A) allele rs53576 allele had significantly higher OXTR expression levels in several brain regions, including frontal cortex, compared with tissues from GG homozygous  in the OXTR rs53576 minor allele carriers, suggest that higher OXTR expression levels may correlate with improved efficacy of intranasal oxytocin in ASD individuals.Among the studies listed in Table OXTR SNPs, OXTR mRNA levels seem to be more informative of ASD severity: besides DNA methylation and additional epigenetic modifiers of ASD severity45, they may inform on effects by other non-heritable effectors, such as the gut microbiome, suggested to affect ASD47. Based on our findings, we suggest that higher peripheral OXTR expression as detected in PBMCs may reflect differential brain expression and allow improved response to prescribed oxytocin and/or future oxytocin derivatives/agonists. The same considerations may apply also for the prognostic value of higher expression levels of AVPR1A and IGF1 . Parents of participants provided written informed consent. All methods were performed in accordance with the relevant guidelines and regulations. Children underwent a fasting blood draw in the morning. Control individuals did not have any neurological disorders or developmental delays. Thus, this study reports gene expression and social and behavioral measurements on a total of 63 children, including 43 children with ASD, 11 non-ASD siblings, and 9 unrelated neurotypical control children. This was a subset of the larger cohort of our study of mitochondrial function in children with ASD due to limited availability of PBMC for gene expression analysis. Our recent publications outline the methodology for rating participant characteristics of this participant cohort53, our research staff was trained by a multispecialty team consisting of two licensed psychologists and a speech therapist prior to performing assessments. During the study a research psychologist supervised research staff and provided feedback and retraining if necessary. Observer rated measures included the VABS Survey Interview Form. Parents completed the ABC and the SRS. The VABS is a reliable and valid measure of the ability to perform age-appropriate everyday skills, including communication, daily living, social and motor skills, through a 20–30 minute structured interview with a caretaker54. Of note, functional abilities of children with autism are commonly measured with the VABS55 as it was in this study. Although some have used IQ to distinguish high and low functioning autism, recent studies indicate that it is a poor predictor56. The ABC is a 58-item questionnaire53 that measures disruptive behaviors and has convergent and divergent validity57. The SRS is a 65-item questionnaire that measures the severity of social skill deficits across five domains58 which has been shown to have good correspondence to the gold-standard instrument59. The CBCL includes demographic information and ratings of positive behaviors, academic functioning, social competence, and behavior problems commonly applied for classification of behavior disorders60.As mentioned in our previous study+2/Mg+2-free PBS with 0.1% BSA and 2 mM ethylenediaminetetraacetic acid. Diluted blood was then layered on top of Histopaque-1077  and centrifuged at 400 g for 30 minutes at room temperature. PBMCs were collected, washed twice with wash buffer and counted using a hemocytometer.Samples of 4 ml of venous blood were collected into an ethylenediaminetetraacetic acid-Vacutainer tube, chilled on ice and centrifuged at 1500 g for 10 minutes at 4 °C to separate plasma. Plasma was removed and stored at −80 °C for later analysis. Plasma was replaced with room temperature wash buffer containing CaTotal RNA was extracted using RNeasy mini kit  from 5 million PBMCs by following manufacturer provided protocol. RNA samples were shipped in 100% ethanol at room temperature to Tel-Aviv University. Upon arrival to Tel-Aviv, 10% 3 M Sodium Acetate and 1ul GlycoBlue™  were added and kept overnight at −80 °C. Samples were centrifuged at high speed  for 30 minutes. Pellets were dried and dissolved in 20ul DNAse/RNAse-free water.RPLP0  was used as reference gene.Real-time quantitative PCR (qPCR) reactions were performed with cDNA samples prepared from 1 μg RNA samples using qScript cDNA Synthesis Kit . Reverse transcription was performed using a thermal cycler over three steps . Real-time PCR reactions were done with 10 μl mixtures containing 10 ng of cDNA, PerfeCTa SYBR® Green FastMix Kit  and Integrated DNA Technologies, Inc.  primers (shown below). OXTR and AVPR1A using the GEO2R tool on the NCBI server (https://www.ncbi.nlm.nih.gov/geo/geo2r/). OXTR rs53576 associated brain expression levels were downloaded from the Brain eQTL Almanac website (http://www.braineac.org/). The expression levels corresponding to each OXTR rs53576 genotype  were plotted for six brain regions .The NCBI Gene Expression Omnibus (GEO) was queried for expression data sets derived from ASD blood or PBMC samples containing cohorts of at least 30 ASD children or adults and a similarly sized age and sex matched control cohort. Data sets from human cell lines, postmortem tissues, and mouse ASD models were excluded. The identified data sets were queried for the expression levels of 61. R statistical software was used to test the robustness of correlations between the sum expression levels of OXTR, AVPR1A, and IGF1 and the ABC scores . Normality of data distribution was evaluated using the Shapiro-Wilk test; continuous variables between two groups were analyzed by Student’s t-test; outliers were detected by Grubbs test. Continuous variables between three groups were analyzed by one-way ANOVA test. P-values ≤ 0.05 were considered as significant. Data for correlation analysis were log transformed and Spearman correlation test was performed. P-values ≤ 0.01 were considered significant. P values were adjusted for multiple testing using Benjamini-Hochberg FDR correctionres Fig. . A totalSupplementary figures


The sequence begins with a preparatory stage for measuring relaxation times, and is followed by a multiple pulse sequence for homonuclear dipolar decoupling. Data analysis involves the use of Fourier transform, followed by a one-dimensional inverse Laplace transform for each frequency index. Experimental results acquired on solid samples demonstrate the general approach, and additional variations involving heteronuclear decoupling and magic angle spinning are discussed.A two-dimensional pulse sequence is introduced for correlating nuclear magnetic resonance anisotropic chemical shifts to a relaxation time (e.g., T One 1D) and two dimensional 2D) inverse Laplace transforms (ILTs) have facilitated the characterization of exponential decays in a wide range of diffusion or relaxation nuclear magnetic resonance (NMR) experiments [D and twoD inverseThe one field of applications where such approaches may still be considered nascent are for the characterization of constituents and phases in amorphous and disordered solids. Current approaches to obtaining high resolution chemical shift information of solids in high field laboratory systems often make use of fast magic-angle spinning, hetero- and/or homo-nuclear multiple pulse methods or combinations of these approaches, by selectively averaging a systems’ nuclear spin Hamiltonian(s) in physical and/or spin space . In portIn this work we introduce a new methodology for correlating anisotropic chemical shifts to relaxation times in a 2D experiment of static solids. Homonuclear dipolar decoupling based on multiple pulse methods is introduced together with Laplace inversion of the relaxation data. This 2D pulse sequence is termed static solid relaxation ordered spectroscopy, SS-ROSY. A homonuclear decoupled spectrum is acquired as a function of a relaxation delay . A one-dimensional ILT ,2 is perThe relaxation–chemical shift dispersion experiment is shown in sequence . The SMEsequence ).R. The function G represents the kernel for the relaxation process S. The generalized approach makes use of Fourier transform of the data, followed by a 1D ILT for every frequency index. Experimental results are shown on a single crystal of CaF


Circulating tumor cells (CTCs) have become an established biomarker for prognosis in patients with various carcinomas. However, current ex vivo CTC isolation technologies rely on small blood volumes from a single venipuncture limiting the number of captured CTCs. This produces statistical variability and inaccurate reflection of tumor cell heterogeneity. Here, we describe an in vivo indwelling intravascular aphaeretic CTC isolation system to continuously collect CTCs directly from a peripheral vein. The system returns the remaining blood products after CTC enrichment, permitting interrogation of larger blood volumes than classic phlebotomy specimens over a prolonged period of time. The system is validated in canine models showing capability to screen 1–2% of the entire blood over 2 h. Our result shows substantial increase in CTC capture, compared with serial blood draws. This technology could potentially be used to analyze large number of CTCs to facilitate translation of analytical information into future clinical decisions. Ex vivo methods of circulating tumor cell (CTC) isolation use small blood volumes, limiting sensitivity and introducing analytical inaccuracies. The authors describe a proof-of-concept study of an in vivo aphaeresis system that continuously collects CTCs from a peripheral vein over several hours. Although identified more than 150 years ago2, until recently, CTCs were difficult to detect, enumerate, and characterize. Using modern technologies, several studies have now demonstrated that elevated levels of CTC isolated from a single blood draw are prognostic in patients with metastatic breast, colorectal, prostate, and lung cancers, as well as early stage breast and prostate cancers8. Furthermore, CTC analysis holds promise for predicting benefit from targeted therapies, pharmacodynamic monitoring during treatment, and insight into the biology of metastases10. Indeed, CTC evaluation might be used for early detection of malignancy, if an assay with sufficient sensitivity and specificity could be developed.Cancer metastases arise from circulating tumor cells (CTCs) that are shed from the primary tumor and circulate through lymphatic channels and blood12. However, CTC isolation using these technologies is limited to small blood volumes  due to patient safety concerns, and therefore the absolute number of CTC is small. Moreover, a single blood draw interrogates only those CTC present at the time of venipuncture, and does not take into account temporal differences in CTC release into the circulation. The ability to interrogate larger blood volumes over extended periods of time might enhance the number of CTC available for enumeration, which would therefore increase statistical confidence of sampling for comparison of serial levels14. This approach would also provide more CTCs for molecular phenotyping, genotyping, and further biological characterization.CTCs are extremely rare events. For example, in a single 7.5 mL tube of whole blood drawn from an average patient with metastatic breast cancer, it is unusual to identify more than 10 CTCs within the context of billions of erythrocytes and millions of leukocytes normally present. More than a hundred ex vivo CTC capture devices have been developed to enrich and isolate CTC from whole blood15. However, the accessibility to these sources is limited according to the location of the tumor, and this approach is not practical for routine diagnostic use. Furthermore, despite the considerable number of CTCs detected in samples from the tumor draining vessels, many cells that are disrupted during surgery rapidly undergo apoptosis16, and their biological and clinical impact is unknown.Attempts to increase the volume of blood evaluated for CTC isolation have included using alternative sites of blood collection, including the vessels draining primary cancers accessed at the time of surgery19. Although this strategy enables a substantial increase in detecting CTCs compared with a single blood draw, standard cytopheresis is cumbersome and inconvenient for the patient. Furthermore, cytopheresis products mainly consist of concentrated peripheral blood mononuclear cells, which require an additional high throughput screening step for CTC identification. As with cannulating tumor-draining vasculature, the logistics required for standard leukapheresis/cytopheresis render this approach impractical as a standard diagnostic test, especially for application in a serial fashion.Other investigators have reported isolating CTC in cytopheresis products, either from whole blood or bone marrow, often collected in anticipation of hematopoeitic stem cell transplantation therapy25. However, physiologic variations between patients affecting blood flow and affinity make it difficult to standardize quantitative interpretation of CTCs by time of insertion. Similarly, a recent study has demonstrated in vivo capture of non-small cell lung cancer cells injected into a porcine model, using a flexible magnetic wire (MagWIRE)26. However, the approach requires pre-injection of EpCAM coated magnetic particles to label CTCs which limits its long-term application due to possible systemic exposure of iron overload.Investigators have also reported use of an intravenous gold-coated stainless steel medical wire with a hydrogel layer covalently coupled with antibodies against epithelial cellular adhesion molecule (EpCAM) protein (GILUPI CellCollector)To address the shortcomings of current approaches, we developed a temporary indwelling, intravascular aphaeretic CTC isolation system that can potentially be worn by a patient for several hours and through which a relatively large volume of blood can be interrogated. We demonstrate the feasibility of this device in ex vivo experiments, and we have performed proof-of-principle investigations of its potential clinical application for in vivo CTC capture in a canine model.The overall design of the system is illustrated in Fig. Whole blood is routed into the system from a subject’s peripheral vein with a single cannulation using a dual-lumen catheter Fig.  via the HBGO chip) is designed using functional graphene oxide sheets for sensitive capture and chaotic mixing via herringbone structures for enhanced throughput. It consists of a 24.5 × 60 mm silicon dioxide substrate with patterned gold thin film layer bonded to a polydimethylsiloxane (PDMS) structure containing four bifurcating microchannels with herringbone grooves embedded on their top surface 28 previously reported was used for comparison , 5, 15, 30, 60, and 120 min following injection. The sampled whole blood was then processed through the HBGO chip ex vivo at a flow rate of 100 μL/min. Captured MCF7 cells were quantified by positive CK staining with the absence of canine CD45  expressing MCF7 cells were injected into the cephalic vein of the canine subject, and the system was turned on after 1 min. Blood was then permitted to circulate through the device for up to 120 min. To compare and evaluate the system performance, 1 mL of blood was also concurrently drawn through the same jugular catheter every 20 min and analyzed for MCF7 presence by ex vivo CTC capture  1/4 dilution (25ug/mL)Primary antibodies: Anti-cytokeratin antibody  1/5 dilution, Anti-canine CD45 antibody  1/50 dilution, Anti-canine CD45 antibody  1/10 dilutionSecondary antibodies:  1/250 dilution,  1/250 dilution, 1/250 dilutionThe overall schematics of the entire system are illustrated in Fig. HBGO chip involved two separate processes. First, to fabricate the chip substrate, Cr and Au were evaporated onto a 4 inch silicon dioxide wafer and patterned. The wafer was then diced into individual pieces. Next, to fabricate the PDMS structure, a silicon master mold was created by standard photolithography. Negative photoresist  was patterned on a 4 inch silicon wafer using two separate masks: one for the main fluidic channel (40 µm height) and the other for the herringbone grooves (60 µm). The height of each layer was measured after each process with a surface profilometer (Veeco Dektak 6 M). PDMS pre-polymer mixed with cross linker at a 10:1 weight ratio was poured onto the mold, degassed, and baked in an oven at 65 °C for 24 h. The cured PDMS structure was then carefully peeled off and cut. Finally, two through-holes were punched with a biopsy punch at both ends of the channel to feed and connect the tubing.The production of the 27. The substrates and PDMS replicas were subjected to oxygen plasma treatment and bonded to form the final device. N-γ-maleimidobutyryl-oxysuccinimide ester (GMBS) was flowed through the chip using a syringe pump (Harvard apparatus) and incubated for 30 min. The chip was then flushed with 70% ethanol to pre-sterilize the inner chamber wall. Subsequently, neutravidin and biotinylated anti-EpCAM antibody  was introduced followed by 3% bovine serum albumin (BSA) to block the remaining binding surface.To chemically modify the chip surface, tetrabutyl ammonium hydroxide intercalated graphene oxide nano sheets grafted with phospholipid-polyethylene glycol-amine were prepared and assembled on the gold patterned silicone dioxide substrate by electrostatic attractionTo prevent microbial contamination, all disposable units within direct contact to the blood including the tubes, luer connectors, and syringes were sterilized using heat or ethylene oxide gas sterilization and packaged separately. For the CTC capture chip, the substrate was exposed to UV and the PDMS was autoclaved before assembly. All surface modification steps were performed in a germ poor environment. Each reagent was sterilized and tested for endotoxin level using limulus-amebocyte-lysate (LAL) gel clot assay  before use. After complete functionalization, the device was exposed to UV and the fluid within a subset of chips were sampled, plated on sheep blood agar, and cultured for 2 weeks to detect any bacterial growth.TM (Lonza) and Universal Mycoplasma Detection Kit (ATCC) and were found to be negative. MCF7 and BT474 cells were cultured at 37 °C with 5% CO2 and maintained by regular passage in complete media consisting of Dulbecco’s Modified Eagle’s Media (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution . When cells reached a confluency of 70–80%, they were collected and fluorescently labeled with CellTrackerTM Green CMFDA dye  or CellMaskTM Orange plasma membrane stain  for cell capture experiments. For in vivo cell capture, MCF7 cells purchased from ATCC were transfected with NucLight RedTM  according to manufacturer instructions  and BT474, were purchased from the American Type Culture Collection  and were authenticated by the vendor. Both cell lines were tested for mycoplasma contamination using MycoAlertHBGO chip, a live/dead viability/cytotoxicity assay kit  was used. The chip was washed with 1× PBS  after capturing the cells. Subsequently, a live/dead reagent consisting of calcein AM and ethidium homodimer-1 was prepared according to the manufacturer’s instruction and applied. Following 30 min incubation, cells were imaged and manually counted under a fluorescent microscope for quantification.To measure cell viability after processing samples through the Whole blood was drawn from healthy volunteers after obtaining informed consent for research testing that complied with the ethical regulations under an Institutional Review Board (IRB)-approved protocol at the University of Michigan. All samples were collected into CellSave tubes (Menarini Silicon BioSystems) and processed within 4 h.All canine experiments were performed with approval from the Colorado State University Institutional Animal Care and Use Committee  for animal testing and research that complied with ethical regulations. Young adult male beagles (12–18-months-old) were purchased from a commercial vendor and housed individually in an IACUC-approved facility at the Colorado State University Veterinary Teaching Hospital with standard day/light, watering and feeding schedules. Baseline hematology, clinical chemistry, and coagulation profile were obtained. Animals were randomly chosen for each experiment.For the ex vivo cell capture experiments, cultured MCF7 cells were heparinized, suspended in 5 mL of sterile pyrogen free NaCl and injected intravenously via the cephalic vein. Following injection, peripheral blood was serially collected via jugular venipuncture into CellSave tubes at multiple time points and evaluated for capture efficacy. A minimum of 3 independent experiments were carried out for all ex vivo studies.For the in vivo cell capture experiments, the dog was sedated with butorphanol and acepromazine. The ventral cervical region was clipped and aseptically scrubbed followed by placing a 14-gauge dual lumen central venous catheter  in the jugular vein. The system was connected to the catheter with extension sets and primed. One minute after injection of MCF7 cells via the cephalic vein, the system was activated to allow blood to circulate through the CTC capture module for up to 120 min. Following completion, the central catheter was removed and the dogs were allowed to recover. The following day and 7 days later, blood was submitted for hematology, clinical chemistry, and coagulation profile. The dogs were not screened for the development of cancer since human MCF7 breast cancer cells are xenogeneic to dogs and not expected to colonize in an immunologically intact animal. Each dog was continuously monitored with daily examinations of temperature, pulse, respiration, food and water intake, and the catheter site for one week after the experiment. Then, the dog was off-protocol and eligible for adoption or transfer to other protocols as appropriate.HBGO chip, unlabeled MCF7 cells spiked into blood or injected into dogs were fixed with 4% paraformaldehyde (PFA). Before staining, the cells were permeabilized with 0.1% Triton-X followed by blocking with a blocking buffer containing 2% normal goat serum and 3% BSA. A cocktail of primary antibodies including anti-cytokeratin 7/8  and two types of anti-canine CD45  were diluted in 1% BSA and flowed through the chip. After a quick wash, secondary antibodies conjugated with Alexa Fluor 546 and 488  were prepared in 1% BSA and applied for probing. The cell nuclei were stained with 4’,6-diamidino-2-phenylindole  as the final step before microscopic imaging. Cells were observed under ×10 magnification using the inverted epifluorescence microscope  with an automated motor stage. Images were reviewed blinded and analyzed manually .After capturing on the Further information on experimental design is available in the Supplementary InformationPeer Review FileReporting SummarySource Data


Monoclonal antibody (mAb) therapeutics have a promising outlook within the pharmaceutical industry having made positive strides in both research and development as well as commercialisation, however this development has been hampered by manufacturing failures and attrition. This study explores the applicability of traditional in vitro toxicity tests for detecting any off-target adverse effect elicited by mAbs on specific organ systems using hepatocarcinoma cell line (HepG2) and human dermal fibroblasts neonatal (HDFn), respectively. The mechanism of antibody dependent cytotoxicity (ADCC), complement dependent cytotoxicity (CDC) via complement activation, and complement dependent cellular cytotoxicity (CDCC) were assessed. Major results: no apparent ADCC, CDCC, or CDC mediated decrease in cell viability was measured for HepG2 cells. For HDFn cells, though ADCC or CDCC mediated decreases in cell viability wasn’t detected, a CDC mediated decrease in cell viability was observed. Several considerations have been elucidated for development of in vitro assays better suited to detect off target toxicity of mAbs. Monoclonal antibody (mAb) therapeutics currently dominate many therapeutic areas such as oncology and rheumatism. The non-clinical safety testing of mAbs however are different and more complex when compared to small molecules owing to innate differences in structure, clearance, mechanism of action, and specificity of immune responses elicited . The maiEffector functions of mAbs such as phagocytosis, antibody dependent cytotoxicity (ADCC), complement dependent cytotoxicity (CDC) via complement activation, and complement dependent cellular cytotoxicity (CDCC) are regulated by the interaction between the Fc region of mAbs with the receptors on immune cells ,3.This r® Luminescent Cell Viability Assay  allows for detection of metabolically active cells through quantification of adenosine triphosphate (ATP). Luciferin upon interaction with ATP emits light in a reaction catalysed by firefly luciferase and this can be measured by recording the luminescence. Potential cytotoxicity and reduction in ATP levels of hepatocarcinoma cell line (HepG2) and human dermal fibroblasts neonatal (HDFn) cells following exposure to mAbs were investigated using the methodology described in Different in vitro toxicity assays are in place for assessing toxicity endpoints and these have been described in detail in multiple studies and reviews . Of thesPrevious studies indicate that following immunogenicity, hepatotoxicity and dermal toxicity are the two main adverse effect categories associated with mAbs . This reThe mAbs used in this case study are rituximab and trastuzumab. Rituximab is an AntiCD20 chimeric monoclonal antibody, with an IgG1 heavy chain and kappa light chain, used for therapeutic indications such as Non-Hodgkin’s lymphoma, chronic lymphocytic leukaemia, rheumatoid arthritis, granulomatosis with polyangiitis, and microscopic polyangiitis. It has been shown to elicit hepatobiliary and skin related adverse effects in addition to immunogenicity and hypersensitivity . Trastuzl-glutamine solutions 200 mM and Sodium Pyruvate Solution, tissue culture flask, 96 well F-bottom plates and WST-1 (Cat. No: 05015944001) were purchased from Sigma Aldrich, Dorset, UK. CellTiter-Glo® Luminescent Cell Viability Assay was purchased from Promega, Southampton, UK. All kits will be used as per manufacturers’ instructions.Freshly isolated peripheral blood mononuclear cells (PBMC), human universal AB serum, rituximab (stock 100 mg/mL) and trastuzumab (600 mg/mL) were kindly provided by Alcyomics Ltd., Newcastle upon Tyne, UK. Dulbecco’s Modified Eagle Medium , Fetal Bovine Serum (FBS), Pencillin-Streptomycin , Phosphate Buffered Saline (PBS), Trypsin Ethylenediaminetetraacetic acid (EDTA) solution, Minimum Essential Medium (MEM) non-essential Amino acids, ® HB-8065™) and HDFn (ATCC® PCS-201-010™) cells, kindly provided by the Newcastle University BioBank, Newcastle Upon Tyne, UK. were grown as an adherent culture in complete growth media  in T75 tissue culture flasks at 37 °C with 5% CO2, all of the consumables were purchased from Sigma, Dorset, UK. The cells were subcultured 3 times a week using the following procedure: The spent medium was removed and the cells were given a Phosphate Buffered Saline wash following which 1× diluted Trypsin was added to gently lift the cells. The cells were then re-suspended in 1:15 dilution in T75 tissue culture flasks. HepG2  of ethanol was used as the positive. Target control refers to the target cells (HDFn or HepG2) in media.The HepG2 and HDFn cells were seeded at a density of 5000 cells/well onto a Greiner 96-well F bottom tissue culture plate . 50 µL of rituximab (R) and trastuzumab (H) were added to the test wells at final concentration of 0.1, 1 and 10 μg/mL upon which the plates were incubated for a further 3 h at 37 °C with 5% CO2 [Following incubation with varying concentrations of rituximab and trastuzumab, 50 μL of human universal AB serum were added to the test wells. The plate was incubated overnight at 37 °C with 5% CO2 ,15. The 2 [Following incubation with varying concentrations of rituximab and trastuzumab, 50 μL of PBMCs at a density of 50,000 cells per well were added to the test wells to achieve an effector to target ratio of 10:1. The plate was incubated overnight at 37 °C with 5% CO2 ,15. The 2 [Following incubation with varying concentrations of rituximab and trastuzumab, 50 μL of human universal AB serum and 50 μL of PBMCs at a density of 50,000 cells per well were added to the test wells to achieve an effector to target ratio of 10:1. The plate was incubated overnight at 37 °C with 5% CO2 ,15. The 2. Endpoint measurements of absorbance were taken at 480 nm and 600 nm (background) on FLUOstar® Omega multimode microplate reader . Cell viability was expressed as a percentage of the target control. Following exposure of cells to the test compounds for 24 h, 10 μL of WST-1 reagent were added per well and the plates were incubated for an additional 4 h at 37 °C with 5% CO® Reagent equal to the volume of cell culture medium present in each well  was added. Contents were mixed for 2 min on an orbital shaker  to induce cell lysis. The plate was allowed to incubate at room temperature for 10 min to stabilize luminescent signal. Luminescence was recorded in FLUOStar Omega multiplate reader .Following exposure of cells to test compounds for 24 h, the plate and its contents were equilibrated at room temperature for approximately 30 min. Volume of CellTiter-GloStatistical analysis was carried out using Minitab 17 software  . Statistv/v) of ethanol  was used as the positive control. HepG2 cells were treated with varying concentrations of rituximab and trastuzumab. Cell viability was expressed as percentage of Target control which are wells containing only HepG2 cells. Effector cells/serum blank refer to those wells which contain only PBMCs for ADCC, serum for CDC and PBMCs + serum for CDCC assays to measure background absorbance for the effector cells and/or serum for the respective endpoint. Neither a concentration dependent effect on cell viability nor an effector/serum dependent response were observed for either of the tested mAbs as shown in v/v) ethanol was used as the positive control. Effector cells/serum blank refer to those wells which contain only PBMCs for ADCC, serum for CDC and PBMCs + serum for CDCC assays to measure background absorbance for the effector cells and/or serum for the respective endpoint. HepG2 and HDFn cells were treated with varying concentrations of rituximab and trastuzumab. ATP content was expressed as percentage of target control which are wells containing only HepG2 cells or HDFn cells. A final concentration of 5% (The HDFN cells seem to be slightly more sensitive to the responses evoked by mAbs when compared to HepG2 cells. The PBMCs used in the assay were obtained from four different donors and the corresponding responses were varied and non-specific both in terms of the mAb used as well as dose. The in vitro systems selected in this study were based on the two main adverse effects associated with mAb based therapeutics: hepatotoxicity and dermal toxicity. Rituximab and trastuzumab elicit an immune mediated reaction to neutralize tumour cells via ADCC, CDC, and/or CDCC ,15. The However, as seen from the results shown in Variability in donor responses will be a confounding factor affecting the potential to detect any off target adverse effects, as intra donor variability is quite high owing to the specificity of immune responses elicited . The cliExposure time is another possible reason for the lack of response for these assays. Immune specific reaction could take 5–7 days to develop and this could lead to depletion in the target cell number and hence wouldn’t be feasible in this context without a continuous culture in place. This has been shown in T cell proliferation assays for monoclonal antibodies wherein the early phase effects were identified at 20 h and late phase effects at 7 days . HoweverThe hallmarks of state of the art non-clinical safety testing tools that would facilitate the accelerated growth of the biopharmaceutical market would be: high throughput; rapid and cost effective; highly reproducible and allow for early stage screening. They would also provide an alternative to animal testing considering the various drawbacks of in vivo systems as seen in the case of TGN1412 . The evoTo summarize, this study looked at the applicability of traditional in vitro toxicity tests to assess potential off target hepatotoxicity and dermal toxicity of mAbs using HepG2 and HDFn cell based assays. Though these assays are routinely using for assessing toxicity of compounds, they deemed to be unsuitable in this case due to several factors and thus, no apparent dose or mAbs specific cytotoxicity or decrease in ATP levels were observed. Therefore, novel assays would be more suitable for detecting potential immune related adverse effect elicited by mAbs.


Complete genome duplication is essential for genetic homeostasis over successive cell generations. Higher eukaryotes possess a complex genome replication program that involves replicating the genome in units of individual chromatin domains with a reproducible order or timing. Two types of replication origin firing regulations ensure complete and well-timed domain-wise genome replication: (1) the timing of origin firing within a domain must be determined and (2) enough origins must fire with appropriate positioning in a short time window to avoid inter-origin gaps too large to be fully copied. Fundamental principles of eukaryotic origin firing are known. We here discuss advances in understanding the regulation of origin firing to control firing time. Work with yeasts suggests that eukaryotes utilise distinct molecular pathways to determine firing time of distinct sets of origins, depending on the specific requirements of the genomic regions to be replicated. Although the exact nature of the timing control processes varies between eukaryotes, conserved aspects exist: (1) the first step of origin firing, pre-initiation complex (pre-IC formation), is the regulated step, (2) many regulation pathways control the firing kinase Dbf4-dependent kinase, (3) Rif1 is a conserved mediator of late origin firing and (4) competition between origins for limiting firing factors contributes to firing timing. Characterization of the molecular timing control pathways will enable us to manipulate them to address the biological role of replication timing, for example, in cell differentiation and genome instability. Genome replication must be complete and accurate to generate two faithful copies of the DNA in order to inherit a full set of genes to both daughter cells. Completeness of genome duplication is a particularly complex issue in eukaryotes that require multiple replication origins to duplicate their large genomes within a typical replication period that can last from several minutes in yeasts to some hours in vertebrates. With multiple origins comes the risk of gaps between two replication initiation sites that are too large to be replicated by two replication forks within the duration of an S phase. In order to keep the number of large inter-origin gaps minimal to guarantee completeness of genome duplication two requirements must be met A: (1) orIn most eukaryotes including metazoa the large-gap problem becomes aggravated by the fact that origin positions are not strictly determined by DNA sequence. This generates an element of randomness in the location of origins and, thus, a certain size distribution of small and large inter-origin distances . The larOne important mechanism utilized by eukaryotes to reduce large inter-origin distances that is not discussed in detail in this article is the use of dormant origins . EukaryoIn addition to dormant origins that help reduce size of many inter-origin gaps eukaryotes have also evolved a complex genome replication program to ensure complete genome replication, which is discussed in detail in this review.Some origin firing factors are present in limiting amounts in somatic eukaryotic cells ,13,14,15The timing with which replication domains replicate is an important aspect of eukaryotic genome duplication. Replication timing changes with cell differentiation, as shown by genome-wide replication timing analyses and these timing changes are conserved between different human and mouse cell types, indicating that replication timing is evolutionarily important ,19. MoreReplication domains juxtaposed in the genome appear to mostly replicate successively, meaning once a domain has replicated its neighbour starts (dominos model) B 23,24,,24,25. TFrom this dominos model of higher eukaryotic genome replication follows that the timing with which replication domains replicate is dictated by the replication time of the first replicating domain combined with the rate of spreading of replication through the neighbouring domains B. At TTRThis model of genome replication presented is highly simplified and leaves open many questions. What determines the timing of the first domain in a row (the first domino)? How is spreading of replication achieved and how is it blocked at TTRs? Which chromatin processes and parameters influence replication timing?Some fundamental aspects of replication timing in eukaryotes have been characterised. It has long been known that transcriptionally active open euchromatin replicates in early S phase, whereas the structurally compact heterochromatin replicates late (with some exceptions). Microscopically, early replication becomes visible in mammalian cells as hundreds of foci of active replication scattered throughout the nucleus after pulse-labelling with detectable nucleotide analogues or when expressing labelled PCNA (proliferating cell nuclear antigen) ,28,29,30Interestingly, some genomic regions are subject to specific timing regulation. The heterochromatin at pericentromeres replicates very early in budding and fission yeasts, as does the heterochromatic mating type locus in fission yeast, whereas other heterochromatic regions replicate late. There are specific molecular mechanisms to determine these early and late replication times ,39, provTwo types of control of replication origin firing are necessary in order to avoid large inter-origin gaps and ensure appropriately timed replication in the described domain-wise replication mode: (1) The timing with which the origins within individual domains fire needs to be controlled and (2) once a domain is set to replicate its origins must fire coherently—efficiently and in a narrow time window—to be fully replicated in the typical 45–60 min ,30,40 a All temporal and spatial regulations of replication origin firing must ultimately control the activity of essential origin firing factors. The molecular mechanisms of eukaryotic origin firing are relatively well-known in budding yeast, where genetic screening, cell biology and biochemistry identified the essential origin firing factors and characterised fundamental activities and regulations. Recent biochemical re-constitution experiments then defined the set of core firing factors sufficient for in vitro origin firing, the establishment of bi-directional DNA replication with leading and lagging strand synthesis out of pre-replicative complex (pre-RCs) : the McmBRCA1 C-terminal repeats) [pre-initiation complex (pre-IC) [Eukaryotic replication initiation has two steps, origin licensing and origin firing. Licensing is the loading of the replicative helicase comprising the six Mcm2-7 subunits onto origin DNA. Licensing occurs exclusively under low-CDK conditions during mitotic exit and in the G1 phase of the cell cycle . The loarepeats) ,70  ,70 it isrepeats) , whilst repeats) . We here(pre-IC) ,72, alth(pre-IC) ,64 and c(pre-IC)    with the(pre-IC) ,74, the (pre-IC) ,75  chromatin organisation in structural units determines if and when a domain fires its origins. For this, origin firing factors, for example TMT, CDK or DDK, need to be granted access to or allowed to be active within the confines of the domain at the right time A. (2) FiIntuitively, origin coupling could involve that neighbouring origins influence each other. The unfired origins in a cluster could inhibit each other until at least one origin fires, relieving the inhibitory signal. Alternatively, firing of the first origin in a cluster could create an activating signal and stimulate firing of its neighbours.Alternatively, timing could be controlled by an intrinsic property of origins rather than by active regulation by external factors. For example, origins of early domains could respond more sensitively than late origins to limiting origin firing factors, for example due to higher accessibility or higher affinity and therefore fire earlier. It was proposed that the different firing probabilities between origin clusters resulting from such specific sensitivities to firing factors could largely explain replication timing programs like those seen in eukaryotes .Models 1 and 2  are not That stable chromatin units may underlie replication timing was proposed based on the finding that replication in metazoan cells occurs in form of individual physical chromatin units, today called replication domains. Early studies analysing isolated DNA fibres from mammalian cells on which replication had been labelled using radioactive nucleotides showed that origins do not fire scattered over the DNA but that juxtaposed origins often fire nearly simultaneously in clusters (reviewed in Reference ). MicrosXenopus egg extracts. In nuclei isolated from cells in mitosis or G1 before the TDP (up to 1 h after anaphase onset), the different genome regions did not replicate in a defined order but in a random fashion typical for Xenopus embryonic extracts. In contrast, chromatin isolated more than 2 h after mitosis replicated in the same order as in the cells of origin. They had passed the TDP. The TDP coincided with the time of re-establishment of an interphase-like chromatin architecture out of the mitotic chromatin. The authors therefore suggested that the establishment of interphase chromatin domains in G1 may specify replication timing in the subsequent S phase. Later genome-wide proximity studies of genome regions in cells by HiC showed a correlation of genome structure with replication timing [A specific replication timing program was proposed based on the observation that the same replication foci patterns  re-occurred in every S phase ,27,28,30n timing ,99. It tn timing . Re-formn timing .Xenopus egg extracts or by inducing a second replication round in G2 cells [However, direct poof that the structuring of chromatin into folding units underlies the determination of replication timing has not been provided. It has also not been proven that the formation of the microscopically visible replication foci that reflect structural chromatin domains is required to determine replication timing. In fact, genome structure and replication timing do not always correlate: G2 cells retain the overall TAD organisation but replication timing is random when G2 nuclei are forced to replicate either in G2 cells ,102. ConA chromatin domain could form a confined space that excludes or concentrates origin firing factors, thereby controlling firing timing. However, there is little direct evidence to verify this idea.Drosophila can lead to earlier origin firing [A well-established concept is that chromatin structure determines the accessibility of its DNA to DNA binding proteins. Controlled accessibility of DNA for firing factors within a chromatin domain could regulate firing timing. Correlations between high DNA accessibility and early replication activity have been drawn. Genome-wide HiC analysis in cultured cells revealed a good correlation between the nuclear compartment containing open, transcriptionally active chromatin and early S phase replication, whereas the compartment containing closed heterochromatin replicates late . Moreoven firing ,106,107.n firing . Here, oAnother model for how chromatin domain formation determines firing timing is that domains could constitute structural units to control DNA position in the nucleus. Re-positioning of domains could move DNA between nuclear regions with high or low concentrations of firing factors. It was suggested that localisation of late replicating telomeric DNA close to the nuclear periphery may withdraw it from regions with high firing factor concentrations in the nuclear interior . HoweverIt has emerged in recent years that yeast cells utilize distinct molecular pathways to control origin firing timing of different genome regions. There is even more than one pathway to control the firing of separate groups of early and late origins, respectively. For example, both pericentromeric heterochromatin and a large proportion of the transcribed euchromatin replicate early. Yet pericentromeric origins require the kinetochore protein Ctf19 for early firing whereas many euchromatic origins rely on forkhead transcription factors ,111. DifIn addition to such active firing control pathways, competition between origins for limiting firing factors is important to determine firing time. In mutant yeast and mammalian cells, in which firing of early origins is delayed, the firing of later origins is advanced and vice versa ,115. AppTogether, these insights suggest that eukaryotes employ several distinct regulation pathways specific for the different genome regions to advance the firing of some origins and delay others. Combined with competition for limiting firing factors these pathways establish the replication timing program . What neThe forkhead transcription factors Fkh1 and Fkh2 are required in budding yeast for early replication of about 30% of origins  Figure . This waIn addition to these structural effects of forkhead factors on chromatin Fkh1/2 also directly recruit DDK kinase to early origins to control their firing . Early fmatK locus but not the subtelomeric heterochromatin, show early origin firing. Pericentromeres and matK recruit DDK dependently on the heterochromatin protein Swi6, the fission yeast orthologue of metazoan heterochromatin protein 1 (HP1) [Despite the typically late replication of heterochromatin, the heterochromatin at pericentromeres replicates in early S phase in yeasts . In fiss 1 (HP1) . DDK recIn budding yeast, early replication of centromeres relies on DDK recruitment to pericentromeric origins dependently on the Ctf19 kinetochore protein . Thus, fCtf19-dependent DDK recruitment to pericentromere origins occurs in telophase and G1 phase, indicating that DDK recruitment precedes the firing of the respective origins in the subsequent S phase . This isThe presence of DDK at origins in G1 potentially poses a problem. If the G1-recruited DDK was active, the cell would run the risk of firing these DDK-bound origins prematurely in G1, inevitably leading to re-licensing and re-firing. Eukaryotes control origin firing by DDK and CDK that both increase in activity upon S phase entry to mediate S phase specificity of firing. The requirement for two distinct S phase kinase pathways is considered a safety mechanism against premature firing and bypassing one control pathway likely compromises robustness of this regulation. This means either that origin-bound DDK needs to be kept inactive or that other firing factors need to be strictly suppressed in G1. It was suggested that a protein complex formed by Rif1 and protein phosphatase 1 (PP1) may keep DDK bound to G1 origins in check until the time is right for firing in S phase . This waTreslin is bound to early origins in G1 phase [Treslin interaction with pre-RCs [Despite this Rif1-dependent DDK inhibition in G1, there is indication that DDK is active in G1 phase at some early firing origins in budding yeast. Thorough ChIP experiments showed that Sld3G1 phase ,73. This pre-RCs ,63,64. TCtf19-mediated early origin firing may have evolved to meet a specific requirement of the pericentromeric genome region. The recruited DDK does not only allow pericentromeric origins to fire early it also recruits the Scc2-Scc4 cohesion loading complex to pericentromeres . In the In theory, the late firing of the heterochromatic origins that are not regulated by Swi6/Ctf19 could be a passive process resting on two elements: the competition between origins for limiting firing factors and the low accessibility of heterochromatin for firing factors. These could combine into a delay of firing time. Although this passive process likely contributes to late firing, yeasts employ active regulation to mediate late firing of origins, for example in subtelomeric heterochromatin . The latAnother class of late firing origins also depends on Taz1 . These oYet another class of late firing origins in chromosome arms depend on Rif1 but not Taz1 ,113,121.Together, it appears that yeast Rif1, Taz1 and PP1 constitute a set of versatile firing time regulators that integrate various signals to mediate late firing of different classes of origins.Our picture of the molecular pathways that control origin firing timing in higher eukaryotes is far less complete than in yeasts. Rif1 could be confirmed as an important determinant of late replication. Mouse Rif1 knock-out cells and Rif1 siRNA-treated cultured human cells showed gross changes in genome-wide replication timing profiles ,122. TheXenopus Rif1 and PP1 cooperate in counteracting DDK phosphorylation of Mcm2-7 subunits at replication origins, decreasing their firing efficiency [Two molecular mechanisms have been suggested for how Rif1 mediates late firing in metazoa. (1) Rif1 may delay origin firing by targeting PP1 to late firing origins, similar to yeast. In line with this model, mouse Rif1 associates with PP1 . Moreoveficiency . Howeverficiency . After R+), the other one characterised by high Rif1 levels but low lamin B (Rif1/LB−) [- domains were the late regions that changed their replication time to early upon Rif1 inactivation, whereas Rif1/LB+ domains did not. This means that Rif1 is required for late replication of one class of domains, Rif1/LB−. In Rif1/LB+ domains, Rif1 is either not required to regulate timing or it acts redundantly with another timing control mechanism.Also metazoa seem to have more than one molecular pathway to determine replication timing, since upon Rif1 deletion not all late replication domains advance replication time . Two claif1/LB−) . Rif1/LBA third class of late domains may be controlled by polymerase theta . In polyIn conclusion, a model is emerging that, like yeasts, higher eukaryotes utilise distinct mechanisms to control replication timing of different genome regions.Sld3-MTBPSld7-TopBP1Dpb11, is a regulation platform for origin firing in metazoa. Its counterpart in yeast, the SDS complex, Sld3Treslin-Sld7MTBP-Dpb11TopBP1 together with Sld2RecQL4, mediates cell cycle and DNA damage checkpoint regulations and these factors are subject to subtle control of levels and activity to ensure complete and accurate genome replication [Dpb11 and Treslin/TICRRSld3 have also been implicated in other regulations, for example the coupling of genome replication to sufficiently high nutrient levels [Sld3 contains metazoa-specific N- and C-terminal domains that do not perform essential core origin firing functions but are required for normal DNA replication in human cells [MTBP does not seem to be a very important signal integrator has apparently evolved into an origin firing regulator in metazoa. Apart from the Sld7-homologous functions, it contains a metazoa-specific domain that is important for complete genome duplication in human cells [The TMT complex, Treslin/TICRRlication ,127,128.lication ,84 and Tt levels . The TMTan cells ,94,95  of the origin firing reaction must be switched on both sharply and locally. This switch could involve any of the molecular mechanisms used to control the localisation or activity of firing factors discussed above. Intuitively, linking the origins of a domain, for example, by physical clustering, like suggested for forkhead factors , could bRecQL4 and Sld3Treslin could induce their switch-like binding to Dpb11TopBP1 in a manner, mediating coherent origin firing [Sld3 is sufficient for relatively high affinity binding to TopBP1Dpb11, Treslin/TICRRSld3 contains dozens more CDK consensus sites that could regulate this binding event.Mathematical modelling suggested that, in yeast, the multisite phosphorylation of Sld2n firing . This isn firing . In analEukaryotes have evolved distinct molecular pathways to control firing timing of distinct sets of origins. This indicates that appropriate replication timing serves to adapt genome replication to the specific requirements of the different genomic regions. These active timing control pathways combine with the competition of origins for limiting firing factors to establish the temporal program of genome replication. During competition between origins the much-discussed intrinsic firing propensity of individual origins likely plays an important role in determining which origins win the competition and fire first.Pre-IC formation is the step at which firing timing regulation occurs. In yeasts, the control of DDK activity is a common theme among firing control pathways. In higher eukaryotes, the TMT regulation platform is a good candidate to mediate firing time control. Rif1 is conserved from yeast to man as a mediator of late firing. Much research is required, particularly in metazoa, to uncover and understand how origin firing time is determined.Once the molecular mechanisms are known it will be possible to directly address how important replication timing control is by manipulating these pathways using genetics and chemical inhibitors. As mentioned, replication timing changes with cell differentiation and tumorigenesis and has been linked to genome instability but the nature of these links—e.g., if timing changes are cause or consequence and what the cellular mechanisms are—is mostly unknown. Thus, investigating the relevance of replication timing control is important for our understanding of how cells work and also for human health.


Objective: Epigenetic mechanisms have been described in several mental disorders, such as mood disorders, anxiety disorders and schizophrenia. However, less is known about the influence of epigenetic mechanisms with regard to personality disorders (PD). Therefore, we conducted a literature review on existing original data with regards to epigenetic peculiarities in connection with personality disorders.Methods: Systematic literature review using PRISMA guidelines. Search was performed via NCBI PubMed by keywords and their combinations. Used search terms included “epigenetic,” “methylation,” “acetylation” plus designations of specified personality traits and disorders according to DSM-IV.Results: Search yielded in total 345 publications, 257 thereof with psychiatric topic, 72 on personality disorder or traits, 43 of which were in humans and epigenetic, 23 thereof were original studies. Lastly, 23 original publications fulfilled the intended search criteria and were included. Those are 13 studies on gene methylation pattern with aggressive, antisocial and impulsive traits, 9 with borderline personality disorder (BPD), and 2 with antisocial personality disorder (ASPD). The results of these studies showed significant associations of PD with methylation aberrances in system-wide genes and suggest evidence for epigenetic processes in the development of personality traits and personality disorders. Environmental factors, of which childhood trauma showed a high impact, interfered with many neurofunctional genes. Methylation alterations in ASPD and BPD repeatedly affected HTR2A, HTR3A, NR3C1, and MAOA genes.Summary: Epigenetic studies in PD seem to be a useful approach to elucidate the interaction of co-working risk factors in the pathogenesis of personality traits and disorders. However, the complexity of pathogenesis leads to divergent results and impedes an explicit interpretation. Differing methylation patterns within the selected PD could indicate subgroups which would benefit from patient-oriented therapeutic adjustments. They might play a major role in the future design and observation of early therapeutic intervention and thus could help to prevent severe dysfunctional conduct or full-blown personality disorder in risk subjects. The epigenetic view on genes presumably associated with psychiatric disorders is gaining increasing academic interest and enables auxiliary insights in the pathogenesis of a particular disease. Severe psychiatric axis-I disorders like major depressive disorder (MDD) are currently widely investigated at the epigenetic level, and results account for a substantial pathogenetic impact of gene epigenetic modifications.Epigenetic mechanisms in general function to homeostatically control gene accessibility and transcriptional functionality. Thus, gene function can be organized in both a highly programmed and life-enduring, but also in an environmentally-responsive way . The traNR3C1) in the hippocampus (BDNF) promoter in the prefrontal cortex (AVP)  in rats led to increased hippocampal glucocorticoid receptor (GR) expression, disturbed hypothalamus-pituitary-adrenal (HPA) axis functionality, and changed DNA methylation of the GR gene NRC1 in thel cortex  and of tex (AVP) . In human brains . Hithertn brains , 8, and n brains , since pn brains –11.AVP promoter is regulated by the methyl-CpG-binding protein 2 (MeCp2), which is phosphorylated and activated by depolarization of hypothalamic neurons and in turn moderates demethylation of the BDNF promoter  in humans.Literature search was performed as a systematic review according the Preferred Recording Items for Systematic Reviews and Meta-Analyses (PRISMA-P) guidelines . We baseFor the search keywords were inserted in a double or triple combination to yield comprehensive hits. The following keywords were utilized: “personality,” “personality disorder,” “personality trait,” each of them combined with “epigenetic,” “methylation,” “acetylation,” “phosphorylation,” “ubiquitation,” “sumoylation,” “microRNA,” “chromatin” and “chromatin remodeling,” respectively, as well as with one of the keywords “aggression,” “anankastic,” “antisocial,” “anxious,” “avoidant,” “borderline,” “dependent,” “eccentric,” “emotionally unstable,” “histrionic,” “passive-aggressive,” “impulsive,” “narcissism,” “narcissistic,” “paranoid,” “schizoid,” and “schizotypal,” respectively. The search included publications until May 15th 2018.n = 257], (2) personality disorder or specified personality trait [n = 72 of (1)], (3) human study subjects [n = 61 of (2)], (4) epigenetic analyses [n = 43 of (3)], and (5) original study [n = 23 of (4)]. Following these selection criteria, it remained 23 articles according to the intended objective of this review  psychiatric topic , and thHigh requirements in the technical methods and in an expedient study design make the disentangling of personality-specific methylation patterns a sensitive challenge, especially since there might be more than one way to (epi-)genetically forward the development of a PD. This also might be a reason for the diversity of the final clinical epiphenotypes of these disorders. Conversely, the variety of pathogenesis and of the individually developed clinical shapes impedes the attribution of possibly identified genetic or epigenetic aberrances to a specific personality trait or disorder.If the functional attribution of epigenetic aberrations to personality disorders is so highly impeded, which benefit can we gain in doing so?The possibility of epigenetic subtyping helps to assess the individual pathophysiologic condition as well as to improve deductive therapeutic approaches. The identification of someone's epigenetic risk profile could initiate efforts to establish standards for intensified clinical watching in afflicted children, and methods for early parental coaching or for premature intervention in order to prevent further development of a full-blown clinical picture of a PD. As impressively shown by Cecil et al., divergent epigenetic conditions within psychopathologically similar subjects can elicit different implications for the individual treatment requirements .APBA3 and MCF2 were predictive for psychotherapy outcome (As in other mental disorders, epigenetic patterns can help to predict medical response. Thus, in depression, selected methylation aberrations could predict different therapy response to escitalopram and between escitalopram and nortriptyline , 52. In  outcome .In cancer, advanced therapy today comprises epigenetic drugs targeting i.e., on silenced tumor suppressing genes. Due to the plurality and individual diversity of involved genes in psychiatric disorders, the use of demethylating drugs cannot be targeted to distinct genes. Adverse effects with general hypomethylation might conflict with the aimed benefits. But there is evidence that methyldonor components as valproic or folic acid have positive therapeutic effects in mental disorders by themselves as well as by increasing the effect of fluoxetine –57.Aside from their primary neurobiological effect, psychotropic drugs can further exert direct epigenetic effects. Studies on antidepressants and antipsychotic drugs i.e., evidenced their possibility to modulate the epigenome by acetylation of gene-associated histones, by methylation changes in dopamine pathways , by incrThese merely exemplary findings suggest a high dynamic in epigenetic processes in mental disorders and their course. The interaction of psychotropic drugs and other therapeutic interventions with epigenetic remodeling seems still understudied. Finally, the fact of this interaction then could be utilized as an intraindividual control of lasting therapy response.The pathophysiological principle of gene-environment-interaction not only explains the obvious differences in the severity or combination of a person's personality traits but further implicates the existence of genetic and epigenetic risk and protective factors during the formation of the ultimate personality structure. Hitherto epigenetic studies underline the impact of early life adversity on the multifactorial pathway to PD. They reveal several relevant gene loci that are epigenetically affected in PD but differ in between the studies. This can partially be explained by the multifactorial and multi-step genesis of each PD, leading to different pathogenetic subtypes.Epigenetic analyses in connection with PD represent a complex, but suitable amendment in the elucidation of personality development, and pose as valuable diagnostic step in the specification of an individual's premorbid risks, and for the development of individually tailored therapeutic strategies. They might play a valuable role in the future design and observation of early and personalized therapeutic intervention and thus could help to prevent the unfolding in severe dysfunctional conduct or personality disorder in risk subjects.DG conducted the full literature search process, read all found articles, wrote the manuscript. KK critically overworked focus and manuscript. TH and HF provided expert advice in epigenetics. JK and TF reviewed the manuscript and collaborated in the interpretation of the results and for the discussion.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.