CaKmf) was assessed following 45Ca administration to the dam and subsequent radiolabel counts within the fetuses. Placental expression of calcium pathway components was measured by Western blot. Data (median) are lightest placenta expressed as percentage of the heaviest within a litter and analyzed by Wilcoxon signed-rank test. In WT mice having normally grown fetuses, CaKmf, per gram placenta near term, in the lightest placentas was increased  in association with reduced fetal calcium accretion earlier in gestation , that was subsequently normalized near term. Increased placental expression of calbindin-D9K, an important calcium binding protein, was observed in the lightest placentas near term . There was no difference in fetal calcium accretion between male and female littermates but a trend toward higher CaKmf in females (P = 0.055). These data suggest a small, normal placenta adapts calcium transfer according to its size, as previously demonstrated in a mouse model of FGR. Fetal sex had limited influence on this adaptive increase. These adaptations are potentially driven by fetal nutrient demand, as evidenced by the normalization of fetal calcium content. Understanding the regulatory mechanisms involved may provide novel avenues for treating placental dysfunction.Appropriate placental transport of calcium is essential for normal fetal skeletal mineralization. In fetal growth restriction (FGR), the failure of a fetus to achieve its growth potential, a number of placental nutrient transport systems show reduced activity but, in the case of calcium, placental transport is increased. In a genetic mouse model of FGR this increase, or adaptation, maintains appropriate fetal calcium content, relative to the size of the fetus, despite a small, dysfunctional placenta. It is unknown whether such an adaptation is also apparent in small, but normally functioning placentas. We tested the hypothesis that calcium transfer would be up-regulated in the lightest vs. heaviest placentas in the same C57Bl/6J wild-type (WT) mouse litter. Since lightest placentas are often from females, we also assessed whether fetal sex influenced placental calcium transfer. Placentas and fetuses were collected at embryonic day (E)16.5 and 18.5; the lightest and heaviest placentas, and female and male fetuses, were identified. Unidirectional maternofetal calcium clearance ( Active placental calcium transport from mother to fetus in normal pregnancy maintains a higher calcium concentration in fetal compared to maternal plasma , the activity of many placental nutrient transporters, including System A and System L amino acid transporters, is reduced, per gram placenta  mouse  litter, as reported previously for System A and glucose  Act of 1986, under the authority of a UK Home Office project license (PPLs 40/3385 and 70/8504), and were authorized by the Animal Welfare and Ethical Review Board of the University of Manchester. The methods stated in this section adhere to the ARRIVE guidelines  female (10–16 weeks old) and male (12–26 weeks old) mice were mated and discovery of a copulation plug was used to define embryonic day (E) 0.5 (term = E19.5). Mice were provided with nesting material and communally housed  in individually ventilated cages under a constant 12 h light/dark cycle at 21–23°C with free access to food  and water . Pregnant female mice were euthanized by cervical dislocation alone, or terminal anesthesia followed by exsanguination (cardiac puncture) and cervical dislocation  appropriate under ASPA schedule 1. Following euthanasia a laparotomy and hysterotomy was performed. All fetuses were rapidly killed by cervical dislocation.N = 20 litters; n = 139 placentas and fetuses) and E18.5 , pregnant WT females were euthanized and fetuses and placentas were rapidly harvested, blotted and wet weights measured. Median litter sizes were 7 [range 4–9] and 8 [4–10] at E16.5 and E18.5, respectively. Following the identification of the lightest and heaviest placentas in a litter, all placentas and fetuses  were snap frozen and stored at −80°C  and at E18.5 (n = 16 lightest vs. n = 16 heaviest), and between litter averages for females and males at E16.5  and at E18.5 . There are fewer litters in the male vs. female comparisons as litter averages were calculated only for those litters with at least 2 males and 2 females.On E16.5  and NDS   was assessed across the intact placenta at E16.5 (N = 10 litters) and E18.5 (N = 9 litters) as previously described  into the tail vein of the anesthetized dam, a cardiac puncture was performed 2–3 min post-infusion which exsanguinated the dam. All fetuses and placentas were collected, assessed for total radiolabel accumulation and compared to a maternal plasma 45Ca disappearance curve (see below). CaKmf was calculated as previously described  and at E18.5 (n = 9 lightest vs. n = 9 heaviest); and between litter averages for female and males at E16.5  and at E18.5 . Only those litters with at least 2 males and 2 females were included in the female vs. male comparisons.Unidirectional maternofetal N = 13 litters) or E18.5 (N = 13 litters) and fitted to a one-phase exponential decay model (r2 > 0.5), as described previously  and E18.5 (N = 8 litters) by inductively coupled plasma atomic emission spectroscopy  following ashing of fetuses overnight at 800°C in a Gallenkamp Furnace . Comparisons were made between: the lightest and heaviest placentas from each litter at E16.5 (n = 9 lightest vs. n = 9 heaviest) and at E18.5 (n = 8 lightest vs. n = 8 heaviest); and between litter averages for female and males at E16.5  and at E18.5 . Only litters with at least 2 males and 2 females were included in the female vs. male comparisons.As previously described  and E18.5 (N = 7 litters) were homogenized and processed as described previously  and calcium ATPase isoform 1 . β-actin  was used as a loading control; no difference was observed in β-actin expression between groups. Negative controls were by omission of primary antibody. Immunoreactive species were detected with fluorescent-conjugated secondary antibodies  and membranes imaged using an Odyssey Sa Infrared Imaging System (Li-COR). Signal density was measured using Image Studio Lite (Li-COR). All signals were in the linear range of detection. Membrane fractions were used for assessing the expression of TRPV6 and PMCA1 and the cytosolic fraction was used for assessing calbindin-D9K expression. Comparisons were made between the lightest and heaviest placentas at E16.5 and at E18.5 separately.The lightest and heaviest placentas harvested at E16.5 , and also as the litter average of females as a percentage of the litter average of males , or median [range] where the experimental P < 0.05 was considered statistically significant.The solid line on graphs represents the median value. To calculate sample sizes per experiment, we utilized data from previous studies  and E18.5 (N = 16), the weights of the lightest placentas were 78 and 79% of the heaviest placentas (Table P < 0.01) and E18.5  but were still within the normal range of fetal weight . Fetal weight: placental weight (F:P) ratio was used as a proxy of placental efficiency (gram of fetus produced per gram of placenta) and was higher in the lightest vs. heaviest placenta group at E16.5 and E18.5.At E16.5, 75% (15/20) of the placentas in the lightest groups were from females; this figure increased to 100% at E18.5. At E16.5, 75% (15/20) of the placentas in the heaviest group were from males; this figure was 81% (13/16) at E18.5. At E16.5 (N = 16) and E18.5 (N = 14), placental weights from female fetuses  were 93 and 92% of those from male fetuses (Table P < 0.0001) and at E18.5 . F:P weight ratio was similar in males and females at E16.5 but higher in females at E18.5 (P < 0.01).At E16.5 (P < 0.05). At E18.5, fetal calcium content was comparable between the lightest compared to the heaviest placenta group. There was no significant difference in fetal calcium content at either gestational age between female and male littermates  was not different between the lightest and heaviest placenta groups at E16.5 but was significantly higher in the lightest compared to the heaviest placentas at E18.5 . In the protein expression studies, the lightest placentas were all from female fetuses whilst the heaviest placentas were predominantly from male fetuses (5/8 63%] at E16.5 and 5/7 [71%] at E18.5). At E16.5, there was no difference in the expression of calcium transporters, TRPV6 and PMCA1, and the calcium binding protein, calbindin-D% at E16.9K expression, is an example of a placental adaptation which promotes fetal calcium acquisition despite a relatively small placental size. The normalization of fetal calcium accretion, relative to fetal size, by E18.5, following a reduction at E16.5, may be indicative of a fetus signaling to its placenta, by as yet unknown mechanisms, to increase maternofetal transfer of calcium. This adaptation by a small placenta is also likely to prevent fetal bone mineralisation being compromised; this is of particular importance as poor fetal provision of calcium in utero has been linked with an increased risk of developing osteoporosis later in life  mouse litter had a higher maternofetal clearance of calcium (per gram placenta) than the heaviest placenta near term. We suggest this increased transfer efficiency, coinciding with increased placental calbindin-DCaKmf in females vs. males at E18.5 (P = 0.055). This indicates that the lightest placentas' adaptive increase in unidirectional maternofetal calcium transfer, in apparent response to reduced fetal calcium content, is unlikely to be solely explained by the disparity between males and females that make up the lightest and heaviest groups.As the lightest placentas were predominantly from female fetuses (75% at E16.5 and 100% at E18.5), it is possible that the effects seen were due not just to differences in placental size, but also due to sex-dependent effects. To investigate this, we compared female and male fetuses within each litter to determine whether fetal sex had an impact on the adaptive changes observed in placental calcium transfer. On average, females had lower fetal and placental weights than their male littermates. Females also demonstrated a higher F:P ratio vs. males, indicating greater placental efficiency in female fetuses; this may account for the comparable birthweight to males near term. Fetal calcium content and placental calcium transfer, however, were comparable in females and males at both gestational ages, although there was a trend toward increased In WT mouse litters, the lightest placentas were, on average, more than 20% lighter than the heaviest at both gestational ages, whilst the difference in the weight of fetuses with the lightest compared to heaviest placentas fell with increasing gestational age; a reduction of 11% at E16.5 and 6% at E18.5. This is indicative of a fetus catching up to its sibling close to term, likely due to increased rates of placental nutrient transfer, as described in this and another study . In those litters that were not comparisons of male and female but were female to female comparisons (3/8 litters at E16.5 and 2/7 litters at E18.5), calbindin-D9K expression in the lightest as a percentage of the heaviest placenta was 51%, 106% and 115% at E16.5, and, 108% and 117% at E18.5. These values, at E18.5 especially, appear broadly in line with the male vs. female comparisons but we cannot discount the possibility that the increased expression of calbindin-D9K in the lightest placentas at E18.5 may be due, in part, to fetal sex.In rodents, increased placental calcium transfer and fetal accrual toward term (E15–19) is accompanied by a 14 and 5 fold increase in TRPV6 and calbindin-D9K protein expression , indicating calbindin-D9K contributes to an adaptive response in FGR, in common with the current study. Like P0 mice  mouse  knockout mouse. PTHrP knockout mice demonstrated increased maternofetal calcium flux as measured in vivo and using a dual perfusion model (Bond et al., in vivo, novel culture systems which mimic the placental barrier in vitro may prove useful in this regard (Blundell et al., Calbindin-DDespite the predominance of female fetuses in the lightest placenta group, fetal calcium content did not alter according to fetal sex. However, there was a trend toward increased unidirectional maternofetal calcium transfer in females vs. males near term, consistent with the increased F:P ratio at E18.5 in females vs. males. This increased F:P ratio suggests that functional adaptations in the placenta prevent female fetuses being smaller than their male littermates near term and that, whilst not quite significant in terms of calcium, other nutrient transport systems, such as system A, are worthy of investigation. The current study and recent collated data Clifton,  demonstrThis study has shown that calcium transfer across lightest placentas in WT litters is adaptively up-regulated so that all fetuses, whether with lighter or heavier placentas, demonstrate an appropriate level of calcium accretion relative to their size. This will likely have important implications in terms of appropriate fetal skeletal mineralisation. The timeline of this adaptation is similar to those changes in placental nutrient transfer observed in P0 knockout mice (Dilworth et al., Conception or design of the work: CH, CS, SG, and MD. Acquisition, analysis or interpretation of data for the work: CH, LR, CS, SG, and MD. Drafting the work or revising it critically for important intellectual content: CH, LR, CS, SG, and MD. All authors approved the final version of the manuscript and agree to be accountable for all aspects of the work ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed.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.


ENESTfreedom is evaluating treatment-free remission (TFR) following frontline nilotinib in patients with chronic myeloid leukemia (CML) in chronic phase. Following our primary analysis at 48 weeks, we here provide an updated 96-week analysis.4.5 [BCR-ABL1 ≤ 0.0032% on the International Scale (BCR-ABL1IS)], and sustained deep molecular response (DMR) during a 1-year consolidation phase. Patients restarted nilotinib following loss of major molecular response .Attempting TFR required ≥ 3 years of nilotinib, a molecular response of MR4.5 by the data cut-off. Ninety-six-week TFR rates were 61.3, 50.0, and 28.6% in patients with low, intermediate, and high Sokal risk scores at diagnosis, respectively. Patients consistently in MR4.5 during consolidation had higher TFR rates (50.6%) than patients with ≥ 1 assessment without MR4.5 during consolidation (35.0%). In a landmark analysis, 96-week TFR rates for patients with MR4.5, MR4 (BCR-ABL1IS ≤ 0.01%) but not MR4.5, and MMR but not MR4 at TFR week 12 were 82.6, 23.1, and 0%, respectively. There were no reports of disease progression or death due to CML; overall adverse event frequency decreased following TFR. Within the follow-up period, TFR did not adversely affect disease outcomes.Ninety-six weeks after stopping treatment (3.6-year median prior nilotinib duration), 93 of 190 patients (48.9%) remained in TFR. Of 88 patients who restarted nilotinib following loss of MMR, 87 regained MMR and 81 regained MRThese results demonstrate the feasibility and durability of TFR following frontline nilotinib and emphasize the importance of sustained DMR for TFR. For patients with chronic myeloid leukemia in chronic phase (CML-CP) who have achieved a stable deep molecular response (DMR) using BCR-ABL1 tyrosine kinase inhibitors (TKIs), treatment-free remission (TFR) following TKI cessation is an emerging goal ] than imatinib  . This is comparable to TFR rates following imatinib treatment, despite a shorter duration of prior TKI therapy (median of 3.6 years in ENESTfreedom vs ≈ 5–8 years in prior imatinib studies)  than frontline treatment with imatinib , ≤ 2 assessments between MR4 and MR4.5, and MR4.5 in the last assessment, could enter the TFR phase. Patients in the TFR phase were monitored by RQ-PCR every 4 weeks during the first 48 weeks, every 6 weeks during the next 48 weeks, and every 12 weeks thereafter up to 264 weeks after the last patient entered the TFR phase. Nilotinib reinitiation was triggered by any single assessment showing BCR-ABL1IS > 0.1% (loss of MMR). Patients in the reinitiation phase were monitored every 4 weeks for the first 24 weeks and every 12 weeks thereafter  to assess molecular response.ENESTfreedom is an ongoing, single-arm, phase 2 study NCT01784068) that has been previously described  was defined as the time from TFR start to the loss of MMR, nilotinib reinitiation for any reason, progression to accelerated phase/blast crisis (AP/BC), or death from any cause and was estimated using the Kaplan–Meier method. For patients without any such event, TFS was censored at the date of last assessment. Molecular response rates in patients who entered retreatment due to loss of MMR in the TFR phase were calculated as cumulative incidences.4.5 stability during the consolidation phase and calculated TFR rates at 48 and 96 weeks in each subgroup. Patients’ Sokal risk scores at diagnosis were not collected at study entry but rather retrospectively following a protocol amendment; therefore, data on Sokal risk at diagnosis were not available for all patients. MR4.5 stability during the consolidation phase was defined based on patients’ responses during the 4 protocol-mandated RQ-PCR assessments collected during that phase. TFR rates were evaluated in patients with MR4.5 in all consolidation phase assessments vs those with ≥ 1 assessment worse than MR4.5. To assess the association between BCR-ABL1IS levels at week 12 of the TFR phase and future TFR maintenance, we stratified patients remaining in the TFR phase for ≥ 12 weeks by BCR-ABL1IS level at week 12  and calculated TFR rates at 48 and 96 weeks. Patients with missing BCR-ABL1IS values at week 12 were excluded from this analysis.To investigate factors potentially associated with TFR, we stratified all patients attempting TFR by Sokal risk score at diagnosis and by MRUpdated safety data included a characterization of AEs across the consolidation phase and the first and second 48 weeks of the TFR phase for patients remaining in the TFR phase for > 48 weeks. The musculoskeletal-pain AE grouping included events reported using the preferred terms of myalgia, arthralgia, bone pain, spinal pain, pain in extremity, and musculoskeletal pain. Time to first musculoskeletal-pain event among patients entering the TFR phase was estimated using the Kaplan–Meier method. AEs were assessed according to the Common Terminology Criteria for Adverse Events version 4.03.This study was designed and conducted in accordance with the ethical principles of the Declaration of Helsinki and the ICH Harmonized Tripartite Guidelines for Good Clinical Practice. An independent ethics committee or institutional review board for each study center reviewed the study protocol and its amendments. All patients provided written informed consent before any study procedures and in accordance with local laws and regulations.n = 190) had a median age at study entry of 55 years (range 21–86 years), median total nilotinib duration prior to entering the TFR phase of 43.5 months (range 32.9–88.7 months), and a median time from first MR4.5 until entering the TFR phase of 30.4 months (range 12.3–83.0 months). The Sokal score at diagnosis was low in 62 patients (32.6%), intermediate in 50 patients (26.3%), high in 28 patients (14.7%), and unavailable for the remaining patients. The median duration of follow-up in the TFR phase at the 96-week data cut-off date was 75.9 weeks (range 8.4–133.0 weeks). At the data cut-off date, of the 190 patients who entered the TFR phase, 93 (48.9%) remained in the TFR phase, 88 (46.3%) reinitiated nilotinib, and 9 (4.7%) discontinued from the study while in the TFR phase , while the remaining 2 discontinued from the study without MMR loss (1 each due to patient decision and loss to follow-up). In addition to the three patients no longer in MMR at 96 weeks, a fourth lost MMR at 120 weeks; all four of these patients had lost MR4.5 and MR4 in the first 48 weeks of TFR. The estimated TFS rate at 96 weeks was 50.9% (95% CI 43.6–57.8%), and the Kaplan–Meier-estimated median TFS was 120 weeks .At week 96 of the TFR phase, 93 of 190 patients [48.9% (95% CI 41.6–56.3%)] remained off treatment and in MMR; 88 were also in MRely Fig. , the sin4.5 in all four assessments during the consolidation phase, while the remaining 20 had ≥ 1 assessment worse than MR4.5. The TFR rate in the patients with MR4.5 in all assessments vs those lacking MR4.5 in ≥ 1 assessment was 52.9 vs 40.0% at 48 weeks and 50.6 vs 35.0% at 96 weeks, respectively.At both week 48 and 96 of the TFR phase, patients with low Sokal risk scores at diagnosis had higher TFR rates  than those with intermediate (50.0 and 50.0%) or high (32.1 and 28.6%) scores at diagnosis , the overall frequency of AEs decreased from 85.0% during the consolidation phase to 76.0% during the first 48 weeks of the TFR phase and 62.0% during the second 48 weeks of the TFR phase. In this group of patients, the most frequent all-grade AEs reported during the second 48 weeks of TFR were nasopharyngitis (9.0%) and back pain (5.0%). Overall, among these 100 patients, cardiovascular events were reported in 3 during the consolidation phase, 2 during the first 48 weeks of TFR, and 1 during the second 48 weeks of TFR  were predictive of successful TFR (Hochhaus et al. 4.5 consistently throughout the consolidation phase both had numerically higher TFR rates. In agreement with this, the STIM1 study reported that low and intermediate Sokal risk scores were associated with lower molecular recurrence rates (Etienne et al. We previously performed multivariate logistic regression analyses to evaluate whether patient baseline characteristics (including sex, age, and duration of nilotinib treatment or MR4.5 at 12 weeks, close monitoring is especially critical, since they may have an increased risk of molecular relapse. This is consistent with the fact that many TFR studies reported that most molecular relapses occurred early in TFR (Etienne et al. 4 during the first 12 weeks of the TFR phase went on to lose MMR within the first 48 weeks. We have demonstrated that loss of MMR is a safe and reproducible trigger for retreatment with nilotinib; for patients in whom BCR-ABL1IS levels rise to above 0.01% within the first 12 weeks, diligent follow-up is critical to ensure that treatment is resumed as soon as loss of MMR is evident.We additionally found that a deeper molecular response at 12 weeks of TFR appeared to be predictive of maintaining TFR through 96 weeks. Thus, for patients with MMR but not MR4.5 throughout the year prior to nilotinib cessation or low Sokal risk scores at diagnosis have numerically higher TFR rates. Strong predictors of successful TFR after stopping nilotinib remain to be identified. Overall, these findings support the use of frontline nilotinib in patients with newly diagnosed CML-CP for whom TFR might be a future treatment goal.The updated results from ENESTfreedom presented here demonstrate the durability of TFR following frontline nilotinib and continue to demonstrate the safety of TFR. The results suggest that patients with consistent MR


To assess the effect of applying a protocol for image selection and the number of images required for adequate quantification of corneal nerve pathology using in vivo corneal confocal microscopy (IVCCM).IVCCM was performed in 35 participants by a single examiner. For each participant, 4 observers used a standardized protocol to select 6 central corneal nerve images to assess the inter-observer variability. Furthermore, images were selected by a single observer on two occasions to assess intra-observer variability and the effect of sample size was assessed by comparing 6 with 12 images. Corneal nerve fiber density (CNFD), branch density (CNBD) and length (CNFL) were quantified using fully automated software. The data were compared using the intra class correlation coefficient (ICC) and Bland-Altman agreement plots for all experiments.The ICC values for CNFD, CNBD and CNFL were 0.93 (P<0.0001), 0.96 (P<0.0001) and 0.95 (P<0.0001) for inter-observer variability and 0.95 (P<0.0001), 0.97 (P<0.001) and 0.97 (P<0.0001) for intra-observer variability. For sample size variability, ICC values were 0.94 (P<0.0001), 0.95 (P<0.0001), and 0.96 (P<0.0001) for CNFD, CNBD and CNFL. Bland-Altman plots showed excellent agreement for all parameters.This study shows that implementing a standardized protocol to select IVCCM images results in high intra and inter-observer reproducibility for all corneal nerve parameters and 6 images are adequate for analysis. IVCCM could therefore be deployed in large multicenter clinical trials with confidence. The use of corneal confocal microscopy (CCM) for the assessment of neuropathy has grown considerably over the last decade. Many studies have shown corneal nerve pathology in patients with a range of peripheral neuropathies –7. IndeeVagenas et al.  have shoImages from 35 randomly selected participants with obesity who had previously undergone detailed assessment of neuropathy and corneal confocal microscopy were included. Informed written consent was obtained from all patients before their participation in the study. This research adhered to the tenants of the declaration of Helsinki and was approved by Greater Manchester Central and North Manchester research ethic committees. Participants were excluded if they had corneal dystrophy or cystic corneal disorders.Corneal confocal microscopy (CCM) was performed in both eyes of participants using a Heidelberg Retinal Tomograph III with Rostock Cornea Module (HRT III RCM)  according to our previously established protocol . The illTo evaluate the inter observer variability when selecting CCM images; four independent observers from the University of Manchester, United Kingdom participated in the study. Observer 1 (OB1) and observer 2 (OB2) were experienced optometrists in identifying and extracting CCM images and considered ‘experts’. Observers 3 and 4 (OB3 and OB4) were medical doctors without any experience of working with CCM and had minimal experience in selecting and analyzing images of the corneal sub-basal nerve plexus and were therefore considered ‘beginners’. All four observers’ independently selected six images of the sub-basal nerve plexus from the central cornea (3 per eye) for each subject and followed a standardized protocol detailed below .Selected images were required to be from the center of the cornea. Image choice from the central cornea was based on both the live image on the CCD camera as well as the orientation of corneal nerves. The red reflex, (the reflex of the laser beam on the cornea), was required to be in the center of the eye in the live image for the selected image. With regard to the orientation of the nerves they are usually vertical in the center and become oblique in the peripheral cornea, therefore only images with vertically oriented nerves were selected.Images of differing numbers of main nerves were selected such that images with the least, intermediate and highest number of main nerves were selected. This is especially relevant when images are selected from control subjects with many nerves, compared to patients with severe neuropathy with very few nerves.Images were required to be of high quality with paucity of pressure lines, optimal contrast and with an absence of overlap, and where part of the image was from either the epithelial or stromal layers in addition to the sub-basal layer . Fig 1, To evaluate the intra-observer variability; OB2 exported 6 images per participant on two separate occasions (OB2a and OB2b) with a one-week interval in between .To evaluate whether the image sample size affects corneal nerve quantification, OB1 exported 6 images on one occasion (OB1a) and then 12 images on a separate occasion (OB1b) for each participant.2), corneal nerve fibre length  (mm/mm2) and corneal nerve branch density  (no./mm2).Images were analyzed using purpose designed fully automated nerve analysis software  (ACCMetrics)   to quanthttps://figshare.com/s/125f0c92e3535bcc8f5f“.Statistical analysis was performed using IBM SPSS v19.0 . The Shapiro-Wilk normality test was employed to assess whether the data is normally distributed or not. Paired sample t test and repeated measure ANOVA was used to assess the differences between measurements. Intraclass correlation coefficient (ICC) was measured to identify inter and intra-observer repeatability and were considered as excellent if 0.80–1.0. The Bland-Altman agreement plots  were use2 and the neuropathy disability score (NDS) was 2.12 ± 2.78.Thirty-five subjects (18 men and 17 women) with diabetes and/ or obesity were included in this study. The average age was 49.97 ± 12.47 years, HbA1c was 47.77 ± 21.35 mmol/mol, BMI was 41.19 ± 15.24 kg/mAmong 4 observers  the ICC values with confidence intervals (CI) were 0.93 (95% CI- 0.93–0.97) for CNFD, 0.96 (95% CI-0.95–0.98) for CNBD and 0.95 (95% CIs-0.95–0.98) for CNFL . The ICC2 for CNFD, 2.75 ± 8.9/mm2 for CNBD and 0.16 ± 1.15/mm2 for CNFL. ICC values and mean difference of CCM parameters among all observers are shown in There was no significant difference between CNFD , CNBD  and CNFL  for an expert on the first and second occasion (OB2a and OB2b), respectively. The ICC values for OB2 on both occasions were 0.95 (95% CI-0.91–0.97) for CNFD, 0.97 (95% CI-0.94–0.98) for CNBD and 0.97 (95% CI-0.95–0.98) for CNFL . Fig 3A–To measure the optimal image sample size, CNFD, CNBD and CNFL were measured by OB1 on two separate occasions (6 images on the first occasion and 12 images on the second occasion). CNFD , CNBD  and CNFL  did not differ significantly on the first and second occasions. ICC value between OB1a and OB1b were 0.94 (95% CI-0.88–0.97) for CNFD, 0.95 (95% CI-0.89–0.97) for CNBD and 0.96 (95% CI-0.92–0.98) for CNFL. The quantification of corneal sub-basal nerve morphology has evolved to become an important technique for assessing the severity and progression of diabetic neuropathy , 10, 17.This is the first study showing the utility and reproducibility of a standardized protocol for image selection. We demonstrate excellent inter- and intra-observer concordance for each nerve fibre metric. The highest intra-observer and inter-observer concordance was found for CNFL and the highest inter-observer concordance amongst all observers was found for corneal nerve branch density (CNBD). Previously Hertz. et.al. (2011) have concluded that CNFL is the best biomarker to identify patients with and at risk of sensorimotor polyneuropathy, whereas Petropoulos et.al. showed that corneal nerve fibre density is the most repeatable corneal nerve parameter , 21. CerAlthough intra class correlation was excellent for both experiments comparing the results measured by both experts and 2 experts versus 2 beginners, the mean of the difference was lower when comparing the results between 2 experts. This suggests that the level of expertise will affect the test retest variability as previously suggested by Petropoulos et al 2013 [In conclusion, we show that the application of a standardized method for selecting and analyzing images of the corneal sub-basal plexus with automated quantification provides excellent inter and intra observer repeatability irrespective of investigator experience and image number.S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.


Non-invasive prenatal testing (NIPT) for aneuploidy using cell-free DNA in maternal plasma has been widely adopted. Recently, NIPT coverage has expanded to detect subchromosomal abnormalities including the 22q11.2 deletion. Validation of a SNP-based NIPT for detection of 22q11.2 deletions demonstrating a high sensitivity (97.8%) and specificity (99.75%) has been reported. We sought to further demonstrate the performance of a revised version of the test in a larger set of pregnancy plasma samples.Blood samples from pregnant women (10 with 22q11.2-deletion‒affected fetuses and 390 negative controls) were successfully analyzed using a revised SNP-based NIPT for the 22q11.2 deletion. The sensitivity and specificity of the assay were measured.Sensitivity of the assay was 90% (9/10), and specificity of the assay was 99.74% (389/390), with a corresponding false positive-rate of 0.26%.The data presented in this study add to the growing body of evidence demonstrating the ability of the SNP-based NIPT to detect 22q11.2 deletions with high sensitivity and specificity. Non-invasive prenatal testing (NIPT) using cell-free DNA from maternal plasma has altered the landscape of prenatal screening, owing to its improved performance over traditional screening techniques , 2. ClinWith a reported population-wide frequency of 1 in 3,000–6,000 live births, 22q11.2 DS is the most common microdeletion syndrome . SeveralFor individuals with 22q11.2 DS pregnancy, changes in management such as delivery at specialized neonatal care centers may be advised and early therapeutic intervention can positively affect clinical outcomes  but apprPreviously, validation of a targeted single–nucleotide-polymorphism (SNP)-based test targeting the 22q11.2 deletion (~3 Mb A–D region) demonstrated an analytical detection rate of 97.8% and a false positive rate of 0.76% . Herein,Pregnant women were enrolled at six collaborating prenatal centers and all samples were collected in accordance with the following institutional review board (IRB)-approved protocols: Columbia University IRB , The Children's Hospital of Philadelphia Research Institute IRB Children's Hospital of Philadelphia, PA), Western IRB  and Biomed IRB . A signed informed consent was obtained from all enrolled participants.Two tubes of blood (approximately 20 ml) were obtained from study participants who underwent amniocentesis or chorionic villus sampling; the study groups consisted of 10 women with a confirmed molecular diagnosis of fetal 22q11.2 deletion  and 409 women with a confirmed unaffected pregnancy based on molecular analysis (with no ultrasound findings suggestive of the disorder). For each sample, information regarding maternal age and gestational age of the patient at the time of the blood draw, and time between invasive procedure and blood draw was requested. Out-of-specification samples , and SNP allele ratios were determined  to generate a fetal copy number for the 22q11.2 region. Risk status for the 22q11.2 deletion was assigned as “high risk” (≥0.95 confidence for the presence of the 22q11.2 deletion), “low risk” (≥0.90 confidence for the absence of the deletion), or “risk unchanged”/”no calls”  [6 reads/sample) to confirm “high-risk” status. For cases with a fetal fraction of 2.8–6.5%, the sample was evaluated only for the presence or absence of the paternally-inherited haplotype.Maternal plasma samples were analyzed at Natera’s Clinical Laboratory Improvement Act-certified, and College of American Pathologists-accredited laboratory using an updated SNP-based screening methodology. The protocol uses a single set of pooled targeted primers to perform a massively multiplexed PCR (mmPCR) amplification targeting SNPs covering chromosomes 13, 18, 21, 22, X, and Y. The target set contains 13,926 distinct genetic loci, including 1,351 SNPs spanning a 2.91 Mb section of the 22q11.2 region that constitutes ~87% of all deletions detected in individuals with the 22q11.2 DS. Following amplification, samples were sequenced using the NextSeq 500 platform  at a standard depth of read  (4 affected samples), or massively multiplexed PCR (mmPCR)/NGS analysis  [To validate the performance of the updated SNP-based technology in detecting the 22q11.2 deletion, 419 pregnancy plasma samples with known fetal copy number for the 22q11.2 region were evaluated. The study included 10 samples with a confirmed affected fetus  and 409 samples)  on fetalAmong the 10 affected samples, the deletion was confirmed by array or mmPCR to occur in the common A–D region in six samples and deletion sizes ranged from 2.55–3.16 Mb. The remaining four affected samples were confirmed by FISH and were conservatively treated as having the full ~3 Mb A–D deletion . In 8 ofThe mean maternal age for the affected samples was 28.1 weeks compared to 26.0 weeks for unaffected samples . The gestational age range was higher  for the affected samples compared to the unaffected samples  with a p-value of 0.005. The affected samples also had a higher fetal fraction distribution  compared to the unaffected samples  with a p-value of 0.04 .Of the 10 affected pregnancy samples, 9 were identified as high-risk (test positive) and 1 was identified as low-risk (test negative), generating a sensitivity of 90%  . All higPreviously, we reported that the SNP-based NIPT detects the fetal 22q11.2 deletion (~3 Mb A–D deletion) with a sensitivity of 97.8% and a false positive rate of 0.76% . FinallyThe false positive rate with the revised methodology was 0.26% compared with 0.76% in the previous validation study . HoweverA limitation of this study is the higher gestational age distribution of the affected pregnancy cases compared with those typically observed in a clinical cohort. The higher gestational age is attributed to the delay in identifying and procuring samples from affected pregnancies. As gestational age and fetal fraction are positively correlated, higher fetal fractions among the affected pregnancies would be expected to affect the assay’s performance. Note that in our previous validation study, which used mixture samples, the fetal fraction distribution of the affected samples was specifically designed to approximate that of a clinical cohort , and in The relative rarity of the 22q11.2 deletion makes it challenging to assemble a patient cohort closely resembling a clinical population for use in a validation study. Historically, validation studies for rare conditions required alternative approaches—e.g., testing maternal plasma diluted with DNA from an affected individual , 21. As Subchromosomal abnormalities occur in >1% of pregnancies and are often associated with severe phenotypes . NIPT ha


Background: This study was conducted to find out the age when tibiofemoral angle starts to be in valgus and reaches maximum angle. The differences of the angles between genders were also studied.Methodology: This cross sectional study on tibiofemoral angle was conducted among 160 normal healthy children using clinical measurement method. The children between 2 18 months to 6 years old were assigned to 5 specific age groups of 32 children with equal sex distribution.Result: This study had shown a good inter-observer reliability of tibiofemoral angle measurement with intraclass correlation coefficient (ICC) of 0.87 with narrow margin of 95% confident interval . The mean tibiofemoral angle for children at 2 , 3 , 4 , 5 and 6 years old were 2.25° (SD=0.53), 8.73° (SD=0.95), 7.53° (SD=1.40), 7.27° (SD=1.14) and 6.72° (SD=0.98) respectively. The age when they achieved maximum valgus tibiofemoral angle was 3 years old. The maximum mean (SD) tibiofemoral angle for boys, girls and all children were 8.91° (SD=1.17), 8.56° (SD=0.62) and 8.73° (SD=0.95)respectively. The mean tibiofemoral angle showed no statistically significant difference between girls and boys except for the 5-year-old group, in which the mean TF angle for girls was 7.560 (SD=0.95) and for the boys was 6.970 (SD=1.26) with p-value of 0.037.Conclusion: Measurement of tibiofemoral angle using the clinical method had a very good inter-observer reliability. The tibiofemoral angle in Malay population was valgus since the age of 2 years with maximum angle of 8.730 (SD=0.95) achieved at the age of 3 years. Then the knee angles become valgus at 2 to 3 years with maximum mean valgus of 120 at 3 years old. The valgus angle of knee decreased to between 50 and 60 in children between 7 and 12 years. However, there have been contradicting data of normal ranges of knee angle in relation to age, transition time from varus to valgus and the maximum peak valgus of different populations and ethnic groups6.The normal development of tibiofemoral angle evolves through a number of stages in a normal growing child. Salenius and VankkaThe aim of this study was to test the reliability of clinical method for measurement of tibiofemoral angle. We investigated the pattern of tibiofemoral angle. among Malay children using this clinical measurement.This cross-sectional study was conducted from January 2011 till January 2012 in Kelantan, Malaysia. Children age ranged from 2 to 6 years were put into 5 specific-age-groups consisting of 32 children with equal sex distribution. The chronological age of the subject was rounded off to the nearest integer. For example, children from the age of 1 years and 7 months to the age of 2 years and 6 months were included in the age group of 2 years. All the children must be with the weight and height within 2nd and 98th centile. Children with skeletal and neuromuscular disorder and Limb Length Discrepancy more than 2 cm were excluded from the study.et al4, δ (a difference in population means) = 1.76, σ (is the within group standard deviation) = 2.2, the sample size required was 160 children after considering 20% dropout rate.The sample size was determined by using independent t-test calculation method in PS Software . Parameters that had been used were :α (Type 1 error) = 0.05; power (probability of correctly rejecting the null hypothesis) = 0.8. ; m  = 1. Based on the previous study done by Saini et al3. Angle between the two lines was measured as tibiofemoral angle using goniometer while the children in standing position with patella pointing forward and either both knees or both ankles were just touching each other. The knees and hips joints were positioned in full extension with arm placed behind the back to increase the stability of posture. The first line was the line transacting anterior superior iliac spine and the mid-point of patella . The second line was the line transacting midpoint of ankle . Measurements on other children were done by a single person at two different intervals for each child and mean of two measurements was taken as final tibiofemoral angle.Tibiofemoral angle was measured according to the method described by Arazi All data entry and analysis was performed using the Social Science and Statistical Package (SPSS) version 20.0 which are licensed to Universiti Sains Malaysia, Kubang Kerian Kelantan. The means, standard deviations, and 95% confidence intervals (95% CI) were calculated for each age and gender group. The mean tibiofemoral angle for both boys and girls were tested using the independent t-test. Inter-rater reliability of tibiofemoral angle measurement was measured using with 95% confidence intervals to gauge the precisions of the ICCs.This study was approved by Ethical Committee, School of Medical Sciences, Universiti Sains Malaysia, Kubang Kerian, Kelantan.Between two observers was 0.87 indicating a very good reliability.o (SD= 0.53), 8.73o (SD= 0.95), 7.53o (SD= 1.40), 7.27o (SD= 1.14) and 6.72o (SD= 0.98) respectively (o (SD=1.17), 8.56o (SD=0.62) and 8.73o (SD= 0.95) respectively. Independent t-test showed no significant difference of mean tibiofemoral angle between boys and girls except for those at the age group of 5 years (There were 160 children (320 limbs) involved in this study. The mean of tibiofemoral angle for children at the age groups of 2 years, 3 years, 4 years, 5 years and 6 years were 2.25ectively . The patectively . The max 5 years .The value of normal physiological range of the tibiofemoral angle in specific population would help clinicians to decide on further treatment plan without having to expose children to unnecessary radiations, orthotics or braces.1. Many investigators chose the clinical method of measurement with goniometer because it was radiation-free, cheap and easily performed7. Mathew et al7 had found the clinical measurement of the tibiofemoral angle to have minimal intra-observer variability and high inter-observer reliability. In this study we also found a high inter-observer reliability by with intra-class correlation coefficient (ICC) of 0.87. By having an equal large sample of 64 for each age group and 32 for each gender subgroup, this study would be reliable to give conclusion on the timing of maximum valgus angle occurrence and differences between genders in the study group.Clinical measurement of tibiofemoral angle had been shown to have good correlation with roentgenographic measurement8. Salenius and Vankka1 postulated that when a child starts to learn how to walk, he/she would increase the stability by holding the feet wide apart. This posture will increase the pressure over the lateral part of epiphyseal growth plate resulting in a slower growth process as compared to the medial part of epiphyseal growth plate, thus leading to genu valgus. Oyewole et al9 found that varus shape tibiofemoral angle decreased abruptly in the first 3 months of life and decreased slowly after that until it turned to valgus. Unfortunately in our study we did not include timing when the children started to walk.We focused on children between age group of 2 to 6 years because most of the changes occur between these age groups. The age group of 2 years includes children from 18 months and all of them were able to walk. Before walking age, most children have physiological genu varus12.The presence of valgus alignment of tibiofemoral angle in Malay from age of 2 to 6 years, as found in this study, is similar to Finland, Chinese, American, Turkish, Nigeria, Korean, Indian and Saudi children13. There were two other studies reported a statistically significant differences between the gender on children between 5 to 6 years old6. Similar to our study, Rahman et al6 found difference of tibiofemoral angle of less than one degree with more valgus in girls. Reason for differences to occur at this age group remained to be explored.Our study showed that there was no significant difference in the development of the tibiofemoral angle between boys and girls between 2 to 6 years old except for 5 years old group. Many other studies reported no tibiofemoral angle difference between genders in children between 2 to 6 years old12. In contrast, maximum tibiofemoral angle was shown to occur at 6 years old in female and 7 years old in male Turkish children3. In Indian children, it occurred at 5 years old in boys and 6 years old in girls7.In our study, we found that the age when maximum valgus angle achieved in Malay children was 3 years old. The children from Finland, China, America, Nigeria, Korea, and Saudi Arabia also achieved maximum valgus angle between 3 to 4 years old0 (SD=0.95) in this study was comparable with Chinese, Turkish, American, Korean, Indian and Saudi Arabian children12. A slightly lower maximum valgus angle was found in Nigerian children (7.10) and South Indian children (6.70 to 7.30)11. We did not compare our finding with the study by Salenius and Vankka1 who showed higher maximum valgus angle of 120 as they measured the tibiofemoral angle by using radiograph.The mean maximum valgus angle of 8.70(SD=0.95) achieved at age of 3 years. The TF angle in boys and girls only had no significant difference except at age of 5 years.This study had shown that measurement of TF angle using the clinical method had a very good inter-observer reliability. The pattern of tibiofemoral angle in Malay children population between 2 years old to 6 years old is comparable to children elsewhere. The TF angle in Malay population was valgus since age of 2 years with the maximum angle of 8.73


Preoperative imatinib mesylate (IM) treatment has not yet been standardized. Here, we aim to further explore such therapy on patients with gastrointestinal stromal tumors (GIST) retrospectively.Patients experiencing preoperative IM were identified from January 2009 to February 2015.P < 0.001) in patients receiving operations. Three in 7 cases of rectum GIST underwent abdominoperineal resection, and four others adopted sphincter-sparing resection. Partial gastrectomy was performed in four patients.A total of 28 GIST patients were identified. The patients received preoperative IM treatment for a median length of 13.5 months, ranging from 5 to 37 months. PR and SD were observed in 24 (85.7%) and 4 (15.3%) patients, respectively. The tumor shrinkage occurred predominantly within 6 to 12 months, and slight tumor shrinkage could be observed after 12 months in certain patients. Nineteen patients (67.9%) received surgery, and R0 resection was acquired in 18 (94.7%) patients. The initial mean maximum diameter was 10.5 (5.2 to 19.0) cm and decreased to 5.9 (2.7 to 19.0) cm after preoperative treatment with a median length of 12 (ranging from 5 to 36) months (IM prior to surgery can effectively prevent tumor rupture and facilitate surgery with low surgical morbidity for GIST patients. Tumor shrinkage following IM occurred predominantly within 6 to 12 months, and slight tumor shrinkage could be observed after 12 months in certain patients. In selected patients, prolonged exposure to IM is seemingly advisable under close radiological surveillance. Gastrointestinal stromal tumors (GIST) are the most common mesenchymal tumors of alimentary tract, accounting for approximately 1 to 3% of all malignant gastrointestinal neoplasms , let aloKIT or PDGFRA encoding receptor tyrosine kinase, which revolutionizes the management of the disease with its high efficacy, primary resistance observed in only approximately 10% patients [Imatinib mesylate (IM) is applied in GIST patients gradually based on the discovery that GIST is initiated by mutational proto-oncogene patients . SurgicaHowever, the duration of preoperative IM is still unclear. The NCCN guideline recommends a maximal response time 6 to 12 months after IM onset. Multidisciplinary team members should collaborate to determine whether operation is needed when early response is acquired in some cases without giving definite time frame in unresectable or locally advanced primary GIST . Both 6 Patients experiencing preoperative IM were identified from West China Hospital, Sichuan University, over a continuous period, from January 2009 to February 2015. The following is the inclusion criteria: (1) patients were pathologically confirmed as primary or recurrent/metastatic GIST stained positive for CD117 and/or Dog-1, whose specimens were obtained by fine needle biopsy or surgery or endoscope; (2) GIST were diagnosed to be unresectable, or more mutilating surgery was needed due to locally advanced or metastatic disease or rupture tendency, assessed by a multidisciplinary team of experienced radiologists, oncologists, and surgeons; and (3) patients with recurrent/metastatic disease who had no history of taking IM. The following is the exclusion criteria: (1) GIST showed primary resistance to IM in gene analysis or radiographic imaging, and (2) patients had poor performance status due to other serious diseases. The Institutional Review Board and Ethics Committee of the West China Hospital of Sichuan University deemed that an ethical review was not needed for this retrospective research.The response of target lesions to IM treatment was defined in our study cohort according to the revised RECIST guideline (version 1.1) . PlateauIM was administrated as a first-line drug treatment at a dose of 400 mg once daily. Surgical excision of tumors was performed as tumor progressed or tumor downsizing plateaued, or multidisciplinary team deemed improved surgery  possible.Relevant demographics, tumor characteristics, pathological reports, and treatment protocols were collected. Consecutive baseline computed tomography (CT) or magnetic resonance imaging (MRI), abdominal ultrasonography, and full hematological test were performed on every patient after the initiation of IM treatment during the follow-up period. If liver parenchyma was invaded by GIST, liver function assays were also included. Imaging examination intervals depended on the attending physician, or the patient and results would be recorded in detail. The last follow-up was in October 2015 or the date when patient died of GIST progression or other causes.t test was used to analyze difference of tumor size as appropriate. Difference of follow-up periods between operation and non-operation groups was identified by Mann-Whitney U test. PFS and OS were analyzed by the statistical method of Kaplan and Meier, and univariate analysis was performed using the log-rank test. A two-sided P value less than 0.05 was considered statistically significant. Death resulting from other causes rather than GIST was counted as censoring. The statistical software of SPSS 17.0  was used to analyze all raw data.Count data were shown as percentages while measurement data as mean or median with the range from the minimum to the maximum. The paired sample or independent sample A total of 28 patients were included with male to female ratio being 21:7. Maximum tumor diameter was calculated with CT and MRI, and all exceeded 5 cm with >8.3 cm accounting for 75% prior to IM treatment. The majority of tumors were primarily located in the small intestine (32.1%), followed by the stomach (28.6%), the rectum (21.4%), other (10.7%), and the colon (7.1%). There were eight patients carrying multiple GIST lesions, and four were accompanied by synchronous liver metastases among them. Risk stratification was done according to the Modified NIH criteria . All patP < 0.001). No primary drug resistance was documented. No severe drug-related side effect took place in this series. No obvious association between the mean tumor size reduction and location of tumor  and 4 (15.3%) patients, respectively. The mean maximum diameter of tumor decreased from 10.7 (5.2 to 19.0) cm to 5.9 (2.7 to 19.0) cm . All of the 19 patients were given adjuvant IM due to high recurrence risk after surgery.Nineteen patients (67.9%) finally received surgical resection of tumor following preoperative IM with a median length of 12 (5 to 36) months. PR and SD were demonstrated in 16 and 3 patients, respectively. The initial mean maximum diameter was 10.5 (5.2 to 19.0) cm and decreased to 5.9 (2.7 to 19.0) cm after preoperative treatment (P < 0.001).The remaining 9 patients refused operation and chose only IM treatment with a median length of 24 (8 to 37) months. The mean maximal diameter of tumor reduced from 11.0 (9.0 to 15.6) cm to 5.9 (3.7 to 8.0) cm  primary and 6 (50%) recurrent/metastatic patients experienced surgical procedure. R0 resection was acquired in 18 (94.7%) patients, and minor lesions in the liver could not be removed completely in patient no. 4. No tumor rupture and blood transfusion at operation happened. Three in 7 cases of rectum GIST underwent abdominoperineal resection because the anus was too adjacent to the tumor to be preserved, and sphincter-sparing resection of tumors was adopted in 4 (57.1%) cases with excision margins pathologically negative, which was impossible initially. Partial gastrectomy was realized in 4 (100%) patients with huge mass in the stomach which could not be resected or potentially resectable with total or subtotal gastrectomy, before taking IM, judged by radiological findings or exploratory laparotomy. The remaining eight patients with tumors in other parts of the body were also performed organ-preserving or less-mutilating surgery as anticipated. Pulmonary infection and anastomotic fistula occurred separately in two cases, which were managed well through prolonged hospitalization care and fistula repair. Detailed information is recorded in Tables P = 0.980, 0.741 respectively), PFS and OS as well . Median PFS and OS had not been reached at the last follow-up. Detailed information is recorded in Table No patients were lost in the follow-up period, and the median follow-up was 21 (8 to 75) months from the onset of IM treatment. The rate of 2-year PFS was 76.5% Fig. . No deatPreoperative IM treatment is playing an indispensible role in locally advanced or recurrent/metastatic GIST by contributing to complete tumor resection with the least cost. Until now, multiple studies have delineated benefits from the treatment, for example, low surgical morbidity and mortality and function-preserving –13. It iThe optimal duration of preoperative IM or other kinase inhibitors remains controversial. The major concern is missing a surgery window if long-term IM treatment allows tumor to progress, while short-term IM treatment cannot achieve optimal efficacy. During the past years, reported durations of IM prior to surgery tended to be inconsistent, varying between a few days and several months. The RTOG 0132 trial probed into preoperative IM effectiveness after a period of 8 to 12 weeks, followed by 2-year adjuvant IM for advanced primary (group A) and recurrent/metastatic patients (group B), with 2-year PFS of 83 and 77% for a separate group, and the results of the trial were updated in 2012 , 15. TheOur patients in this study experienced a longer IM duration before operation  compared to earlier reports. Figure Cmin), which is affected by transit time and PH in the gastrointestinal tract, is related to the prognosis of advanced GIST. IM Cmin of patients experiencing total or subtotal gastrectomy was lower than that of patients experiencing partial or no surgery [Cmin may be achieved. Of note, laparoscopic surgery by experienced surgeons should be available after preoperative IM treatment.Preoperative IM could promote safety of GIST surgical resection by facilitating tumor shrinkage and loss of vascularity , 21. As  surgery . TherefoP = 0.513) and OS (P = 0.077) in our study at the time of last follow-up. However, the previous research revealed that surgery following IM could improve both PFS and OS  in locally advanced non-metastatic GIST patients [Cmin. Of course, randomized controlled trial of large sample accompanied by long-term follow-up should still be warranted to further investigate preoperative IM.In this series, a 2-year PFS rate of 76.5% was demonstrated, which was similar to previous reports . Nine papatients . An inteIn summary, tumor resection was R0 in more than 90% patients after IM for a median length of 12 months and low surgical morbidity was observed. Accordingly, we suggest preoperative IM when GIST is large in size or undesirable in location. In selected patients, prolonged exposure to IM is seemingly advisable under close imaging surveillance.


Cixian is one of the high‐risk areas for upper gastrointestinal cancer in China and the world. From 2005, comprehensive population‐based screening for upper gastrointestinal cancers has been conducted in Cixian. The aim of this study was to investigate population‐based cancer survival from 2003 to 2013 and to explore the effect of screening on upper gastrointestinal cancer survival in Cixian. Observed survival was estimated using the life table method. The expected survival from the general population was calculated using all‐cause mortality data from the population of Cixian with the EdererII method. Cixian cancer registry, with a total coverage of 6.88 million person years, recorded 19,628 cancer patients diagnosed during 2003–2013. In Cixian, from 2003 to 2013, there were 19,628 newly cancer cases and 13,984 cancer deaths, with an incidence rate of 285.37/100,000 and mortality rate of 203.31/100,000. The overall five‐year relative cancer survival for patients diagnosed in Cixian in 2003–2013 was 22.53%. The relative survival for all cancers combined in Cixian had an overall upward trend from 2003 to 2013. Among upper gastrointestinal cancer in Cixian, the five‐year relative survival for cardia gastric cancer was highest at 30.42%, followed by oesophageal cancer at 25.37% and noncardia gastric cancer at 18.93%. In 2013, the five‐year relative survival for oesophageal cancer, cardia gastric cancer, and noncardia gastric cancer patients aged 45–69 years was 39.97% (95% CI: 34.52–45.43%), 51.74% (95% CI: 42.09–60.86%), and 37.43% (95% CI: 26.93–48.17%), respectively, the absolute values increasing 14.11%, 16.71%, and 14.92% compared with that in 2003. There is an increasing trend in overall survival for upper gastrointestinal cancer with early screening and treatment of cancer in Cixian. Cixian is one of the high‐risk areas for upper gastrointestinal cancer in China and the world. The Cixian Cancer Registry was the first population‐based cancer registry in Hebei Province and was established in 1974. Studies on cancer incidence and mortality trends in Cixian have previously been reported in detail Hebei Provincial Cancer Registry Centre is responsible for compiling cancer data collections, evaluations, and publications from local population‐based cancer registries. The population‐based Cixian Cancer Registry was established in 1974 and covered a population of 6,878,097 among the 11 years. Cancer information was reported to cancer registries by local hospitals and community health centers, including the Basic Medical Insurances for Urban Residents and the New‐Rural Cooperative Medical System.Data for patients diagnosed with cancer were obtained from the Cixian Cancer Registry. Individuals with the first occurrence of primary cancer and multiple primary cancers were selected. There were 19,628 new cancer patients diagnosed during 2003–2013. Cancer records were excluded if they were based only on a death certificate or unknown vital status. After exclusions, 18,786 patients were included in the survival analysis (95.7% of those eligible). The rate of follow‐up was 97.2%. Data on the population used to calculate relative survival were obtained from the Cixian Bureau of Statistics.A mix of active and passive follow‐up methods was used to identify the vital status and cause of death for all registered patients from the date of diagnosis until 31 December 2016. Passive reporting relies on the periodical linkage of cancer registration records with death records from any cause from the mortality surveillance system. Active reporting involves registry personnel examining the sources of data and abstracting the required information on vital status on special forms from different types of hospitals and health insurance systems. Home visits or telephone contacts with next of kin were used for patients whose vital status could not be ascertained by other methods.The national screening for upper gastrointestinal cancer program has been available in Cixian since 2000. From 2005, the first round of comprehensive population‐based screening for upper gastrointestinal cancer was conducted in Cixian. In the past decade, more than 50,000 high‐risk residents aged 45–69 years among the whole county were screened with endoscopy. The positive rate is between 2.2% and 3.0%, and the early diagnostic rate is between 85.0% and 92.0%. Moreover, the treatment rate is very high with 90.0%. The screening for upper gastrointestinal cancer can improve the survival for high‐risk patients.According to “Guideline of Chinese Cancer Registration,” we checked the data quality using the inclusion criteria from “Cancer Incidence in Five Continents Volume X,” which was required by the International Agency for Cancer Registry (IACR) and International Agency for Research on Cancer (IARC). The quality and completeness of the cancer registry data were assessed with IARC‐crgTools to identify errors, inconsistencies, and unusual combinations of cancer site, morphology, sex, and age at diagnosis Relative survival is the ratio of the observed proportion surviving in a group of patients to the expected proportion that would have survived in a comparable group of people from the general population. Observed survival was estimated using the life table method In Cixian, from 2003 to 2013, there were 19,628 newly cancer cases and 13,984 cancer deaths, with an incidence rate of 285.37/100,000 and mortality rate of 203.31/100,000. During this decade, incidence and mortality fluctuated over time.By statistical analysis, the overall one‐year, three‐year, and five‐year relative cancer survival for patients diagnosed in Cixian in 2003–2013 was 63.59% [95% confidence interval (CI) 62.87–64.31%], 33.84% (95% CI: 33.12–34.57%), and 22.53% (95% CI: 21.87–23.19%), respectively. For males, the overall five‐year relative survival for all cancers combined [20.31% (95% CI: 19.48–21.16%)] was 20.70% lower than that for females [25.61% (95% CI: 24.56–26.66%)]. According to age at diagnosis, the overall five‐year relative survival for cancer patients younger than 45 years, 45–69 years, and older than 70 years was 28.80% (95% CI: 26.53–31.10%), 24.63% (95% CI: 23.82–25.45%), and 14.59% (95% CI: 13.35–15.88%), respectively. The relative cancer survival for patients younger than 45 years was the highest at all times. The overall relative survival for all cancers combined declined with age. , 30.95% (95% CI: 29.77–32.15%), and 32.05% (95% CI: 30.64–33.47%) in different period: 2003–2006, 2007–2010, and 2011–2013, respectively. There is an obvious increase trend.The five‐year relative survivals for the five most common cancers  in Cixian were lower than 30% in both sexes. Female breast cancer had the highest survival 40.21%), followed by cancers of the colorectum (26.07%), esophagus (25.37%), and stomach (25.03%), with lung and liver cancer, respectively, having the poorest survival (12.52% and 7.64%). In males, the five‐year relative survival for the five most common cancers ranged from 7.06% (95% CI: 5.37–9.06%) for liver cancer up to 25.26% (95% CI: 23.42–27.14%) for stomach cancer. In females, the five‐year relative survival for female breast cancer was the highest at 40.21% (95% CI: 35.85–44.55%), which was higher than 30%. The five‐year relative survival for the five other most common cancers  ranged from 8.73% (95% CI: 6.22–11.79%) for liver cancer to up to 30.42% (95% CI: 25.60–35.42%) for colorectal cancer , followed by esophageal cancer at 25.37% (95% CI: 24.24–26.51%), and noncardia gastric cancer at 18.93% (95% CI: 16.98–20.98%) , 51.74% (95% CI: 42.09–60.86%), and 37.43% (95% CI: 26.93–48.17%), respectively, the absolute values increasing 14.11%, 16.71%, and 14.92% compared with that in 2003 , 37.08% (95% CI: 34.70–39.46%), and 36.37% (95% CI: 33.48–39.29%), respectively. For cardia gastric cancer patients aged 45–69 years, the overall five‐year relative survival was 40.74% (95% CI: 35.71–45.79%), 43.47% (95% CI: 39.36–47.56%), and 44.64% (95% CI: 39.64–49.58%) in 2003–2006, 2007–2010, and 2011–2013, respectively, while they were 26.98% (95% CI: 22.54–31.65%), 27.68% (95% CI: 23.26–32.31%), and 34.72% (95% CI: 28.89–40.69%), respectively, for noncardia gastric cancer patients aged 45–69 years in these three periods.The Cixian Cancer Registry, an important component of the National Central Cancer Registry (NCCR) of China, has been collecting cancer data since 1974. In 1996, the Cixian Cancer Registry joined the International Association of Cancer Registries (IACR). Because of the accurateness of the Cixian cancer data, the incidence and mortality data were published in Cancer Incidence in Five Continents . With the importance of cancer surveillance increased, data with the follow‐up information required by NCCR for surveillance of cancer survival were collected by the Cixian Cancer Registry. Survival data from Cixian were included in CONCORD‐2 This research showed that the one‐year relative survival for all cancers combined in Cixian was 63.59% in 2003–2013. This value had significantly increased by 73.70% compared to the one‐year relative survival in 2000–2002 (36.61%) By comparing the relative survival of common cancers between Cixian and rural Chinese areas, the five‐year relative survival for both lung cancer and liver cancer in Cixian were, respectively, 11.79% and 21.27% higher than average levels in rural areas. However, the five‐year survival for both colorectal cancer and female breast cancer in Cixian was lower than in Chinese rural areas. Early screening and treating with an enteroscope for colorectal cancer and the Two Cancers Screening Project for females must be strengthened in Cixian.Upper gastrointestinal cancers were the most common cancers in Cixian, China. A recent study showed that the five‐year relative survival for oesophageal cancer and stomach cancer was 20.9% and 27.4%, respectively, in China for 2003–2005. The five‐year relative survival for oesophageal cancer and stomach cancer was 19.1% and 32.5%, respectively, in urban Chinese areas, and 21.2% and 24.9%, respectively, in rural Chinese areas Cardia gastric cancer is currently classified as gastric cancer. Lower cardia gastric cancer has been given much attention because of its increasing incidence, which is in striking contrast with the steady decrease in distal stomach cancer. In China, cardia gastric cancer has unique epidemiological features distinguishing it from distal stomach cancer. Cardia gastric cancer should be considered a distinct entity In this study, we analyzed the relative survival for esophageal cancer, cardia gastric cancer, and noncardia gastric cancer in Cixian. The five‐year relative survival for cardia gastric cancer is the highest among upper gastrointestinal cancers. Regarding the time trend, the survival curve for noncardia gastric cancer changed slightly, but the relative survival for cardia gastric cancer and esophageal cancer increased. Moreover, the five‐year relative survival of patients aged 45–69 years significantly increased from 2003 to 2013. Our data showed that the five‐year relative survival for esophageal cancer, cardia gastric cancer, and noncardia gastric cancer patients aged 45–69 years was 39.97%, 51.74%, and 37.43%, respectively, in 2013, all of which were higher than in 2003. Obviously, early screening and detection for cardiac cancer and oesophageal cancer were significantly effective for patients’ survival. Upper gastrointestinal cancer was accounted for 58.1% of all cancers in Cixian. With the efforts of screening for upper gastrointestinal cancer in Cixian, the survival for upper gastrointestinal cancer presented the obviously up‐trend, which had contributed substantially to the increase in overall cancer survival in Cixian.The relative survival for cardia gastric cancer is the highest among upper gastrointestinal cancers, which is consistent with that in Linxian As mentioned above, Cixian has been chosen as a demonstration base for the early detection and treatment of upper gastrointestinal cancers for 17 years. Increased efforts have been invested into screening and early detection to identify precancerous lesions in upper gastrointestinal cancers, especially esophageal epithelium dysplasia, gastric intraepithelial neoplasia, preinvasive carcinoma, and gastrointestinal mucosal carcinoma. The national screening program, which uses endoscopy with mucosal iodine staining and index biopsy combined with pathological examination to confirm the disease, has been available in Cixian since 2000. From 2005 to 2014, the first round of comprehensive population‐based screening for upper gastrointestinal cancer was conducted in Cixian. Residents aged 45–69 years throughout the county were screened with endoscopy, and the second round of screening started in 2014.Endoscopy with mucosal iodine staining was not only a sensitive technique for identifying clinically relevant upper gastrointestinal cancers but also an effective preventive technique. The sensitivity and specificity of endoscopy were approximately 80% and 93.4%, respectively Upper gastrointestinal cavity, especially esophageal and cardiac cavities, could be clearly seen by the endoscope. Pathological tissue of the esophagus and cardia can also be identified for relatively more precise diagnosis by biopsy and treatment decisions. Screening with an endoscope can prevent the development of upper gastrointestinal cancers and death in high‐risk individuals, thereby contributing to life expectancy and increasing survival for upper gastrointestinal cancer, especially oesophageal cancer and cardia gastric cancer. Our study also validated that early detection and treatment of precursor lesions with an endoscope has made progress with the disease; tumor stages were changed and survival time for esophageal and cardia gastric cancers was particularly prolonged in the screened age group.This is the first time that survival data based on the population of Cixian, Hebei Province were fully analyzed. However, there are some limitations to the study. Although the overall quality of cancer data was reliable and the long‐term survival trend can be fully shown, cancer patients in Cixian were followed until 2016. We did not obtain the five‐year survival for cancer patients diagnosed after 2011. We just analyzed the survival with period approach now. But with the development of research methods and improvements in cancer data quality, more accurate and complete survival data from Cixian will be reported.Although there was an upward trend in cancer survival after long‐term efforts for cancer prevention and control in Cixian, the five‐year survival from all of the five most common cancers  was below 30%. A commentary highlighted that there are substantial barriers to health care that are faced by the Chinese population, which relate to access to care, fatalism about cancer, and traditional medicine, each of which is likely to provide additional explanations for the disparities in survival between China and more developed countries The authors declare no potential conflicts of interest.Figure S1. Survival methods used in the present study.Click here for additional data file.


AbstractPelophylaxlessonae   and P.ridibundus  , and their natural hybrid P.esculentus  of various ploidy and genome composition . Tetraploids RRLL were found (4n = 52) in juveniles. We applied cytogenetic techniques: AgNO3, chromomycin A3, PI and fluorescent in situ hybridization with a 28S rDNA probe. Results obtained by silver staining corresponded well with those stained with CMA3, PI and FISH. As a rule, NORs are situated on chromosomes 10. The number of Ag-NORs visible on metaphase plates was the same as the number of Ag-nucleoli present in interphase nuclei of the same individual. In all analyzed metaphases, NORs exhibited variations in size after AgNO3 and CMA3 stainings. Sixty-six individuals  were polymorphic for the localization and number of NORs. Fifty-one diploids had NORs only on one chromosome of pair 10. Three triploids (LLR and RRL) displayed two NORs, and two other triploid RRL individuals displayed one, instead of expected three NORs. In ten individuals extra NORs were detected on chromosomes other than 10 (chromosomes 2 and 9).We studied water frogs from a complex composed of two species:  They are the only genes that can be recognized in genomes on the basis of chromosome structure and thereby are useful as cytogenetic markers. These chromosomes differ from others by the presence of secondary constrictions where NORs are situated. The number and position of NORs are species specific, although inter-individual variability of these regions has also been observed within species. In anuran amphibians, NORs detected by silver staining (Ag-NOR) revealed that most species, both from primitive and derived families, have only one pair of NOR-bearing chromosomes in their diploid karyotypes . The lochromatin . Exceptihromatin . Variatihromatin . Using sDNA loci . The varribution . Similarolfersii , Hypsiboypsiboas , Physalacuvieri , and in sonianum .3 staining   and P.ridibundus  , and their natural hybrid P.esculentus  of various ploidy and genome composition   or all-hybrid (P.esculentus- P.esculentus) composed of diploid and triploid individuals  is discarded from the germ line before meiosis and the other one is duplicated and clonally transmitted into gametes  . Hybridsividuals . Reproduoduction . During  gametes . Some hy gametes  and the  gametes .i.e. silver, chromomycin A3 and Propidium Iodide (PI) staining, and fluorescent in situ hybridization with 28S rDNA probe commonly used in comparative studies of cold-blooded vertebrates: fish  were collected from 11 natural populations in Central Europe  were obtained from in vitro artificial crosses. Their parents  were collected from natural populations and served for 28 crosses resulting from various parental combinations . Shortly before tissue preparation, the frogs were anesthetized with 0.25% water solution of 3-aminobenzoic acid ethyl ester . The intestine was dissected, hypotonized in distilled water (20 min for adults and 10 min for juveniles), and fixed in fresh-made fixative ethanol:glacial acetic acid (3:1) according to Twenty-four hours before dissection, adults were injected intraperitoneally with 1 ml, and juveniles with 0.5 ml of 0.3% colchicine  2.5 h before dissection, femur bones were removed from euthanized frogs, epiphyses were clipped off, and bone marrow cavities were immediately flushed with 0.075 M KCl solution applied with a syringe at 37°C. Bone marrow tissue was pressed through a small-mesh gauze, flushed out with 0.075 M KCl solution, then placed in a centrifuge tube filled up to 7 ml with 0.075 M KCl solution, and incubated at 37°C for 20 min. Hypotonic treatment was stopped by fixation in absolute methanol:glacial acetic acid (3:1), and cell suspension was centrifuged at 1,500 rpm for 10 min. The supernatant containing fat droplets was discarded with a Pasteur pipette and fresh fixative was added up to 5 ml. The pellet was re-suspended by agitation and kept in a freezer for 20 min, then centrifuged again at 1,500 rpm for 10 min. The procedure  was repeated 3 times. The suspension was then transferred to a 1-ml syringe, dropped onto slides , and finally air-dried. Chromosome preparations obtained with either protocol were suitable for all staining methods used in this study. However, chromosomes prepared from bone marrow cells were more uniformly condensed and thus more suitable for FISH than the chromosomes obtained from gut epithelium prints that varied in the degree of condensation (Chromosome preparations of three ensation .3 (CMA3) and AgNO3. All individuals that displayed another number of NORs than one per a haploid set after Ag-NOR staining were examined by PI or CMA3. For 22 individual  we applied the FISH method with 28S rDNA as a probe.The nucleolus organizer regions (NORs) were stained in all individuals by the silver nitrate technique (Ag-NOR). Chromosome slides of 15 randomly selected individuals were sequentially stained with chromomycin ASilver staining. We followed the protocol of 3/1ml H2O/0.5ml gelatin PageBreaksolution, i.e.1g gelatin/0.5ml formic acid/50ml bi-distilled water) were applied to each preparation. Slides were covered with a nylon mesh and incubated in a humid chamber at 60°C for 1min, washed in distilled water and air-dried. If the chromosomes were poorly visible, they were counterstained with DAPI. The number of active rDNA loci was documented by simultaneous use of fluorescence and incandescent light.3 (CMA3).Chromomycin A The method was used according to 2 for 10 min and stained with CMA3 solution (0.5 mg/mL buffer and 2.5mM MgCl2) for 15 min in the dark, briefly rinsed in buffer and counterstained with methyl green for 15 min (0.175g methyl green/50ml buffer). After washing in buffer, slides were stained with DAPI solution (0.5µg/ml) for 10 min in the dark and briefly rinsed with buffer. Tissue prints were mounted in 50% glycerol and analyzed using fluorescence illumination. After CMA3 staining, preparations were faded and cleaned in xylene and benzene, each bath for 2 min, and then stained with AgN03.Propidium Iodode staining after denaturation of chromosomes (PI). Tissue prints were dehydrated in 70%, 85% and 95% ethanol (30 sec each wash), air-dried at room temperature and denatured in 70% deionized formamide in 2xSSC at 70°C for 3.5 min. Slides were immediately dehydrated in chilled 70% ethanol from a freezer for 2 min, then in 85% and 95% at room temperature for 30 sec. Chromosomes were stained with PI (200ng/ml).We applied the FISH method with 28S rDNA as a probe, according to the protocol of PageBreakSlides were incubated in 100 µg/ml DNase-free RNase A in 2x SSC for 1 h at 37° C in a humid chamber and then washed twice in 2xSSC at room temperature for 5 min, dehydrated in ethanol series  and air-dried. Chromosome preparations were denatured in 70% deionized formamide, 2x SSC at 70° C for 3.5 min, dehydrated immediately for 2 min in ice-cold 70% ethanol, then in 85% and 95% ethanol for 30s at room temperature, and finally air-dried. The 28S rDNA probe was dissolved in hybridization mixture consisted of 100% deionized formamide and 20% dextran sulfate in proportion 1:1. The mixture was denatured at 90° C for 5 min and then immediately placed on ice for 3 min. 20 µl of the probe was applied to each slide and covered with a coverslip. Hybridization lasted overnight in a dark humid chamber at 37° C. After hybridization, the coverslip was removed by rinsing with 50% formamide in 2xSSC at 42°C twice for 7 min and the slide was washed 3 times (7 min each) in 1xSSC at 42°C and then in 2xSSC at room temperature for 30 s. Chromosomes were counterstained with DAPI in Vectashield antifade buffer .Chromosomes were analyzed in Olympus Provis AX 70 or Carl Zeiss Axioskop 20 microscopes equipped with fluorescence lamp HBO50 and appropriate filters. Images were recorded with Olympus DP30BW CCD and cooled Carl Zeiss AxioCam HRc CCD cameras and processed using AxioVision and Lucia ver. 2.0 (Laboratory Imaging) softwares.i.e. 354 diploids , 48 triploids (30 adults and 18 juveniles RRL and LLR), and 5 tetraploids RRLL. The results are summarized in Table 3 staining, one NOR per a haploid set was identified in secondary constrictions on long arms of chromosomes 10 in 341 individuals (83.8%), and in these individuals the number of NORs reflected the ploidy level. Two NORs were detected in 302 (85.3%) diploid individuals belonging to all studied taxa , regardless of their capture site and taxonomic status. In all analyzed metaphases of the species (P.lessonae and P.ridibundus) and hybrids (P.esculentus 2n and 3n) , NORs exhibited variations in size after AgNO3 and CMA3 stainings, bands at one of the homologs were slightly thicker than at the other one , and four Ag-NORs were recorded in all metaphase plates of five tetraploids (RRLL).The number of NORs was visualized for all (407) studied individuals  and 43 diploid juveniles  had NORs only on one chromosome of pair 10 , the number of NORs differed from the expected values. Lack of NORs was relatively more frequent  than extra NORs , regardless of genomic compositions  and origin of individuals. Eight diploid adult individuals  we observed 16 individuals with two NORs and 21 individuals with only one NOR. Each individual was stained with AgNOR and seven of them were confirmed by FISH. Thus, despite that the genomes of ova were the same , the females produced in fact two types of gametes – with and without NORs  located within the secondary constriction on the long arm of chromosome Onychodaxtylusfischeri . Various sizes of AgNORs in frogs were described by 3 stained chromosomes and the intensity of CMA3 signals which were proportional to the amount of rDNA in NORs. We also found variability of NOR sizes in chromosomes 10, however The variability of AgNOR sizes in individuals from the same populations may reflect different amount of rDNA (Ranacatesbeiana (Lithobatescatesbeianus) that displayed from two to seven Ag-positive small NORs per haploid karyotype, apart from one “standard” NOR on the chromosome 10 . We observed a lack of one NOR (2n and 3n) or two NORs (only in 3n), both in metaphase plates and interphase nuclei. Lack of NORs in amphibians was also reported by Schimd (1982) in Hylachrysoscelis and H.versicolor   set with NORs whereas the set without NOR is transmitted without recombination .The lack of one NOR was detected in both RRL and LLR triploid females and was associated with the  in LLR) . Because in LLR) . This inbaturae , 2012. Tlessonae and ridibundus genomes, and – as expected – was observed in individuals regardless of their taxonomic position , ploidy level , genomic constitution , and collection site. The number of active silver-stained NORs reflected ploidy levels: two in diploids, three in triploids, and four in tetraploids, and therefore we believe that there is no diploidization of polyploids in water frogs, as has been observed in some of polyploid amphibians (for review see Finally, we conclude that polymorphism concerning the number and localization of NORs in water frogs was characteristic of both PageBreakThe study was financially supported by grants 3490/B/P01/2007/33 and 3946/B/P01/2009/36 provided by the Polish Ministry of Science and Higher Education. We are grateful to Katarzyna Lipiec-Sidor for technical help with figures arrangement and setup.


Kaplan–Meier survival curves and the log-rank test demonstrated that higher BRD4 expression was an adverse predictive factor for survival in GC. Multivariate analysis by Cox proportional hazards regression revealed that BRD4 expression was an independent worse prognostic factor in GC. In conclusion, BRD4 could act as a potential biomarker for prognostic assessment of GC.Expression of bromodomain protein 4 (BRD4) has been reported to predict a worse prognosis in solid tumors. However, its expression profile and prognostic value in gastric carcinoma (GC) remains unknown. Here we investigated BRD4 expression in GC and explored its association with patient survival. Tissue samples were obtained from 95 GC patients who underwent surgical resection to remove the primary tumor from January 2009 to December 2010. Immunohistochemistry was used to detect the expression of BRD4 in GC tissues and adjacent normal tissues. Kaplan–Meier survival curves and Cox proportional hazards regression were used to analyze the data of BRD4 expression profile and clinicopathological characteristics. Immunohistochemical analysis revealed BRD4 was overexpressed in GC tissue compared with adjacent normal tissue. BRD4 expression was significantly associated with TNM stage ( Gastric carcinoma (GC) is one of the most common malignant tumors of the digestive system. GC is the third most frequent cause of cancer-related death worldwide and affects approximately one million people every year . On a glExisting treatments for GC, including invasive techniques such as laparoscopic resections combined with chemotherapy or radiotherapy, have proven to be effective in prolonging the overall survival time mainly in patients at an early stage . For advin vitro studies, two BRD4 inhibitors (JQ1 and I-BET762) have been found to effectively inhibit the proliferation and growth of cancer cells such as melanoma, pancreatic cancer, lung cancer, multiple myeloma, acute myeloid leukemia, and Burkitt's lymphoma [The bromodomain (BRD) and extra terminal domain (BET) family is composed of three ubiquitously expressed proteins, BRD4, BRD3, and BRD2, and a testis-specific protein, BRDT. The BET protein family members bind to acetylated lysine residues in histones and function as epigenomic readers at the interface between chromatin remodeling and transcription regulation , 10. Parlymphoma , 21–23. In the present study, we hypothesized that high expression of human BRD4 protein may promote the proliferation and invasion of GC cells. To test this hypothesis, we investigated BRD4 expression in GC by immunohistochemistry and explored its association with prognosis in patients with GC.This study included 95 patients who had undergone surgical resection of GC (and whose diagnosis was confirmed as GC by more than two pathologists). In total, 41.1% of included patients were aged less than 60 years and 58.9% were 60 years or older. Among these, 73.7% were men and 26.3% were women. Approximately half (51.6%) of the patients had a smoking or drinking history. Detailed clinicopathological characteristics of the included GC patients are listed in Table In terms of histologic grade, a few cases were well differentiated (8.4%), while most cases were moderately (49.5%) or poorly differentiated (42.1%). For the T classification, T1, T2, and T3 accounted for similar proportions (~22% each); the majority of cases were T4 (35.7%). Lymphatic permeation was present in 64.2% of the patients. Among the six different tumor shapes, the ulcerative type was most common (76.8%). The vast majority (90.5%) were classified as adenocarcinoma, while the remaining cases were signet-ring cell carcinoma. Approximately 28.4% of the patients had distant metastasis. High EGFR and BRD4 expression was recorded in 21.5% and 47.4% of the patients, respectively. After surgery, 41.1% of the patients were alive at the end of the follow-up.To detect the expression level of BRD4 in human GC, tissue samples were selected from 95 GC cases (confirmed following surgery) and examined by immunohistochemistry. We determined the immunoreactive score by the sum of BRD4 expression extension and intensity . Immunohp < 0.001), vital status at the end of follow-up (p < 0.001), and lymphatic permeation (p < 0.05). No significant difference in BRD4 expression was observed with respect to gender, age, drinking or smoking status, histologic type, or EGFR expression (p > 0.05).The relationship between clinicopathological characteristics and BRD4 expression in the GC patients is shown in Table p < 0.001), lymph node metastasis (p < 0.001), lymphatic permeation (p < 0.001), and TNM stage (p < 0.001) were significant prognostic factors for poor survival (Table p < 0.001) and advanced TNM stage  were independent worse prognostic factors for overall survival in GC patients  between January 2009 and December 2010 were included in this study. TNM stage and histology were classified according to the 2002 AJCC staging system . Exclusi2O2 [Tissue samples were collected from resected GC patients, fixed in 10% neutral formalin, and then embedded in paraffin. Paraffin sections (4 μm thick) were deparaffinized in xylene and rehydrated in grade alcohol, followed by boiling in 10 mmol/L of citrate buffer (pH 6.0) for antigen retrieval. The sections were blocked with 2% bovine serum albumin for 30 min and incubated overnight at 4°C with primary monoclonal rabbit anti-human BRD4 antibody  after the inhibition of endogenous peroxidase activities for 30 min with methanol containing 0.3% H2O2 . After b2O2 , a metho2O2 , the intp values < 0.05 were regarded statistically significant. Computerized statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS), version 18.0 .The chi-square test was used to examine statistical associations between clinicopathological characteristics and BRD4 expression in GC patients. The relationship between BRD4 expression and overall survival was evaluated by the Kaplan–Meier method, and the differences between subgroups were evaluated by the log-rank test. Univariate and multivariate Cox proportional hazards regression model analyses were used to evaluate the prognostic value of known categorical variables and BRD4 expression. All statistical analyses were two-sided, and


Currently 23 million U.S. adults have been diagnosed with diabetes  were calculated for the overall population and by selected sociodemographic characteristics. P values were calculated from chi-squared tests and were considered significant at <0.05. Final survey weights were applied to the data to adjust for various probabilities of selection and household nonresponse. Statistical software was used to account for NHIS’s complex sampling design.A total of 3,519 respondents aged ≥18 years reported having diabetes, including 211 classified as having type 1; 3,210 classified as having type 2 ; and 98 classified as having “other” type. In 2016, the overall crude prevalence of diagnosed diabetes among U.S. adults was 9.44% (95% CI = 9.01–9.88). The prevalences of type 1 diabetes, type 2 diabetes, and other diabetes types were 0.55%, 8.58%, and 0.31%, respectively . The weiEstimated crude prevalence of type 1 diabetes among U.S. adults did not significantly vary by age group (p = 0.54) or education (p = 0.14) . The preIn 2016, the estimated prevalences of diagnosed type 1 and type 2 diabetes were 0.55% (corresponding to 1.3 million U.S. adults) and 8.6% (corresponding to 21.0 million U.S. adults), respectively. Type 1 and type 2 diabetes accounted for approximately 6% and 91% of all cases of diagnosed diabetes, respectively. Because the prevalence of type 2 diabetes is so much higher than that of type 1, current diabetes surveillance data that do not distinguish diabetes type are more reflective of persons with type 2 diabetes. Recent analysis of diagnosed diabetes prevalence indicates a plateauing among adults aged 20–79 years  when estimating diabetes type in these data (There is no reference standard for classifying prevalent type 1 diabetes or type 2 diabetes cases in public health surveillance. The presence of autoantibodies against the beta cells of the pancreas and the lack of endogenous insulin secretion are biologic markers of type 1 diabetes. However, beta cell autoantibodies disappear with time and might even be absent at the time of type 1 diabetes diagnosis (The findings in this report are subject to at least three limitations. First, the data were self-reported and underestimate the total number of adults with diabetes. Second, data were not validated, which could have led to misclassification of diabetes type. Adults with self-reported type 1 diabetes who did not report insulin use were reclassified as having type 2 diabetes, which might have resulted in misclassification if they actually used insulin but did not report use. However, self-reported use of insulin is highly specific: <0.02% of persons who reported insulin in a medication log failed to report using it when asked (Despite these limitations, this first study to estimate the prevalence of diagnosed type 1 and type 2 diabetes based on self-report and current insulin use among U.S. adults provides information to track prevalence of diabetes by type to monitor trends and assess the burden of disease for education and prevention programs. Knowledge about national prevalences of type 1 and type 2 diabetes might facilitate assessment of the long-term cost-effectiveness of public health interventions and policies aimed at improving diabetes management and help to prioritize national plans for future type-specific health services.The two most common forms of diabetes are type 1 and type 2. Previous national diabetes prevalence estimates did not distinguish between types among U.S. adults.New data allowed estimation of diagnosed diabetes by type. In 2016, the prevalence of diagnosed type 1 diabetes was 0.55%, representing 1.3 million U.S. adults; the prevalence of diagnosed type 2 diabetes was 8.6%, representing 21.0 million U.S. adults. Non-Hispanic white adults had a higher prevalence of diagnosed type 1 diabetes than did Hispanic adults. Non-Hispanic blacks had the highest prevalence of diagnosed type 2 diabetes. Diagnosed type 2 diabetes prevalence estimates increased with age and decreased with increasing levels of educational attainment.Knowledge about national prevalence of diagnosed diabetes by type might be helpful in monitoring trends, assessing the burden of disease for education and management programs, and guiding and prioritizing national plans for future type-specific health services.