Computed tomography (CT) is widely used in the diagnosis of giant paraesophageal hernias (GPEH) but has not been utilised systematically for follow-up. We performed a cross-sectional observational study to assess mid-term outcomes of elective laparoscopic GPEH repair. The primary objective of the study was to evaluate the radiological hernia recurrence rate by CT and to determine its association with current symptoms and quality of life.All non-emergent laparoscopic GPEH repairs between 2010 to 2015 were identified from hospital medical records. Each patient was offered non-contrast CT and sent questionnaires for disease-specific symptoms and health-related quality of life.The inclusion criteria were met by 165 patients . Total recurrence rate was 29.3%. Major recurrent hernia (> 5 cm) was revealed by CT in 4 patients (4.3%). Radiological findings did not correlate with symptom-related quality of life. Perioperative mortality occurred in 1 patient (0.6%). Complications were reported in 27 patients (16.4%).Successful laparoscopic repair of GPEH requires both expertise and experience. It appears to lead to effective symptom relief with high patient satisfaction. However, small radiological recurrences are common but do not affect postoperative symptom-related patient wellbeing. The term giant paraesophageal hernia (GPEH) is used when at least one third of the stomach is situated above the diaphragm. GPEHs constitute approximately 2 to 5% of all paraesophageal hernias . SymptomThe risk of a major medical emergency associated with GPEH is approximately 1% annually and leads to a lifetime risk of approximately 18% at 65 years . Due to The long-term outcomes and durability of laparoscopic repair are debated –12. CompIn previous studies, the imaging of choice has usually been barium esophagogram , 18. .HoHealth-related quality of life (HRQoL) has been measured individually in previous studies with generic or disease-specific instruments. Minimal correlation between radiological findings and HRQoL has been shown , 18, 22.The objective of this study was to evaluate the treatment outcomes of elective laparoscopic GPEH repair in a single tertiary care centre. The primary outcome was radiological recurrence in CT and its association with current symptoms and HRQoL. We also evaluated possible pre- and perioperative factors that could be linked to unsatisfactory outcomes or GPEH recurrence.This was a consecutive case-series study on laparoscopic GPEH repair with follow up for radiological surveillance of hernia recurrence and patient postoperative HRQoL.The patient cohort included patients diagnosed with GPEH and subsequently operated electively with laparoscopy for GPEH in a single tertiary care hospital between 2010 and 2015. Patient information was collected retrospectively from electronic medical records (EMR) and included date of operation, preoperative symptoms, comorbidities, smoking, body mass index (BMI), preoperative proton pump inhibitor (PPI) use, pre- and postoperative laboratory and radiological tests, length of hospital stay, complications, reoperations, mortality, readmissions, intensive care unit stay, duration of operation, urgency of operation, possible additional procedures, referring hospital, operating surgeon, and follow-up data. The size of the hernia was based primarily on the original radiologist’s report of preoperative CT, or if unavailable, the assessment in the operation report or preoperative gastroscopy.The operations were performed laparoscopically using five ports with a Nathanson liver retractor. During the operation, the hernia sac is dissected circumferentially and reduced from the mediastinum. The esophagus is mobilised to obtain > 2 cm of tension-free esophagus beneath the diaphragm. A hiatoplasty is performed with sutures and the diaphragmatic crura are reinforced with mesh if needed. A fundoplication is commonly performed, predominantly a floppy Nissen fundoplication with a thick nasogastric tube as a bougie. In patients not suitable for fundoplication, a simple gastropexy is performed. The operation reports were reviewed to obtain more detailed information about the surgery. We examined the reports for possible fundoplication, use of mesh reinforcement, lengthening of esophagus, and other features.The identified patients were sent an invitation to participate in the study with a letter of information, study consent, and questionnaires for generic 15D HRQoL and disease-specific Gastroesophageal Reflux Disease-Health Related Quality of Life (GERD-HRQL) instruments. The 15D is a generic, 15-dimensional self-administered quality of life instrument . The 15DThe patients who consented to participate were offered a non-contrast CT scan of the chest and upper abdomen. A radiologist’s report was received to determine the recurrence of the hernia. A recurrent hernia was defined as a hernia with > 2 cm of the stomach above the diaphragm. The hernia was considered small, or minor recurrence, if it was 2 to 5 cm and a large recurrent hernia, or major recurrence, was > 5 cm, as defined in prior studies , 22, 27.p-value was < 0.05.We analysed results with statistical tests using IBM SPSS Statistics Version 24. Chi-squared or Fisher’s exact test were used between two categorical variables. The Mann-Whitney test was used for analyses with interval independent variables and categorical dependent variables. The Kruskal-Wallis test was used for analyses with interval independent variables and ordinal dependent variables. Spearman correlation was used for analyses with two interval variables. A simple logistic regression was used for the analyses with categorical independent variables and interval dependent variables. Results were considered statistically significant when the two-tailed The study was submitted to and approved by the Research Ethics Committee of the Faculty of Medicine of Helsinki University (code 419/13/02/02/2015) and by the Institutional Review Board (IRB) of the Helsinki University Hospital Heart and Lung Centre (decision 8/2016).. A total of 227 patients underwent laparoscopic repair of paraesophageal hernia between 2010 and 2015. We excluded open procedures, both planned and converted, patients who had been previously operated on (n = 29), emergency operations (n = 20), patients with non-giant paraesophageal hernia (n = 9) and 4 patients who had been operated at a regional hospital. We included 165 patients in our study. The total number available for QoL analysis was 123 and a total of 93 for radiological analysis. The median follow-up time from the operation to receiving the questionnaires was 33 months (range 10 to 72 months).Patient demographics are presented in Table 1n = 134, 81.2%) were performed by one surgeon (JR) and altogether five different surgeons performed these operations. Mesh reinforcement was used in 8 patients (4.2%) and absorbable mesh was used in all except one of them. Esophageal lengthening was considered necessary after mobilization in none of the patients. A fundoplication was performed in 149 patients (90.3%). The mean duration of operation was 125 min . Robot-assisted surgery was used for 9 patients (5.5%).Most operations (Complications after laparoscopic operation were reported in 27 patients (16.4%); 4 patients had more than one complication. The complications were classified according to the Clavien-Dindo classification , 29. TheNine deaths occurred during follow up. There was one postoperative death within 30 days. This patient was considered high risk preoperatively, with an age-adjusted CCI of 6. According to autopsy, death was due to cryoglobulinemic vasculitis which caused intestinal perforation. The other eight deaths were not directly related to GPEH and occurred a mean of 22 months (SD ± 14.6) after operation.n = 3), gastric paralysis (n = 2), small intestine strangulation (n = 1), suspected bleeding (n = 1), gastric perforation (n = 1), small intestine perforation (n = 1), and leakage at the GE junction (n = 1). The reoperations were carried out mainly using open technique, either laparotomy or thoracotomy. The patient with suspected bleeding was reoperated laparoscopically and for two patients endoscopic intervention with PEG was sufficient. The causes for a later reoperation were hernia recurrence and in one case gastric strangulation.In total, 16 patients (9.7%) required reoperation. Of these, 10 (6.1%) occurred within 30 days of the primary operation, with reasons including recurrent hernia .Of the 165 operated patients, 158 (95.8%) were symptomatic preoperatively. Disease-specific pre- and postoperative symptoms are presented in Table p = 0.001). The unsatisfied patients had a median score of 19 (range 6 to 54) and the satisfied patients had a median score of 1 (range 0 to 19).A total of 118 patients (71.5%) answered the question regarding current overall satisfaction. Seven (5.9%) patients reported overall dissatisfaction for symptom control postoperatively. The GERD score correlated with satisfaction (p = 0.001). The mean 15D total score was slightly better in the group with radiological recurrence (0.89) than in patients without recurrence (0.84) (p = 0.03). The only dimension with a statistically significant difference between patients with and without recurrence was in the bladder and bowel functions (excretion) (p = 0.012).The 15D quality of life survey was returned by 121 patients (73.3%). The results are presented in Fig. p = 0.124) . The median follow-up period from operation to CT was 39 months (range 12–79). Major recurrence, as defined in the Methods section, was revealed by CT in 4 patients (4.3%). The total recurrence rate in our patients, including the patients reoperated for recurrence preceding our follow up, was 29.3%. The symptoms evaluated by overall GERD-HRQL scores did not correlate with radiological findings (p = 0.036), a poorer quality of life in the 15D score (p = 0.011) and greater symptoms in GERD-HRQL (p = 0.002). Older age and longer operating time correlated with a prolonged length of stay (both p = 0.001). We did not identify any factors from patient or operation characteristics that could predict hernia recurrence.Known preoperative pulmonary disease was associated with postoperative complications  had major recurrent hernia, which may still be susceptible to the risks associated with giant paraesophageal hernia, most notably incarceration, volvulus and perforation of the stomach. Recurrent hernia does not seem to affect patient HRQoL, which may explain the minimal reported need for reoperations in the literature for recurrent hernia (approximately 0.5 to 4.4%) even though radiological recurrence is common , 31, 32.In initial studies, the radiological recurrence rate for GPEH was often higher in laparoscopic than in open procedures, with laparoscopic recurrence rates from 13% up to 42% . DespiteOur relatively high recurrence rate may depend on several factors. First, we performed follow up with CT, which may be more sensitive to detect recurrent hernias than a barium swallow test, especially true paraesophageal and sliding hernias. Second, we did not perform Collis gastroplasty, which may increase the number of radiological recurrences. We avoided lengthening procedures and mesh reinforcement to reduce additional risks. These include leakage and later stricture formation at the site of gastroplasty and erosion, stenosis, and dysphagia caused by mesh. The value of recurrence prevention by the most commonly currently used biological meshes in long-term follow up has been questioned . As our In our data, the HRQoL as defined by the 15D score was close to the population average. A slight inferiority was detected in breathing, and bladder and bowel functions. The reasons for this result remain unclear and further examination would require a much larger series. The 15D score was slightly higher in the group of patients with recurrent hernia. While this group had fewer problems in bladder and bowel functions, the association with GPEH is uncertain. The preoperative quality of life was not measured here, but overall HRQoL improves with a laparoscopic GPEH repair .Symptom relief (as measured by the GERD-HRQL score) after laparoscopic GPEH repair was good in our series. This has also been shown in previous studies, with good to excellent results reported by 77 to 90% of patients , 18, 32.Perioperative mortality was low (0.6%), similar to the 0 to 1.6% previously reported. The 30-day reoperation rate was 6.1%. The complication rate (16.4%) was comparable to the rates reported in previous studies (9 to 19%). Most complications were minor (Clavien-Dindo grade II), with wound infections being the most common. There were some cases of leakage and perforation, which is comparable to other series , 18, 31.The strength of our study was that we reached a high percentage of our patients for follow-up surveys and CT scans. This study also has some limitations. This was a retrospective cross-sectional study with a single timepoint surveillance for imaging joined with HRQoL questionnaires. In addition, this study was limited to laparoscopic operations that were successfully completed; conversions to open repair were excluded. Thus, we did not use an intention-to-treat analysis, but rather concentrated on the end results of laparoscopy. We also acknowledge that there were no structured questionnaires for both HRQoL and symptoms preoperatively, which could cause recall bias in the presentation of symptoms. Despite a significant portion of patients willing to participate in this study, some patients declined an additional follow-up CT scan, which could cause selection bias in the results.In conclusion, although laparoscopic repair of GPEH is a complex procedure, when executed by an experienced surgeon it appears to lead to effective symptom relief with high patient satisfaction. Minor radiological recurrence is common but does not affect HRQoL or satisfaction. These findings suggest that the laparoscopic approach is a feasible first-line surgical strategy for GPEH repair. Further research with longer follow-up times and wider, population-based studies are required to determine the optimal approach to repair GPEH.


Treatment with direct-acting antiviral agents (DAAs) offers an opportunity to eliminate hepatitis C virus (HCV) endemic among people who inject drugs (PWID) and people enrolled in opioid agonist therapy (OAT) programs. The objective of this study was to estimate and to compare HCV treatment uptake after the introduction of DAAs among patients receiving OAT in Sweden and Norway. We also aimed to evaluate predictors of DAAs treatment among OAT patients in both countries.This observational study was conducted with data from The Swedish Prescribed Drug Register and The Norwegian Prescription Database. We studied dispensed medications to calculate HCV treatment among OAT patients from 2014 to 2017 in Sweden and Norway. HCV prevalence was estimated from primary and secondary sources. Dispensations of medicines from different therapeutic areas, which served as proxy for co-morbidities in 2017, were conditionally adjusted for age, gender, and OAT medications. Logistic regression was used to evaluate these parameters.In total 3529 individuals were identified with dispensed OAT in the Swedish cohort and 7739 individuals in the Norwegian cohort. HCV treatment was utilized by 407 persons in Sweden and 920 in Norway during the study period. Annual HCV and DAA treatment uptake increased in both countries. The estimated cumulative HCV treatment uptake at the end of 2017 was 31% in Norway and 28% in Sweden. DAA treatment was associated with increased age  and the dispensation of drugs used for diabetes  in Sweden. In Norway, lipid modifying agents and antibacterials were associated with decreased odds .An increase in DAA treatment and HCV treatment uptake was observed among Swedish and Norwegian OAT patients whilst introducing new direct-acting antiviral treatment regimens. However, more than two thirds of the OAT population in Norway and Sweden were untreated at the beginning of 2018. A further scale-up is crucial in order to control and eliminate the HCV endemic among OAT patients. Treatment of chronic hepatitis C virus (HCV) infection has been subject to vivid changes in the last few years with the introduction of direct-acting antiviral agents (DAAs) . The ambThe scale of the HCV endemic among people who inject drugs (PWID) is tragic and is a result of years of failing health policies for vulnerable populations. The HCV prevalence is around 50%, or more, among PWIDs , and aroThe coverage of preventive interventions and harm reduction services varies among PWIDs. Although the distribution of needle and syringe programs is relatively poor , opioid calculate HCV treatment annually and cumulatively after the introduction of DAAs among patients receiving OAT in Sweden from 2014 to 2017compare DAA treatment between Sweden and Norway among patients receiving OAT from 2014 to 2017 and estimate the HCV treatment uptakeevaluate if various dispensed drugs (proxy for comorbidities), age, gender and OAT medication is associated with DAA treatment among OAT patients in Sweden and Norway in 2017Therefore, this observational study aims to:This is an observational study among patients on OAT in Sweden and Norway from 2014 to 2017. Data were extracted from The Swedish Prescribed Drug Register and The Norwegian Prescription Database. The registries cover the entire Norwegian and Swedish populations and record all drugs dispensed from pharmacies. All drugs are classified according to The Anatomical Therapeutic Chemical (ATC) classification system . HCV preThe study population included all individuals aged 18 to 75 years who received OAT in Sweden and Norway. OAT was defined as being dispensed at least one defined daily dose (DDD) per day per calendar year of buprenorphine, methadone, buprenorphine-naloxone, or levomethadone by summarizing all annually dispensed OAT DDDs divided by 365.25 days.Moreover, OAT medication per individual was noted as the last dispensation per calendar year. To avoid including other medical indications than OAT, we excluded methadone preparations on the basis of route of administration (injections and tablets), and introduced a dosage criteria in order to make sure that actual patients on OAT were captured. The dosage criteria was set at minimum one DDD daily throughout each calendar year as an inclusion criteria. The study populations were thus chosen annually for both countries and it was possible for an individual to be included in more than one calendar year. See additional file N is the size of the study population, δ is the rate of spontaneous HCV clearance, ϕ is the proportion of OAT patients who are PWID, πPWID and πNonPWID are the anti-HCV prevalence estimates among PWID and non-PWID, respectively, and τ is the number of HCV treatments given. Using the above formula, we calculate the expected number of chronic HCV infections in 2014–2017 for Norway and Sweden, with uncertainty in this quantity arising only from the uncertainty in spontaneous clearance. The chronic HCV prevalence was then calculated by dividing the expected number of chronic HCV infections by the total population size in that particular year and setting (i.e. Norway or Sweden). HCV treatment uptake was then estimated by dividing the HCV treatments in each year by the estimated number of chronic HCV infections in that same year, yielding a percentage of chronic HCV infections that were treated per year. The cumulative HCV treatment uptake was then calculated as the sum of HCV treatment uptake across years.HCV treatment was defined as being dispensed either one or more types of pegylated interferon alpha in combination with ribavirin, or one or more of the DAAs per calendar year during the study period , age, gender and various dispensed drugs (yes vs. no) from different therapeutic areas that were used as proxies for co-morbidities. All dispensations were recorded at the second ATC level (therapeutic subgroup), except for drugs affecting the nervous system.p < 0.05 level.All data analyses was conducted in STATA SE 16.0 . Descriptive data was presented as frequencies, percentages, and means, with corresponding 95% confidence intervals where appropriate. Logistic regression was used to estimate whether DAA treatment uptake was associated with gender, age, OAT medication, and dispensations of other drugs in 2017. Statistical significance was set at the All data were received pseudonymised from registry administrators and subsequently analyzed, therefore, no written consent was obtained from any of the individuals in the study. The study was approved by the Regional Ethical Review Committee in Stockholm, Sweden, (no 2018/2080–31/1) and the Regional Committee for Ethics in Medical Research (no. 2018/939) in Norway. Furthermore, the study was conducted in accordance with the Helsinki Declaration and as an observational study in accordance with international accepted STROBE guidelines .In Sweden, 3529 individuals receiving OAT were identified. Around 70% were male, with a mean age of approximately 44 years and 45 years in 2014 and 2017, respectively. See additional file For Sweden, chronic HCV prevalence was estimated to range from 55.6%  53.3 to 58.8) in 2014, to 53.1 (UI: 50.8–56.3) in 2017. In Norway, prevalence was estimated from 54.4 (UI: 52.1–57.5) in 2014 to 50.0 (UI: 47.7–53.1) in 2017. The cumulative HCV treatment uptake was thus projected to be 31% in Norway and 28% in Sweden for the study period  received DAA treatment whereas in Sweden, 123 (4.5%) individuals received treatment. Variations in treatment within countries were few, except for drugs used for diabetes  or the International Classification of Primary Care (ICPC), rather, it was estimated from published reports and modelled where adequate data sources were missing. Thus, there is some uncertainty in the denominator of people with HCV in need of treatment. The predictors for DAA treatment were limited to the main dispensed drugs and sociodemographic variables and so did not fully acknowledge that there could be other vital reasons why access to DAAs would be limited in this group of patients.Finally, PWID are a heterogeneous group of individuals, and one should be careful not to generalize OAT patients to include all PWIDs.This study indicates a large scale-up in DAA treatment among Swedish and Norwegian OAT patients. Cumulative HCV treatment uptake was around one-third from 2014 to 2017 in both countries, attributed by a complete shift to DAA treatment regimens. Amidst a HCV endemic among PWIDs, it seems that two-thirds of OAT patients in need of treatment were untreated in the beginning of 2018. Coupled with the prospect of HCV elimination, there is a need for further scale-up of the most effective HCV treatment strategies, by identifying possible predictors of treatment and to establish more accurate surveillance systems in order to provide better care to this group of marginalized people.Additional file 1. Methodology description: estimating chronic hepatitis C (HCV) prevalence among people on opioid agonist therapy (OAT) in Norway and Sweden:Additional file 2. Flowchart of study populations in Norway and Sweden from 2014 to 2017. According to World Health Organization Collaborating Centre for Drug Statistics Methodology, ATC/DDD Index. OAT = opioid agonist therapy, DDD = defined daily dose. The DDDs are the assumed average maintenance dose per day for a drug used for its main indication. *Therapeutic subgroup, chemical subgroup, or chemical substance. **Including clonazepam. ***Excluding clonazepamAdditional file 3. Opioid agonist therapy and hepatitis C treatment in Norway and SwedenAdditional file 4. Anatomical Therapeutic Chemical Classification and defined daily dose for OAT medications. OAT = opioid agonist therapy; SD = standard deviation; Sources: The Swedish Prescribed Drug Register (SPDR), The Norwegian Prescription Database (NorPD). *Last registered OAT medication each calendar yearAdditional file 5. Basic characteristics of patients receiving OAT from 2014 to 2017 in Sweden and Norway. OAT = opioid agonist therapy, HCV = hepatitis C virus infection, DAA = direct-acting antiviral agent, CI = confidence interval. Sources: The Swedish Prescribed Drug Register (SPDR), The Norwegian Prescription Database (NorPD). *Exluding Ribavirin (J05AP01)Additional file 6. Annual and cumulative DAA and HCV treatment among patients receiving OAT from 2014 to 2017. OAT = opioid agonist therapy, DAA = direct-acting antiviral agent, OR = odds ratio, aOR = adjusted odds ratio, CI = confidence interval. Source: The Norwegian Prescription Database (NorPD) and The Swedish Prescribed Drug Register (SPDR). *N05BA01, N05BA04, N05BA06, N05BA12, N05CD02, N05CD03, N05CD08, N03AE01. **N05CF01 and N05CF02. ***N03AA, N03AB, N03AF, N03AG, N03AX. ****N06AA, N06AB, N06AF, N06AG, N06AX. *****N05AA, N05AB, N05AC, N05AD, N05AE, N05AF, N05AG, N05AH, N05AL, N05AN, N05AX. For ATC codes see additional file 4Additional file 7. Logistic regression on factors associated with DAA treatment among patients receiving OAT in 2017. OAT = opioid agonist therapy. Sources: The Swedish Prescribed Drug Register (SPDR) and the Norwegian Prescription Database (NorPD). *Methadone, levomethadone, buprenorphine and buprenorphine-naloxone


The clinical presentation of idiopathic normal pressure hydrocephalus (iNPH) may overlap with progressive supranuclear palsy (PSP). The Magnetic Resonance Parkinsonism Index (MRPI), MRPI 2.0, and the interpeduncular angle (IPA) have been investigated to differentiate PSP from healthy controls (HC) and other parkinsonisms. We aimed to assess equivalences and differences in MRPI, MRPI 2.0, and IPA in iNPH, PSP, and HC groups.t tests (TOST) procedure was used to assess these markers in iNPH, PSP, and HC.We retrospectively recruited 99 subjects  from two institutions. MRI exams, acquired on either 1.5 T or 3 T scanners, included 3D T1-weighted images to measure MRPI, MRPI 2.0, and IPA. Inter- and intra-rater reliability was investigated with the intra-class correlation coefficient (ICC), and the two one-sided For all the three measures, intra-rater and inter-rater ICC were excellent (range = 0.91–0.93).p < 0.01 in all cases) and no equivalence  were found at TOST. iNPH and PSP MRPI showed no difference (p = 0.06) and no equivalence (p = 0.08). MRPI 2.0 was not equivalent (p = 0.06) and not different (p = 0.09) in the same two populations. PSP and HC IPA proved equivalent (p < 0.01) while iNPH IPA was different (p < 0.01) and not equivalent (p = 0.96 and 0.82) from both PSP and HC.In the comparison of iNPH and PSP with HC, differences for MRPI and MRPI 2.0  contains supplementary material, which is available to authorized users. Idiopathic normal pressure hydrocephalus (iNPH) is a potentially treatable syndrome characterized by a variable combination of impaired gait, cognition, and urinary dysfunction (urgency and incontinence) . iNPH caClinically, PSP could be distinguished from iNPH based on other specific features, in particular the typical ocular motor dysfunction, characterized by supranuclear gaze palsy or slowing of vertical saccades. Nevertheless, the ocular motor dysfunction can be missing in the early stages of PSP, especially in non-Richardson’s phenotypes . On the To improve diagnostic accuracy of PSP and iNPH, various neuroimaging indices have been proposed in the last few years. Magnetic Resonance Parkinsonism Index (MRPI) has been introduced by Quattrone et al. in 2008  to recogThese indices proved to be useful in distinguishing PSP and iNPH from healthy controls and from other neurodegenerative diseases, but less useful to distinguish PSP and iNPH between each other .Given the clinical but also radiological similarities between these two diseases, the aim of our study was to evaluate different MRI measurements  between PSP, iNPH, and healthy controls (HCs).The present work has been carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans.We retrospectively analyzed the digital records at two different institutions to find iNPH and PSP patients who underwent MRI exams between January 2014 and December 2018. In all cases, only retrospective, anonymized information was used for the study; therefore, individual written informed consent was waived by the local IRBs . Their inclusion was based on a diagnosis of “probable” disease in accordance with international guidelines –12, madeMR examinations were performed on three different scanners . A complete list of all acquisition details and parameters is available in the supplementary materials.Using 3D T1w isotropic images, MRPI and MRPI 2.0 were calculated as previously described , 5. In pFinally, the IPA was also calculated for all subjects, defined as the angle formed by the posterior half of the cerebral peduncles at the level of the mammillary bodies or immediately below . Two exaImage analysis was performed independently by two raters (both with 8 years of experience).t tests (TOST) procedure corrected for multiple comparisons [p value to determine the result’s significance [The obtained values were analyzed via equivalence testing following the two one-sided parisons , 15. Thiparisons . In TOSTificance .t test was employed. Equivalence bounds to use in the TOST were obtained through a preliminary power analysis with a desired power of 80%, an alpha value of 0.025, the sample size of the smaller group available in each comparison, and a pooled standard deviation obtained with Cohen’s formula. Differences between gender distribution were assessed by Fisher’s exact test, while continuous variables were compared using unpaired t tests.As populations were of different sample sizes, variance was not assumed as equal and Welch’s The intra- and inter-rater reproducibility was calculated using the intra-class correlation coefficient (ICC). To assess the intra-rater reliability, one of the two raters performed a second evaluation after a 4-week washout period.In detail, inter-rater reproducibility was calculated using a single rater, absolute agreement, two-way random effects model while the intra-rater with a single rater, consistency, two-way mixed effects one. The results were interpreted following the scale suggested by Koo and Li: poor (< 0.5), moderate (0.5–0.75), good (0.75–0.9), and excellent (> 0.9) .p value < 0.05 was considered statistically significant with corrections for multiple comparisons when necessary.All analyses were conducted using the R statistical software  ; a p valp = 0.35–0.50) without significant differences at ANOVA (p = 0.08). Similarly, no significant differences were found in terms of gender distribution (p = 0.10).Demographic data and MRI indices for the three groups are reported in Table Both intra-rater and inter-rater agreements proved to be excellent . The data for ICC analysis is available in the supplementary materials together with Bland-Altman plots for MRPI, MRPI 2.0, and IPA intra- and inter-rater agreement.Figure p = 0.06) and no significant equivalence (p = 0.08) were found in MRPI score between iNPH and PSP patients. Similarly, MRPI 2.0 was non-equivalent (p = 0.06) and not different (p = 0.09) between these two patient groups. On the other hand, the comparison between iNPH patients and HC, as well as between PSP patients and HC, showed significant differences both for MRPI and MRPI 2.0  and no equivalence . When the IPA measurements were evaluated, this metric proved to be equivalent between PSP patients and HC (p < 0.01), and not different (p = 0.93), while it was significantly higher in iNPH patients compared with both PSP and control groups (p < 0.01 in both cases), being not equivalent .Results of the comparisons performed with the corrected TOST procedure are illustrated in Table In this study we evaluated the possible differences between iNPH, PSP, and HC in terms of different MRI metrics used in clinical practice. We found that both MRPI and MRPI 2.0 scores were not significantly different between iNPH and PSP patients, thus allowing us to suggest that these metrics could lead to a wrong neuroradiological evaluation in clinical practice.iNPH represents the most common form of hydrocephalus in adults with a probable iNPH estimated prevalence of 0.2% in patients aged 70–79 years and 5.9% in patients aged 80 years and older, with no difference between men and women . To dateFrom a radiological standpoint, brain MRI changes in iNPH patients include ventriculomegaly, acute callosal angle, disproportionate changes in subarachnoid spaces with dilated Sylvian fissures, and narrow sulci and subarachnoid spaces at the vertex and medial/parafalcine region, defined as disproportionately enlarged subarachnoid-space hydrocephalus (DESH) , 25. On In the last years, MRPI showed excellent performance in recognizing PSP patients, and in differentiating them from patients with PD, and for this reason, its clinical usage in auxiliary diagnosis of PSP is strongly recommended .Our results show no difference in MRPI between PSP and iNPH patients, leading to several considerations. Firstly, the increased size of the third ventricle in iNPH patients produces a widening of the cerebral peduncles, as demonstrated by the higher IPA values compared with those found in the HC. Given that the mesencephalic measurements are performed on the midsagittal slice, this may lead to an underestimation of the mesencephalic volume in iNPH patients. Even the inclusion of ventricular dilation markers in MRPI 2.0, compared with MRPI, does not solve this overlap in imaging findings, as shown in our results. Volume-based indices might be able to effectively quantify mesencephalic atrophy or superior cerebellar peduncle volume, even though their use in daily clinical practice is still limited . FurtherIn regard to the statistical power of our findings, it should be noted that the equivalence bounds were calculated as to ensure an 80% power. We wish to highlight that the resulting values were above the suggested cut-off proposed by Quattrone and colleagues both for MRPI and MRPI 2.0 in the differential diagnosis between PSP-P and HC (respectively 11.34 and 2.18) . This sup value of 0.049. In our study, the same parameter showed no differences between these groups with a p value of 0.06. This could be explained by the different sizes of their iNPH group (n = 17 vs 30). Nonetheless, their conclusion further supports the imaging overlap between PSP and iNPH as none of the markers analyzed proved reliable in their differential diagnosis. In this setting, the differences we found in IPA between iNPH and both PSP and HC are of particular interest. This finding is further reinforced by the unequivocal equivalence of IPA values in the PSP and HC groups. For this reason, the IPA value might be a useful tool in the radiological evaluation of these patients, in addition to other already established measurements such as the callosal angle.A recent publication by Constantinides and colleagues investigated quantitative and qualitative MRI signs including MRPI in PSP, iNPH, and HC groups . They reThis study has some limitations which have to be pointed out. First of all, iNPH and PSP diagnoses were made by a movement disorder specialist with a “probable” level of diagnostic certainty, and not pathologically confirmed. This may have partially affected the results, since some patients with antemortem diagnosis of iNPH have been noted to have coexisting neurodegenerative pathologies including PSP on neuropathology . DiseaseOur study showed that MRPI and MRPI 2.0 scores may not be helpful in the differential diagnosis between PSP and iNPH, given the overlap of these metrics. On the other hand, IPA was generally higher in iNPH than in PSP patients and in HC; therefore, it demonstrated a useful additional marker to differentiate this potentially treatable condition.ESM 1(DOCX 14 kb)ESM 2(XLSX 10 kb)ESM 3(XLSX 10 kb)Supplementary MaterialFig. 4 (PNG 369 kb)Supplementary MaterialFig. 5 (PNG 370 kb)Supplementary MaterialFig. 6 (PNG 357 kb)Supplementary MaterialFig. 7 (PNG 400 kb)Supplementary MaterialFig. 8 (PNG 370 kb)Supplementary MaterialFig. 9 (PNG 353 kb)


For newly diagnosed breast cancer, estrogen receptor status (ERS) is a key molecular marker used for prognosis and treatment decisions. During clinical management, ERS is determined by pathologists from immunohistochemistry (IHC) staining of biopsied tissue for the targeted receptor, which highlights the presence of cellular surface antigens. This is an expensive, time-consuming process which introduces discordance in results due to variability in IHC preparation and pathologist subjectivity. In contrast, hematoxylin and eosin (H&E) staining—which highlights cellular morphology—is quick, less expensive, and less variable in preparation. Here we show that machine learning can determine molecular marker status, as assessed by hormone receptors, directly from cellular morphology. We develop a multiple instance learning-based deep neural network that determines ERS from H&E-stained whole slide images (WSI). Our algorithm—trained strictly with WSI-level annotations—is accurate on a varied, multi-country dataset of 3,474 patients, achieving an area under the curve (AUC) of 0.92 for sensitivity and specificity. Our approach has the potential to augment clinicians’ capabilities in cancer prognosis and theragnosis by harnessing biological signals imperceptible to the human eye. Determination of estrogen receptor status (ERS) in breast cancer tissue requires immunohistochemistry, which is sensitive to the vagaries of sample processing and the subjectivity of pathologists. Here the authors present a deep learning model that determines ERS from H&E stained tissue, which could improve oncology decisions in under-resourced settings. Patients with hormone-receptor positive tumors often clinically benefit from receiving hormonal therapies, which target the ER signaling pathway. The US National Comprehensive Cancer Network guidelines mandate that hormone receptor status, including ER receptor status, be determined for every new breast cancer patient, as this is critical in clinical decision-making6.More than 2 million women across the world were diagnosed with breast cancer in 2018, resulting in 0.6 million deaths. A large majority of invasive breast cancers are hormone receptor-positive—the tumor cells grow in the presence of estrogen (ER) and/or progesterone (PR)2. Finally, the pathologists’ decision-making process is inherently subjective and can result in human errors3. These factors lead to discordance in ERS determination; an estimated 20% of current IHC-based determinations of ER and PR testing may be inaccurate4, placing these patients at risk for suboptimal treatment.In the current diagnostic workflow, a patient’s sample is thinly sectioned onto microscope slides for staining followed by visual diagnosis by a pathologist. Hematoxylin and eosin (H&E) staining is used for primary diagnosis, and specialized stains for molecular markers can be used for diagnostic confirmation and subtyping. For breast cancer, ERS is always assayed, as it is both a prognostic marker and predictive of endocrine therapy response. ERS is determined by visual inspection of slides stained using molecular immunohistochemistry (IHC) with an antibody targeting the ER receptor Fig. . This prWe find that the morphology of the tumor, captured in the H&E stain, contains predictive signal for the molecular marker status of the tumor and that a machine-learning (ML) algorithm can directly determine ERS from an H&E-stained whole slide image Fig. . The mor8 have shown promising performance for ERS determination from morphological stains, but are based on single-center datasets of tissue microarrays (TMAs). Creating TMAs is a manual process that requires pathologists to select regions of interest (ROIs) from the whole specimen10. In contrast, our method automatically selects ROIs from the total tissue field and demonstrates accurate results on a large, multi-country dataset of WSIs, which would make it feasible for pathologists to augment their standard clinical workflows with no additional manual steps. Other studies using TCGA and whole slide images have reported results on molecular biomarkers such as lung cancer11.Our algorithm is trained with clinical ERS readily available from patient records and requires no additional pixel-level annotations. Recent studiesWorking with H&E has several advantages—it is significantly cheaper than IHC, it exhibits less variability across centers, and it is ubiquitously used in histopathology workflows globally. An automated ERS estimation method has the potential to reduce errors in breast cancer treatment and improve outcomes, and importantly reduce time to treatment decisions. Moreover, an algorithm that identifies discriminative morphological features for molecular markers has the potential to provide biological insights into how hormones drive tumor growth.15. Since pathologists are not clinically trained to determine ERS from H&E images, they cannot provide manual annotations for this particular stain. However, if a tumor has been determined to be ER-negative (ER−) from an IHC stain, we can assume that their H&E WSI contains almost no discriminative features for ER-positivity. Conversely, if the patient has been determined to be ER-positive (ER+), we can assume that at least some regions of their H&E WSI contain discriminative features for ER-positivity. Thus, we can train models using H&E stains as input data, and IHC annotations as input data labels. This problem setup works well with multiple instance learning (MIL)16. MIL has been recently utilized for ML-driven histopathological diagnosis17 and prognosis18.ML-driven histopathology methods have primarily relied on expensive, time-consuming, pixel-level pathologist annotations of whole slide images for trainingThe goal of MIL is to learn from a training set consisting of labeled bags of unlabeled instances. A positively labeled bag contains at least one positive instance, while a negatively labeled bag contains only negative instances. A trained MIL algorithm is able to predict the positive/negative label for unseen bags. We utilize MIL to estimate ERS from a bag of tiles randomly selected from a WSI.19, which we call ReceptorNet . We train ReceptorNet to predict the probability of ER+ status through an iterative learning process. During testing, we sample multiple bags from a WSI and aggregate their probabilities to improve prediction accuracy.ReceptorNet is trained to predict ER+ status from a bag of tiles randomly selected from a WSI at a resolution of 0.5 µm/pixel. The WSI is divided into 256 × 256 pixel-size nonoverlapping tiles. ReceptorNet consists of three interconnected neural networks which are trained together: (1) A feature extractor which converts each 256 × 256 pixel-size tile in a bag into a 512-dimensional feature vector, (2) an attention module that creates a 512-dimensional aggregate of feature vectors from all the tiles in the bag, attention-weighted based on discriminative power, and (3) a decision layer that computes the probability of this bag being positive from the aggregate feature vector. We improve on the existing attention-based MILTo train and test ReceptorNet, we utilize two diverse datasets: the Australian Breast Cancer Tissue Bank (ABCTB) dataset containing 2535 H&E images from 2535 patients and The Cancer Genome Atlas (TCGA) dataset containing 1014 H&E images from 939 patients Fig. . TCGA im4 dataset from a separate study, translate to a PPV of 0.92 and an NPV of 0.683 for concordance between primary institution and central testing on the combined dataset of premenopausal and postmenopausal patients. Validation against gold-standard data using central testing remains an important future direction.We report results using the area under the curve (AUC) for ER+/ER− binary classification, along with its 95% confidence interval (CI) computed using bootstrapping. ReceptorNet obtains an AUC of 0.899 (95% CI: 0.884–0.913) on the cross-validation of the train set and an AUC of 0.92 (95% CI: 0.892–0.946) on the test set Fig. . On the 16. Their performance is inferior to ReceptorNet on the test set . Meanpool obtains an AUC of 0.827 (95% CI: 0.786–0.866) and Maxpool obtains an AUC of 0.880 (95% CI: 0.846–0.912). To evaluate if ERS can be determined from individual patches alone, we created a model with the same basic architecture as ReceptorNet after removing the attention module and trained it on individual patches using binary cross-entropy. This model obtains an AUC of 0.760 (95% CI: 0.726–0.794), with evaluation averaged on 50 patches, indicating that aggregating information from multiple patches using an attention module leads to better performance. We also build a logistic regression model using pathologist-provided histological type and tumor grade, which are clinically determined from H&E. This model obtains an AUC of 0.809 (95% CI: 0.766–0.848), significantly lower than ReceptorNet   on HER2− samples  as compared to HER2+ samples . Moreover, ReceptorNet performs significantly better  on PR+ samples  as compared to PR− samples , which is reflective of the high correlation between ER and PR statuses -status may affect tumor growth and thereby may affect the histomorphological structure of the H&E stained tissue. Since HER2 overexpression is a dominant transformation mechanism in tumorssee Fig. . For comp < 1 × 10−3, F-test), 0.949 for grade 1 (95% CI: 0.925–0.973), 0.810 for grade 2 (95% CI: 0.716–0.888), and 0.865 for grade 3 (95% CI: 0.84–0.887). In contrast, we do not find a statistically significant difference in prediction performance based on whether the tumor was ductal or lobular in origin  in ReceptorNet performance across data from Australia, Germany, Poland, and USA. On the cross-validation of the train set, the AUC on TCGA is 0.861 (95% CI: 0.828–0.893); AUC on ABCTB is 0.905 (95% CI: 0.889–0.921). Moreover, ReceptorNet performance across 42 TCGA tissue source sites is similar and is not dependent on the proportion of training samples collected from a given site  , and tested  ReceptorNet on the data from this site. ReceptorNet obtains an AUC of 0.910 (95% CI: 0.836–0.969) on this cohort, comparable to the AUC obtained on the entire test set (0.920).As additional validation, we trained ReceptorNet only using data from ABCTB and evaluated it on the entire TCGA dataset. ReceptorNet obtains an AUC of 0.850 (95% CI: 0.830–0.868) on TCGA for ERS estimation, a reasonable drop in performance as compared to the combined dataset. TCGA is a harder dataset for prediction as compared to ABCTB, as TCGA has much larger variation in staining and patient demographics. The ABCTB data is obtained from 6 tissue source sites from one state in Australia, while the TCGA data is obtained from 42 different tissue source sites from the USA, Poland, and Germany. Finally, we removed the data from only the University of Pittsburgh from the training set, which is the largest cohort in TCGA  on the TCGA data (demographic information was not available for ABCTB). AUC for postmenopausal women is 0.872 (95% CI: 0.832–0.908). AUC for premenopausal women is 0.838 (95% CI: 0.779–0.893). AUC for African-American patients is 0.859 (95% CI: 0.785–0.921). AUC for the rest of the patients is 0.858 (95% CI: 0.817–0.896) .25 ILC to be predominantly ER+. In contrast, none of the tiles with high ER− discriminative ability displayed characteristic patterns for ILC. In the third ER+ discriminative group, tiles were located within ductal/lobular carcinoma in situ lesions composed of small, uniform tumor cells having modest pleomorphism and without intervening stroma , and a variable number of inflammatory cells. These invasive carcinoma cells had mild to moderate nuclear pleomorphism with interspersed connective tissue, composed predominantly of collagen Fig. . A group30 with ER+ breast cancers rather than ER−, thus providing validation of some features assessed by the network in determining ERS.In sum, ReceptorNet discovers that histomorphological patterns that identify low grade tumors, ILC, and in situ carcinoma are predictive of ER-positivity. These features have been found to be statistically associatedReceptorNet assigned low attention weights to tiles with fat tissue; tiles with connective tissue with no/few tumor cells; tiles with few tumor cells and reactive stroma trapped in-between fat cells; and tiles with macrophages laden with debris and fat Fig. . The mod31, which shows that ReceptorNet learns to separate WSI based on the degree of ER-positivity , tumor grade, tumor origin location , as well as demographic variables , and find that other receptors and tumor grade can significantly influence classifier predictions, while the location of cancer origin and demographic variables considered do not. The ability to determine IHC-derived molecular marker status from H&E stains has the potential to reduce variability in predictions and decrease the cost of pathology workflows. In addition, the time to treatment initiation would be expedited by using a digital workflow, which may affect clinical outcomesWe combined hematoxylin and eosin (H&E)-stained whole slide images from two datasets: the Australian Breast Cancer Tissue Bank (ABCTB) dataset, which contains 2535 H&E images from 2535 patients and The Cancer Genomic Atlas (TCGA) dataset, which contains 1014 H&E images from 939 patients. Both datasets report ER, PR, and HER2 status determined by pathologists from IHC. The WSI were scanned at a resolution of 20× or higher.34 on the WSI thumbnail and discarded the background regions. We then extracted 256 × 256 pixel image tiles from the foreground of WSI at 20× magnification, where an image was considered to be part of the foreground if 1% of the tile was considered to be tissue matter. The tiles were extracted without any overlap between adjacent tiles. The average number of tiles per slide was 19,944, with the number of tiles per slide varying between 949 and 67,368.To train ReceptorNet with slide regions that only contain tissue matter, we first performed segmentation using the Otsu’s method35 without the softmax layer, followed by two fully connected layers with a dropout of 0.5 which convert the 1000-dimensional feature obtained from ResNet-50 to a 512-dimensional feature vector. The ResNet-50 is initialized from ImageNet36-pretrained weights and the fully connected layers are randomly initialized using He initialization35. During one iteration of training, a bag of N image tiles is fed to the feature extractor, which outputs a N × 512 dimensional feature matrix. This feature matrix is fed to the attention module for aggregation. The first part of the attention module contains a linear layer which reduces each feature vector to 128 dimensions and applies an element-wise hyperbolic tangent (tanh(.)) nonlinearity on the output, which scales the features to include values between −1 and 1 and facilitates learning of similarities and contrasts between tiles. The output of this linear layer followed by tanh is multiplied by another linear layer and a softmax function which computes an attention weight between 0 and 1 for a particular tile. So, for a bag of N image tiles, we obtain an N-dimensional vector of attention weights. We then perform an inner product of this attention vector with the N × 512 dimensional feature matrix to obtain an aggregate 512-dimensional feature vector. The 512-dimensional aggregate feature vector is now fed to a decision layer consisting of a 512-dimensional linear layer followed by a sigmoid function, which outputs a probability between 0 and 1 for a bag of N tiles.The ReceptorNet architecture consists of three interconnected neural networks: a feature extractor module, an attention module, and a decision module. The feature extractor is a ResNet-50N = 50 tiles drawn randomly from the pre-extracted tiles from each WSI. We performed extensive data augmentation to help the model learn invariances and to deal with variability in staining methods. Specifically, we (i) randomly flipped the tile from left to right with a probability of 0.5, (ii) randomly rotated the tile by {0°, 90°, 180°, 270°} with equal probability, (iii) performed color jittering, and (iv) performed cutout regularization18 with length 100. We trained the model to minimize cross-entropy loss using Adam optimizer with a learning rate of 1 × 10−5 and weight decay of 5 × 10−5 for 500 epochs, employing hard-negative mining21 during each epoch. Since our dataset contains significant class-imbalance (the number of ER+ samples are 3.7 times the number of ER− samples), we performed balanced sampling to maintain a rough 50–50 proportion of ER+ and ER− samples in each epoch. We found that training the model with a bag containing all the image tiles lead to overfitting. To reduce overfitting, we randomly replaced tiles by an image with all pixel values set to the mean pixel value of the dataset with a probability of 0.75. This mean pixel regularization improved performance substantially. We measured performance using the AUC, PPV, and NPV as metrics for the binary classification task. PPV is defined as / × ). NPV is defined as )/((1 − sensitivity) × prevalence + specificity × ). The sensitivity and specificity are calculated by binarizing the prediction probability of the network using a specific threshold .We trained ReceptorNet by feeding bags of N tiles in a bag are averaged to obtain an aggregate feature representation. In Maxpool, a feature-wise max is obtained for each of the feature dimensions. These methods were trained on the same model architecture as ReceptorNet, except for replacing the Attention module by a Meanpool or Maxpool operation.We compared our method with two widely used MIL methods: Meanpool and Maxpool. In Meanpool, the feature representations of 37. We computed the ER+ fraction for each cluster using the predicted ER status of the slides in each cluster. For tiles in highly ER+ or ER− clusters , we performed k-means clustering on features of individual tiles in the top 1% of each slide according to attention weights. We then sorted these tiles by their distance from cluster centers and displayed one tile each from a different slide, displaying up to five tiles from each cluster. This exercise ensured selection of highly discriminative tiles from slides with similar aggregate visual representation (and hence ER+ probability) according to our trained algorithm. The pathologist manually reviewed these tiles and recorded observations about the cellular morphology and architecture of the tissue field.We selected a set of highly discriminative tiles for ERS estimation for review by a breast subspecialized pathologist (M.F.P.). We first evaluated our trained model on bags of tiles sampled exhaustively from slides in our test set. From each slide, we saved the 512-dimensional aggregate feature vector obtained from the bag of tiles and performed k-means clustering on the features, with k determined by the elbow methodExperiments were performed on USC’s high-performance computing cluster, consisting of Nvidia P-100 Pascal graphics processing units (GPUs). Image tiles were extracted using the Python version of the OpenSlide library (v3.4.1). Each model was trained on a single GPU using the PyTorch library (v0.4.1). Performance evaluation, including AUC calculation and CI estimation with bootstrapping, was done using scikit-learn (v0.20.0) library in Python. Statistical tests were performed in R (v3.6.1); the DeLong test was performed using the Daim R package (v1.1.0). All code was developed using open-source tools.F-test. The numerator variance was computed as the AUC variance across comparison groups. The denominator variance was computed as the pooled variances across comparison groups from 1000 bootstrap AUC values within each group. The numerator degrees of freedom (dof) was taken as the number of groups minus one, and the denominator dof was taken as infinity. The 95% CI calculations were also performed using 1000 bootstrap AUC values within each group. Comparisons between different ERS prediction methods’ AUCs on the same data set were performed using the DeLong method38. Statistical analysis was done using the following Python libraries: Jupyter (v4.4.0), numpy(v1.15.4), scipy (v1.0.0), and pandas (v0.22.0).Statistical tests for differences in AUC were performed using an upper tail We did not curate the dataset, except removing 75 WSI containing excessive pen markings. The dataset faithfully reflects the quality of WSI encountered in real-world clinical scenarios.The study was approved by the Institutional Review Board at the University of Southern California.Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Postoperative pancreatic fistula (POPF) is a life-threatening postoperative complication. The aim of this study was to evaluate the efficacy of the fistula risk score (FRS) and preoperative body composition factors for predicting the occurrence of clinically relevant POPF (CR-POPF) after pancreaticoduodenectomy (PD).In this study, 136 consecutive patients who underwent PD between 2006 and 2018 were enrolled. The risk factors of CR-POPF (grades B and C) were analyzed. Preoperative visceral adipose tissue area (VATA), skeletal mass index (SMI), and subcutaneous adipose tissue area (SATA) were calculated from computed tomography data.The overall 30-day mortality and morbidity rates were 0.7 and 38%, respectively. The incidence rates of grade B and C CR-POPF were 27 and 4%, respectively. A univariate analysis revealed that male sex, habitual smoking, prognostic nutritional index (PNI) < 45, VATA ≥90, VATA/SATA ≥0.9, VATA/SMI ≥ 1.4, and FRS > 4 were significantly associated with the incidence of CR-POPF. A multivariate analysis revealed that PNI < 45, VATA/SMI ≥ 1.4 and FRS > 4 were the independent risk factors of CR-POPF.Preoperative anthropomorphic imbalance, PNI, and FRS were independent risk factors for CR-POPF. Patients with high-risk factors should be closely monitored during the postoperative period. Regardless of surgical technique and perioperative management after patients undergo pancreatic resection, the postoperative complication rate remains high at 20 to 50% –3. AmongIn 2016, the International Study Group on Pancreatic Fistula (ISGPF) redefined POPF  and recoA total of 136 patients who underwent PD between 2006 and 2018 at the Department of Surgery, Onomichi General Hospital, were enrolled in this study. Patients who received palliative surgery were excluded from the study. Clinical and pathological data and preoperative CT findings were collected. Patients with pancreatic ductal adenocarcinoma, adenocarcinoma of the papilla, extrahepatic cholangiocarcinoma, intraductal papillary-mucinous carcinoma, and chronic pancreatitis were considered eligible. The study was approved by the local institutional review board (OJH-201642), and written informed consent was obtained from all the patients.2) to obtain the skeletal muscle index (SMI). The VATA/SMI ratio was defined as high if ≥1.4 and low if < 1.4 , with a section thickness of 2.5–5 mm. The CT slices at the third lumbar vertebra (L3) level were analyzed using Advantage Workshop 4.5  and Virtual Place Fujin . The body composition assessment included calculations of the visceral adipose tissue area (VATA), subcutaneous adipose tissue area (SATA), and skeletal muscle area. CT analysis was performed by trained investigators who were blinded to the patients’ characteristics and clinical outcomes . The tissue Hounsfield unit (HU) thresholds were as follows: − 29 to 150 HU for the skeletal muscle area, − 190 to − 30 for the SATA, and − 150 to − 50 for the VATA. Skeletal muscle area was defined at the L3 level and included the psoas major and minor, paraspinal muscles , and abdominal wall muscles . The skeletal muscle area was normalized for height in meters squared , and a percutaneous drain was inserted under radiological guidance when the collection of infected fluid was suspected on CT.U test. Categorical variables were compared using the Fisher exact test. Logistic regression was used to model categorical outcomes. Variables that showed statistically significant associations in the univariate analysis were entered into a multivariate logistic regression model. Differences between the results of the comparative tests were considered significant if a two-sided p value of < 0.05 was obtained. An FRS of > 4 was defined as high on the basis of the ROC curve analysis .The median age of all the enrolled patients was 71 years . Of the 136 patients enrolled, 94 were male and 42 were female. The overall 30-day mortality and morbidity rates were 0.7% (1/136 patients) and 38% (51/136 patients), respectively. The incidence rates of grade B and C POPF were 27.2% (37/136 patients) and 3.7% (5/136 patients), respectively. The most common pathological diagnosis was pancreatic tumor, followed by tumors in the papilla of Vater and extrahepatic bile duct. Preoperative sarcopenia was diagnosed in 53 patients (39%). The patients’ characteristics are shown in supplemental Table In the univariate analysis of the risk factors of POPF after pancreatectomy, male sex, habitual smoking, PNI < 45, VATA ≥90, VATA/SATA ≥0.9, VATA/SMI ≥ 1.4, and FRS > 4 were identified to be significantly associated with POPF Table . The mulThe results of our study demonstrate that high VATA/SMI, PNI < 45 and high FRS score were independent risk factors of POPF after PD. Patients with three risk factors were significantly more likely to have CR-POPF after PD. Previous reports that examined the relationship between sarcopenia and POPF focused only on skeletal muscle mass and visceral adipose tissue , 13, 15.The underlying mechanism that links body composition and POPF still needs to be elucidated. Lutz et al. showed that the development of visceral adipose tissue is associated with an elevated production of proinflammatory molecules such as leptin, chemerin, resistin, tumor necrosis factor-α, interleukin (IL) 1, and IL-6 . These pFrom a surgical point of view, the presence of abundant visceral adipose tissue may interfere with anastomosis creation, which could result in increased intraoperative bleeding and operation time. Perioperative blood loss should be minimized to protect the patient’s immune system. In addition to high VATA, we determined other risk factors of POPF from pathological findings and postoperative clinical data.The present multivariate analysis identified that the most effective predictor of CR-POPF was VATA/SMI. High BMI has a well-known association with obesity and surgical complications , 21. PatPreoperative nutritional support and rehabilitation programs to decrease the risk of POPF are often implemented for patients with sarcopenia and poor nutritional status. Kaido et al. reported a positive impact of nutritional therapy on prognosis after living-donor liver transplantation especially in patients with sarcopenia . HoweverSeveral limitations of our study should be mentioned. First, this was a single-institution study, and the data were analyzed retrospectively. In addition, only Asian patients were included. Further studies are required to assess the feasibility of VATA/SMI, PNI, and FRS in predicting CR-POPF development.In conclusion, our study demonstrates that preoperative body composition, defined by both visceral adipose tissue and skeletal muscle, are strong predictors of POPF incidence after pancreatectomy. Assessment of preoperative body composition is easy to perform, and reliable measurements can be obtained. Clinicians should closely monitor patients with a PNI < 45 and VATA/SMI ≥ 1.4.Additional file 1: Table 1. Clinicopathological data.


The combination of clinical manifestations and genetic factors in this patient leads to the diagnosis of familial dysbetalipoproteinemia. Implementation of genetic testing into routine clinical practice could not only improve disease diagnostics and management, but also help prevent their development.We present a case of a 40-year-old male with premature atherosclerosis, with evidence of both eruptive and tendinous xanthomas, which could imply an increase in both low-density lipoprotein (LDL) and triglyceride (TG) levels. However, his LDL was 2.08 mmol/l, TG -11.8 mmol/l on rosuvastatin 20 mg. Genetic evaluation was performed using a custom panel consisting of 25 genes and 280 variants responsible for lipid metabolism. A rare ε High level of triglycerides (TG) indicates an increase in the number of atherogenic lipoproteins. In different studies, various mechanisms of atherogenesis of triglyceride-rich lipoproteins (TRLs), which encompass chylomicrons, very low density lipoproteins and their remnants, were proposed and proved. Both clinical and epidemiological data and data from genetic studies (Mendelian randomization and genome-wide association studies) support the independent causal role of TRLs for atherosclerotic cardiovascular disease (CVD) .APOE, APOA5, APOC2, APOB), lipoprotein lipase (LPL), lipase maturation factor 1 (LMF1), glycosylphosphatidylinositol-anchored high density lipoprotein binding protein 1 (GPIHBP1), and others  and hyperlipidemia . The patient had never been diagnosed with pancreatitis. He also had a history of intermittent claudication. His family history was unremarkable. Evaluation of his only child (11-year old girl) is planned. Dietary history was noteworthy for excess carbohydrates, saturated and trans fats together with alcohol abuse (up to eight liters of beer per week).The patient was a smoker with an estimated 25 pack year smoking history. He did not measure his blood pressure (BP) regularly and his maximum level of BP was 160/90 mmHg. Other cardiovascular risk factors included obesity  and 280 variants responsible for lipid metabolism. After a bioinformatics analysis, .bam and .vcf files were obtained. For clinical interpretation only variants with frequencies <0.5% in the database gnomAD were further analyzed according to the ACMG/AMP2015 recommendations.DNA extraction was performed using the QIAamp DNA Blood Mini Kit . Fluorometer Qubit 4.0  was used for DNA quantification. Next-generation sequencing (NGS) was performed using the Ion S5 . Ampliseq libraries were prepared on the Ion Chef System  using the custom panel, created in the Ion AmpliSeq Designer . This panel consisted of 25 genes (2ε1 genotype which was defined by the following variants: heterozygous pathogenic variant rs267606664 [NP_000032.1: p.G145D (p.G127D)] and homozygous pathogenic variant rs7412 [NP_000032.1: p.R176C (p.R158C)]. These variants were confirmed by Sanger sequencing on the 3500 DNA Analyzer . All steps were fulfilled according to the manufacturer's protocols. According to literature, in ε2ε2 subjects some additional genetic factors may contribute to hypertriglyceridemia development. Henneman et al. investigated the variants of APOC3, APOA5, hepatic lipase, and LPL genes in two groups of homozygous ε2ε2 individuals with normal or increased TG levels  was significantly higher in type III hyperlipoproteinemia. Our patient was found to be CC homozygous for this variant. Another variant of APOA5 (rs651821), identified in this patient, is significantly more prevalent in subjects with severe hypertriglyceridemia compared with controls  (APOE plays the most important role for mediating LDL receptor (LDLR)-dependent clearance of TRLs remnants from the circulation into the liver, as it is the ligand for the LDLR family of proteins and heparan sulfate proteoglycans (HSPG). In humans, there are three main isoforms of APOE protein  which arise from three allelic variants [εectively ]. These APOE variants with the lipid profile and CVD is widely investigated. Higher TG levels are associated with ε2 allelic variant   . Amino ateinemia . But HSPteinemia . This ma3ε1 genotype. In this study on cultured fibroblasts, it was shown that this genotype led to reduced binding capacity to LDLR similar to that of APOE2. In another study, a case of hypertriglyceridemic patient homozygous for ε1 was described as having lipid profile changes identical to those seen in patients homozygous for the ε2 allele  which would have allowed to make a full characterization of genotype-phenotype correlations in this family pedigree.One of the pathogenic variants found in this patient (p.G127D) has the highest population allele frequency of 0.0001 (4/31292) according to the gnomAD genome database, dataset version 2.1.1. It was first described in 1984 in a Finnish hypertriglyceridemic patient  with ε3ε2 allele . A familus study . But tha2ε2 individuals was previously described in literature , secondary risk factors , and the appropriate genotype. All together, these indicators lead to the diagnosis of familial dysbetalipoproteinemia. This case of familial dysbetalipoproteinemia demonstrates prominent skin manifestations . Corneal arcus, observed in this patient, was found in a quarter of patients, referred to the lipid clinic due to high plasma TG level . It is oterature . Atherosividuals .2ε2 genotype was first found in a pregnant woman hospitalized with acute pancreatitis, are described in literature  for the publication of any potentially identifiable images or data included in this article.AL was treating the patient, performing his follow-ups, and wrote this paper. AE was treating the patient, supervising all parts of this paper's preparation, and edited this paper. AK performed the genetic testing, wrote the genetic part of this paper, and edited this paper. MD performed the Sanger sequencing. VK calculated the genetic risks scores. AM supervised the genetic testing of the patient and provided valuable comments on this case. OD is the chief of the center who provided valuable comments on this case. All authors contributed to the article and approved the submitted version.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.


Staphylococcus aureus biofilms are a significant problem in health care settings, partly due to the presence of a nondividing, antibiotic-tolerant subpopulation. Here we evaluated treatment of S. aureus UAMS-1 biofilms with HT61, a quinoline derivative shown to be effective against nondividing Staphylococcus spp. HT61 was effective at reducing biofilm viability and was associated with increased expression of cell wall stress and division proteins, confirming its potential as a treatment for S. aureus biofilm infections. Staphylococcus aureus biofilms are a significant problem in health care settings, partly due to the presence of a nondividing, antibiotic-tolerant subpopulation. Here we evaluated treatment of S. aureus UAMS-1 biofilms with HT61, a quinoline derivative shown to be effective against nondividing Staphylococcus spp. HT61 was effective at reducing biofilm viability and was associated with increased expression of cell wall stress and division proteins, confirming its potential as a treatment for S. aureus biofilm infections. Staphylococcus aureus biofilms are commonly associated with chronic infections, particularly of the skin and soft tissue   , 5. The Staphylococcus spp.  and vancomycin (Hospira Inc.) concentrations (0.5 to 128 mg/liter) were compared. HT61 is being developed as a topical agent, and vancomycin has been used extensively as a successful topical treatment for chronic wounds and acute surgical site infections . isolate , was usefections –12. All Planktonic MICs were obtained using the broth microdilution method , and minS. aureus UAMS-1 biofilms compared to vancomycin, demonstrated by a biofilm MBC half that of vancomycin (32 mg/liter compared to 64 mg/liter). At the maximum concentration tested (128 mg/liter), HT61 caused a further 1.3 log reduction in CFUs compared to vancomycin utilized at the same concentration . These HT61 concentrations were chosen because they were below the calculated planktonic and biofilm MBCs. Use of higher concentrations would have been highly bactericidal and would have led to the accumulation of dead cells and unwanted noise within the proteome data sets. Full details of the proteomic methods, including the method of protein isolation and the instrument settings utilized, can be found in the Text S1 in the supplemental material. Briefly, planktonic cultures were grown in TSB for 12 h at 37°C with the appropriate HT61 concentrations. Biofilms were cultured for 72 h as described above prior to replacement of the used media with TSB supplemented with HT61 at the same concentrations. Biofilms were then incubated for a further 12 h before being harvested and suspended into 1 ml HBSS. Following mechanical lysis of the cells, proteins were extracted, purified, and normalized to a final concentration of 0.25 μg/μl in 3% acetonitrile–0.1% formic acid (vol/vol).The cellular response of planktonic and biofilm cultures following treatment with 0, 4, or 16 mg/liter HT61 was then investigated using ultraperformance liquid chromatography-mass spectrometryS. aureus MN8 reference database (accessed 25 January 2018) and further analyzed using a combination of UniProt database searches  and gene ontology analysis using GeoPANTHER  to oxacillin  and 495 16 mg/liter) proteins met the inclusion criteria for quantitative analysis. For HT61-treated biofilm cultures, 461 (4 mg/liter) and 498 (16 mg/liter) proteins met the inclusion criteria . HT61 tr mg/literd-glutamate into cell wall peptidoglycans (Treatment of planktonic cultures with a sub-MIC concentration of HT61 (4 mg/liter) revealed upregulation of MurD and MurI, two cell wall biosynthesis-associated proteins required for the incorporation of oglycans   cluster, a family of genes that are vital for maintaining cell shape and integrity (S. aureus cell membrane, in a manner similar to that seen with the lipopeptide antimicrobial daptomycin (dcw cluster has also been documented in biofilms of Haemophilus influenzae following d-methionine treatment, contributing to altered cell morphology (As well as being part of the CWS stimulon, a number of the differentially expressed cell wall biosynthesis components, DNA synthesis/maintenance genes, and cell cycle components comprise a segment of the division cell wall gene (ntegrity , 23. Preptomycin , 24, 25.ptomycin . In addiptomycin . Alteredrphology . It is pin vitro biofilms of S. aureus, although whether this translates to efficacy in vivo needs to be determined. Furthermore, the safety and tolerated dose of HT61 will need to be evaluated in order to determine whether it is a therapy superior to vancomycin in a clinical setting. We have also shown that HT61 influences the expression of the CWS stimulon and dcw cluster, in line with its predicted mechanism of action. Similarly to other quinoline-like compounds, it may also stimulate the SOS response.To conclude, we have demonstrated that HT61 is more effective than vancomycin at treating Statistical analyses were performed using R version 3.6.0, and figures were plotted using ggplot2 and cowplot –30.–https://doi.org/10.5258/SOTON/D0619.Proteomic data are available at the following URL:


Bisphenol S (BPS) is a common bisphenol A (BPA) substitute, since BPA is virtually banned worldwide. However, BPS and BPA have both endocrine disrupting properties. Their effects appear mostly in adulthood following perinatal exposures. The objective of the present study was to investigate the impact of perinatal and chronic exposure to BPS at the low dose of 1.5 μg/kg body weight/day on the transcriptome and methylome of the liver in 23 weeks-old C57BL6/J male mice.This multi-omic study highlights a major impact of BPS on gene expression (374 significant deregulated genes) and Gene Set Enrichment Analysis show an enrichment focused on several biological pathways related to metabolic liver regulation. BPS exposure also induces a hypomethylation in 58.5% of the differentially methylated regions (DMR). Systematic connections were not found between gene expression and methylation profile excepted for 18 genes, including 4 genes involved in lipid metabolism pathways , which were downregulated and featured differentially methylated CpGs in their exons or introns.This descriptive study shows an impact of BPS on biological pathways mainly related to an integrative disruption of metabolism  and, like most high-throughput studies, contributes to the identification of potential exposure biomarkers.The online version contains supplementary material available at 10.1186/s12864-020-07294-3. Since the ban of Bisphenol A (BPA) in the baby bottles  and the other use restrictions of this substance in relation to these reprotoxic and endocrine disruptor properties, manufacturers may use Bisphenol S (BPS), a structural analogue with similar endocrine disrupting and technological properties . CurrentCpt1a, Sterol regulatory-element binding protein-1c: Srebp1c, Fatty acid synthase: Fas, Glucokinase: Gck, Nuclear factor E2-related factor 2: Nrf2 and Obesity-associated mesoderm-specific transcript: MEST) in rodents and humans [The liver is the key organ for metabolic homeostasis by ensuring the synthesis of most blood proteins, hormone biosynthesis and turnover, protein and bile synthesis, drug and energy metabolism . Many end humans –38.Concerning BPS, there are few data available about its effects on the liver notably at molecular and epigenetic levels. Thus, the study aim was to analyzed transcriptome and methylome (DNA methylation) changes in the liver by performing a perinatal and chronic exposure of C57Bl6/J male mice during 26 weeks at 1.5 μg/kg b.w./day.The list of abbreviations (names of genes) is available in the Additional file p-value < 0.05, 374 genes were found deregulated (140 up-regulated and 234 down-regulated) which represents 1.7% of the total genes investigated , in comparison with two pathway databases, KEGG and Reactome , 40. In The same type of analyses by GSEA carried out with reference to the Reactome database revealed only significant negative NES for metabolism and mitochondrial function pathways Fig. b Additi, with thp-value < 0.05), no significant enrichment has been identified regardless of the database used, suggesting that the gene ontology clustering results rather from several genes that are moderately deregulated, inside relevant cascades, and not a limited number of genes strongly deregulated  and to a lesser extent in amino acids and glycoaminoglycan metabolism , xenobiotic metabolism , urea cycle , steroid metabolism , nucleotide metabolism  and vitamin metabolism  Fig. a , circadian rhythm , nervous transmission , transcription regulation (Polr2g), bone mineralization (Alpl) and intracellular polyamines regulation (Azin1) , mitochondrial function , transcription regulation  and metabolism  , inflammation  and cellular stress  have emerged  level, with a percent methylation difference cutoff of 25% and q-value of 0.01, this BPS perinatal and chronic exposure was associated with a significant hypo or hyper methylation of 1811 DMRs (width of 1000 nucleotides per DMR), which corresponds to 7.3% of DMRs assessed by RRBS approach, in 23 week-old male mice fed a standard diet. Among this DMRs, the percentage of hypermethylated regions was 41.5% against 58.5% for hypomethylated regions , 4.6% within CpG shelves (2–4 kb from CpG island), 9.9% within CpG shores (regions up to 2 kb away from CpG islands) and 79.9% in open sea (isolated CpGs in the genome)   the biological pathways potentially involved - based on differential mRNA expression - ii) genes with differentially methylated cytosine and iii) the connection between mRNA expression and methylation status of genes. Wild-type transcriptomic and genomic methylation profiles were generated from pools of DNA and RNA from three mouse liver samples. Omic analyzes require high quality samples. As a result, the DNA and RNA pools were not composed of the same liver samples. These non-homogeneous pools did not allow a more in-depth analysis linking RNA expression and DNA methylation and can hide inter-individual heterogeneity. However, the current results have highlighted biological pathways or target genes impacted following BPS perinatal and chronic exposure.All the biological pathways identified through biostatistics gene ontology enrichment analyses, revealed an impact of BPS on gene expression of the major liver functions: energy metabolism , detoxification metabolism , bile acids metabolism and synthesis and proteins and hormone biosynthesis and turnover. The genes associated with these biological pathways refer to pathophysiological mechanisms involving alteration of mitochondrial functions (related to oxidative stress increasing), development of inflammatory processes, disruption of circadian rhythms, impairment of intra and extracellular communication (particularly exocytosis phenomena in this study) and an impact on epigenetic regulatory mechanisms (by impacting the epigenetic machinery and one-carbon metabolism for methylation events). Interestingly, in the significant Alcoholism enriched pathway, an important part of leading edge are genes involved in transcription regulation via post-translational modification of histones  or induce the “unmasking” of regions regulating the expression of the adjacent gene (introns) –60. For To our knowledge, this is the first time that the impact of perinatal and chronic exposure to BPS on the mouse transcriptome and DNA methylation pattern has been studied. We have demonstrated through two omic approaches (microarray and RRBS), that BPS impacts genes involved in hepatic metabolism. Like most high-throughput studies, this study contributes to the identification of potential exposure biomarkers.Pregnant C57Bl/6 J mice were acquired from Charles Rivers . Bisphenol S (BPS) was provided by Sigma-Aldrich . The standard diet (SD) 4RF21 was purchased from Mucedola . This diet is certified as estrogen free and estrogenic activities were evaluated. The phytoestrogen level is certified below 4 ppb (parts per billion) according to international standards .Twenty-two pregnant mice were individually housed in a 12 h light-dark cycle at 22 °C in a conventional animal facility and allowed free access to food and water. From the first day of gestation (GD0), the pregnant C57Bl/6 J mice were divided into two groups and either exposed or not to BPS diluted in the drinking water. GD0 was defined from the detection of the vaginal plug. The treatment period was extended during lactation and in pups after weaning up to 23 weeks of age. At weaning, the male offspring were randomly separated into two groups as follows: fifteen in the group exposed to BPS and sixteen in the control group. In each cage was housed a maximum of five mice. After weaning, the litters were mixed randomly to minimize a possible litter effect. In a cage, no mouse came from the same dam. The suitable concentration of BPS was based on the estimated average daily water consumption in C57Bl/6 J mice (about 7 ml / 30 g mouse) to achieve a predicted BPS exposure of 1.5 μg / kg bw / day. Then, we checked the BPS exposure as follows: weekly water intake was evaluated by calculating the difference between the amount of water placed in the water bottle at the beginning and the amount remaining after 7 days. The levels of BPS consumed each week were normalized by day and the number of mice by cage. Thus, we obtained an average BPS intake of 1.13 (±0.084) μg/kg bw/day. The BPS was dissolved in absolute ethanol . The control group were exposed to 0.1% ethanol in drinking water. Bottles and cages were made of polypropylene (bisphenol-free). At 23 weeks old, mice were fasting for 4 h before to be sacrificed in the early afternoon. The euthanasia and the dissections were organized in small sessions, mixing treated and control animals. Anesthesia of the mice was performed with isoflurane before their euthanasia by cervical dislocation. The livers were removed, weighed, frozen in liquid nitrogen and stored at − 80 °C.http://www.ensembl.org/biomart).Liver samples were crushed and homogenized using Lysing Matrix D™ tubes  and the tissue homogenizer Precellys™24 . For each mouse, liver sample was from the medial liver lobe. The extraction of total RNA were performed using Tri-reagent™ . RNA quality (RNA Integrity Number) and quantification were realized by capillary electrophoresis . By using cartridges of Affymetrix Clariom S array hybridization, isolated cRNA of three points per condition were analyzed for global gene expression. The standard validated protocol were performed at the Genomics Platform of the Cochin Institute. Each sample consisted of a pool of RNA prepared from the liver of three separate mice and different litters. The pools were formed according to the physiological characteristics of the mice  and the nucleic acid quality, to have three homogeneous samples. For all samples, equal amounts of nucleic acid were used for each mouse. The genes showing a significant deregulation (p⩽0.05) were converted to ENSEMBL ID with Biomart and mm10 version of the mouse genome  from Affymetrix (Applied Biosystems). The differential mRNA gene expression was represented in the form of pie chart and heatmap.http://www.webgestalt.org/) by using two pathway databases: KEGG and Reactome.A Functional Gene Set Enrichment Analysis (GSEA), for down-regulated and overexpressed genes, was performed with WebGestalt 2019 (WEB-based Gene SeT AnaLysis Toolkit) (The lysis of liver samples was performed as described in 5.3 section. The samples were digested overnight with 20 μl of proteinase K (20 mg/ml) and 9 μl of RNase A (20 mg/ml). Before the DNA extraction, samples were treated with 300 U of RNase T1, 30 min at 37 °C. Then, GenElute Mammalian Genomic DNA Miniprep kit™  were used following the manufacturer’s instructions. DNA concentration was measured using the Qubit® dsDNA BR Assay Kit (Thermo Fisher Scientific) and DNA quality was analyzed using the Fragment Analyzer™ and the DNF-487 Standard Sensitivity Genomic DNA Analysis Kit .The composition of DNA and RNA pools was different as they depended on the respective nucleic acid quality.Mus musculus reference genome (Genome Reference Consortium m38 (mm10)) has been carried out using bismark v0.16.1 [RRBS, an untargeted method allowing the analysis of 70% of CpGs islands of the mouse genome, was executed by Diagenode’s DNA Methylation Profiling (RRBS Service) (Diagenode Cat# G02020000). RRBS analysis were performed as previously described . Briefly v0.16.1 , 76. The v0.16.1 , 78, wit v0.16.1 . The annRaw data and bioinformatics analysis were provided by Diagenode (Cat# G02020000) including percentage of hyper and hypomethylated region (in the form of pie chart and heatmap), CpGs and DMRs genomic distribution, identification and clustering of genes with DMRs in promoter or exons.https://string-db.org/).For genes with an existing association between significant DNA hypo- or hypermethylation and mRNA up- or downregulation, a Pearson’s correlation test was performed by distinguishing DMRs in exon, promoter and intron. Likewise, an ontology analysis was performed using String database v 11.0  in order of appearance.Additional file 2. Functional Gene Set Enrichment Analysis identified by the databases of pathways KEGG (A) or Reactome (B) for down-regulated genes and overexpressed genes using the gene set of genes passed filter criteria .Additional file 3. Gene Set Enrichment Analysis to highlight the network of transcription factor target triggered by BPS treatment using the gene set of genes passed filter criteria .Additional file 4. Gene Set Enrichment Analysis to highlight the alterations of gene functions triggered by BPS treatment.Additional file 5. Gene Set Enrichment Analysis to highlight the network of transcription factor target triggered by BPS treatment.Additional file 6. List of the connections between significant DNA methylation and gene expression deregulations induced by BPS treatment.Additional file 7. Table reporting genomic distribution of differentially methylated CpGs and DMRs.Additional file 8. List of genes of the heatmap in Fig. Additional file 9. Functional Gene Set Enrichment Analysis identified by the databases of pathways KEGG or Reactome for differentially methylated cytosines in exons and/or promoters from liver DNA of C57Bl/6 J male mice exposed to BPS compare to control mice.Additional file 10. Complete list of mRNA differential expression before application of fold threshold in TAC console.Additional file 11. Complete list of DMR in exons and promoters after RRBS analysis.