It is widely acknowledged that HPV prophylactic vaccine could prevent new infections and their associated lesions among women who are predominantly HPV-naive at vaccination. Yet there still remains uncertainty about whether HPV vaccination could benefit to individuals who have undergone surgery for cervical disease.ClinicalTrials.gov, number NCT00779766) conducted in Jiangsu province, China. We evaluate the impact of HPV vaccination on preventing women from subsequent infection and cervical lesions (LSIL+ and CIN2+) after excision treatment.This post-hoc analysis intends to focus on intent-to-treat participants who underwent excision treatment at baseline and the follow-up period in a phase II/III, double-blind, randomized trial  performed excisional treatment in this clinical trial. We observed a significant effect of vaccination on acquiring 14 high-risk HPV (HR-HPV) infection after treatment . The vaccine efficacy against new infections after treatment for 14 HR-HPV infection was estimated as 32.0% , and was 41.2%  for HPV16/18 infection. The accumulative clearance rates of the vaccine group and placebo group were 88.9 and 81.6% for HPV16/18 infection (P = 0.345), 63.4, 48.7% for 14 HR-HPV infection (P = 0.062), respectively. No significant difference was observed on the persistent rate of HPV16/18, 14 HR-HPV infection and occurrence rate of LSIL+ between the two groups.One hundred sixty-eight (vaccine, No significant evidence from this study showed that HPV-16/18 AS04-adjuvanted vaccine could lead to viral faster clearance or have any effect on the rates of persistent infection among women who had excision treatment. However, the vaccine may still benefit post-treatment women with “primary prophylactic” effect. Further research is required in clarifying the effect of using the prophylactic HPV vaccine as therapeutic agents.ClinicalTrials.gov identifier: NCT00779766. Date and status of trial registration: October 24, 2008. Completed; Has Results. Persistent high-risk human papillomavirus (HR-HPV) infection is necessary for the progression of cervical cancer , 2. The Women after treatment for CIN remain at a substantially increased risk of subsequent cervical cancer –8. Both However, few studies evaluate whether the observed effects arise from: (1) “therapeutic effect” of the prophylactic vaccination in leading to faster clearance of the residual infection; or (2) “primary prophylactic” effect of the vaccination on the newly developed lesions caused by new HPV infections after treatment; or (3) “secondary prophylactic” effect of the vaccination in reducing the ability of the residual virus to infect new cells. In this study, we explore the role of a bivalent vaccine in preventing secondary lesions and provide more scientific evidence for the impact of the vaccine on the women who are treated for cervical disease.n = 3026) or aluminum hydroxide (n = 3025) as a placebo at months 0, 1 and 6. Enrolment in this trial started in October 2008 and follow-up lasted for 72 months (14 visits). The trial was conducted according to The Code of Ethics of the World Medical Association (Declaration of Helsinki) and the International Conference on Harmonisation Good Clinical Practice guidelines. The study protocol and informed consent were approved by the ethics committees of the Center for Disease Control and Prevention (CDC) Jiangsu Province and the Cancer Foundation of China. The trial was registered at the ClinicalTrials.gov (number NCT00779766) and adhered to CONSORT guidelines. Before the study-specific procedures, written informed consent was obtained from each participant. The further details of the trial have been described in previous published papers [Women included in the present evaluation were the participants who underwent excision treatment at baseline and the follow-up period in a phase II/III, double-blind, randomized trial conducted in Jiangsu province , China. In this trial, women aged 18–25 years were randomized (1:1) to receive HPV-16/18 AS04-adjuvanted vaccine. And the results were interpreted according to the Bethesda 2001 classification system . ParticiThe automatic analyzer SPF10 PCR-DEIA-LiPA25 version 1  detected HPV DNA of cervical, biopsy samples and tissue specimens including 14 high-risk HPV types  and 11 low-risk HPV types .2 or Fisher’s exact tests, as appropriate. Vaccine efficacy (VE) was calculated as 1-(incidence rate in vaccinated/incidence rate in unvaccinated) and the corresponding 95% confidence intervals (95% CIs) around vaccine efficacy were calculated by exact conditional procedure based on the observed case split between vaccine and placebo recipients, adjusted for the person time in each arm. In the squamous intraepithelial lesion (SIL) analysis, we analyzed the occurrence rate of LSIL+ and HSIL+ stratified by specific-type after excisional procedure. Results of vaccine efficacy were considered as statistically significant if the estimates and corresponding 95% CIs were above zero. Kaplan-Meier method with log-rank test was applied to compare the difference in occurrence of LSIL+ between the two groups. All statistical tests were two-sided and only p-values < 0.05 were considered statistically significant. The statistical analyses were performed using WinPEPI version 4.0 [Comparisons of enrollment characteristic, accumulative clearance rate and persistent infection rate between the two groups were conducted by Pearson χsion 4.0  or SPSS n = 87; placebo, n = 81) received excisional treatment for their first cervical lesion identified at this clinical trial. One woman in each group underwent loop electrosurgical excision procedure (LEEP) at the last visit (visit 14), and thus 166 women had been followed up and were categorized based on cytological results and HPV status at enrollment. Comparison of analytical cohort stratified by study arm revealed the balance regarding to cytology results, and HPV status (HPV type) at enrollment , corresponding to a median of 9 study visits . The median duration between enrollment and treatment was 17 months for those in the vaccine arm (IQR:4.0–30.0 months) and 17 months for women in the placebo arm (IQR:6.0–34.8 months).n = 5; placebo, n = 5) were excluded because of no follow-up after excision treatment  were included in the analysis. There was no significant difference in the distribution of baseline HPV infection between the two groups . The woman was included in this analysis from the day when the women received an excisional procedure (LEEP or cone) for a first cervical lesion to the day of their last follow-up visit, considering the fact that women could be infected by HPV or had abnormal cytological results for several times after excisional treatment. Among all the infections that treated women had, 71.06% were HR-HPV infection and of these 52.49% were the result of new infections. We observed a significant effect of vaccination on acquiring 14 HR-HPV infection , and a nonsignificant but positive vaccine efficacy estimate of 28.1%  for HPV16/18 infection after excision treatment , 81.3, 80.0% for HPV31/33/45 infection(P>0.999), 63.4, 48.7% for 14 HR-HPV infection(P = 0.062), respectively. No significant difference on the persistent infection rate of HPV16/18, HPV31/33/45,14 HR-HPV  was identified between the two groups.Then, we evaluated whether the viral clearance rates or HPV persistent infection rates differed by vaccination status. Results were shown in Fig. When LSIL+ was examined as the outcome, nonsignificant but positive vaccine efficacy was estimated at 45.5%  for 14 HR-HPV infection, and 85.1%  for the newly detected outcome after treatment, respectively. Similar patterns were observed for HPV-16/18 infection as well.n = 7; placebo, n = 8) were excluded because of no cytological results . The distribution of cytological results at baseline was similar between two groups . In this analysis, the follow-up time was defined as the duration from the day of excisional procedure to the day of ascertainment of the subsequent disease end point (LSIL+ incidence), while for women without a subsequent disease end point, until the day of their last follow-up visit. The median follow-up time was 46.0 months among women who were treated. The median follow-up time for vaccine group and placebo group were 48.5 months, 44.0 months, respectively. The vaccine efficacy for the occurrence of LSIL+ was 55.3% , irrespective of HPV DNA types. There was no significant difference between two groups , one with VAIN1, and one with CIN1 post-surgery. Of the two women in the placebo group, one developed CIN2 and one had CIN1 after treatment. The women Case 4: Fig. : Fig. 4 This is the first analysis in China to evaluate the efficacy of HPV 16/18 AS04-adjuvanted vaccination on the relapse of cervical precancerous lesions or cancers among women who underwent excisional procedures for cervical lesions after vaccination. In this study, vaccination failed to lead to viral faster clearance or have any effect on persistent HPV infection. However, the available evidence suggested that the vaccine may still benefit women after excisional treatment for cervical disease by “primary prophylactic” effect, which protects women from new infection after treatment.Former studies have indicated that the prophylactic HPV vaccines have the possibility in benefiting post-treatment women. The post-hoc analysis of the PApilloma TRIal against Cancer In young Adults (PATRICIA) showed that efficacy of the HPV 16/18 AS04-adjuvanted vaccine post-surgery (60 days or more) for the original lesion was 88.2% against CIN2+ and 42.6% against CIN1+ . FurtherThe women who were treated due to HPV-related disease were at high risk for developing subsequent HPV related disease. The persistent or recurrent rate of CIN2+ after excisional treatment was reported as 4% ~ 18% . A meta-The major advantage of our study is that we have detailed information about the margin status of excisional material for each woman treated for the first cervical lesion who subsequently developed lesions after surgery. This made it possible to confirm whether subsequent cervical lesions were associated with HPV genotype infection found in the original lesion. However, there are some limitations in our analysis. The subgroup of women who underwent excision treatment was not a randomized group, so we had limited power to evaluate post-treatment vaccine efficacy. Furthermore, because no any sexual behavior data was collected, we were not able to evaluate the sexual behavior difference between two groups or estimate the effect of vaccination by adjusting the factor. However, a good balance regarding HPV status at enrollment had been achieved between the two arms, which indicated that there may be no significant difference in sexual behavior between the two groups. And we used cytological results as an outcome in the analysis due to not all women after treatment underwent colposcopy, which may have potential bias. Based on previous study , cytologSignificant vaccine efficacy against new infections after treatment for 14 HR-HPV infection was observed. The nonsignificant effect against new infections associated with HPV-16/18 and cervical lesions (LSIL+ and CIN2+) maybe caused by the limited power with smaller number of women. However, the vaccine may still benefit post-treatment women with the “primary prophylactic” effect. Further research is required in clarifying the effect of using the prophylactic HPV vaccine as therapeutic agents.


TBFP (%) was quantified with total body DXA (TBFPDXA). Height (m), weight (kg), and skinfold thickness  at six sites and circumferences (cm) at five sites were measured. In the first step, four anthropometric equations were developed based on the data obtained from multiple regression analyses (TBFPDXA = dependent variable and anthropometric measurements and age = independent variables) with Group A. They were developed on the basis of circumferences (TBFP1), SFTs (TBFP2), circumferences and SFTs (TBFP3), and highly significant circumferences and SFTs (r ≥ 0.6) (TBFP4). In the second step, the newly developed equations were cross-validated using Group B. Three equations  showed the agreement with cross-validation criteria. There were no differences between TBFPDXA and TBFP estimated by these equations (p > 0.05). They showed higher measurement concordance with TBFPDXA; correlation between measured TBFP with DXA and estimated with TBFP1, TBFP2, and TBFP4, respectively, was 0.80 , 0.83 , and 0.84 . Three anthropometric measurements based on predictive equations were developed and cross-validated to satisfactorily estimate the TBFP in adult women.Attempts have been made to estimate body fat using anthropometry, and most of them are country-specific. This study was designed to develop and cross-validate anthropometric predictive equations to estimate the total body fat percentage (TBFP) of Sri Lankan adult women. A cross-sectional study was conducted in Galle, Sri Lanka, with two groups: Group A (group for equation development) and Group B  ( Obesity and overweight are serious health concerns associated with multiple disease conditions among women, both globally and locally , 2. The Even though these anthropometric measurements are used to assess global adiposity in clinical and research settings due to the simplicity and low cost , the reaDual-energy X-ray absorptiometry (DXA) estimates TBFP with a high accuracy and is considered as the gold standard reference method for estimating body composition . HoweverDurnin–Womersley  and JackAccurate assessment of body composition including body fat in South Asians is important since Asians have a higher fat mass for a given BMI compared to Caucasians . FurtherUsing equations developed in populations elsewhere to predict the adiposity is inappropriate since the inherited characteristics of populations may vary . It is eThis descriptive cross-sectional study included 350 community-dwelling women aged 30–60 years, selected from the field study area  of the Faculty of Medicine, University of Ruhuna, Galle, in Southern Sri Lanka using a multistage cluster sampling technique. This area is a semiurban area which has socioeconomic characteristics and disease prevalence similar to national figures . The stun = 175) and two other areas  were selected randomly to recruit women for the study to cross-validate the developed equations . When selecting the women, the distribution of women between different menopausal status  and postmenopausal women (PMW)) and age groups  was considered to be approximately equal.Of the 18 public health midwife's areas in the study area, three areas  were selected randomly to recruit women for the development of anthropometry equations  was calculated as weight divided by square height. Limb circumferences (cm) at midupper arm (MUAC), midthigh (ThC), medial calf (CaC) on the right side of the body, and waist (WC) and hip (HC) circumferences were measured using a nonstretchable plastic measuring tape to the nearest 1 mm  . Body weest 1 mm . Each ciest 1 mm . Each SFDXA was used as the reference standard to quantify the TBFP. TBFP  was measured with DXA scanner  adhering to the manufacturer's guidelines. All scans were performed by the same technician who calibrated the device each scanning day. Analytical software  provided by the DXA manufacturer was used to analyze the TBFP.t-test and the group comparison of categorical data was performed with the chi-square test to evaluate the suitability for cross-validation. p value <0.05 was considered statistically significant.Data were analysed using SPSS version 20.0. Descriptive statistics, means (SD) or frequency (%), were used to describe data. Group comparison of continuous data was performed with the independent sample r) were estimated between TBFPDXA and anthropometric measurements to identify significant associations. The development of equations was based on few criteria:  Step 1: all the correlated circumferences with TBFP  Step 2: all the correlated SFTs with TBFP  Step 3: all the correlated circumference and SFTs with TBFPr ≥ 0.60)  Step 4: highly significant circumferences and SFTs with TBFP (Development of new equations: data from the 175 women in Group A were used for the development of new equations. Pearson correlation coefficients (DXA. The collinearity between variables was verified by the variance inflation factor (VIF) and tolerance (T) values. Thus, VIF values <10 and tolerance values above 0.1 were considered as acceptable [The variables were entered into multiple regression models in a “stepwise” manner in these four steps to remove the weakly associated variables with TBFPceptable . Age, weDXA were determined. Mean differences between TBFP measured and estimated with developed equations were compared by the paired sample t-test. If the TBFP measured and estimated with developed equations was not statistically different, additionally, determination coefficient (R2) and standard error of estimate (SEE) were determined with linear regression analysis for those equations. To consider the equations to be valid, the validation criteria described by Lohman [DXA), SEE should be low (<3.5), and R2 should be high (>0.6). The equations which satisfied the above criteria were further tested for repeatability with Bland–Altman plots, and limits of agreements were calculated (mean difference ± 1.96SD) [Cross-validation of newly developed equations: for the cross-validation of the newly developed equations, 175 study participants assigned to Group B were used. Scatter plots and correlations between the TBFP predicted by the newly developed equations and TBFPy Lohman  were use 1.96SD) .∗ Power software version 3.1.9.2 [In order to assess the adequacy of the sample, power of the study was estimated by post hoc compute achieved power analysis using the G 3.1.9.2 .Ethics Review Committee, Faculty of Medicine, University of Ruhuna, Sri Lanka, granted ethical clearance for the study (Reference number: 24.09.2014:3.2). Each participant signed a written informed consent before providing the information, measuring AIs and body composition.p > 0.05), indicating that two groups were similar with regard to their basic characteristics.Sociodemographic and basic characteristics of measured variables of Groups A and B are shown in Tables r; 0.23, p=0.02) and all anthropometric measurements were studied; weight , height , WC , HC , limb SFT , central SFT , and limb circumferences  showed positive correlations with TBFPDXA. HC  among circumferences and TrSFT among SFTs  (p < 0.001) showed the highest correlations with TBFP.In Group A, age (The equations developed to estimate the TBFP are given in p < 0.05) (r ranging from 0.80 to 0.84) and high coefficients of determination (65% to 72% variation of TBFPDXA) with low SEEs (2.78 to 3.10 kg)  , and mea3.10 kg) . The equ3.10 kg) .  Figure3.10 kg)  was obse3.10 kg) .β error probability) was 1.00 for all the valid equations.In the post hoc power calculation test, to calculate the sample power of valid anthropometry equations was conducted by adopting an error probability of 5% for the sample size used. The sample power  has the highest measurement concordance. However, three equations that met all validation criteria have high accuracy and proved that they are suitable for research and clinical settings. The selection of the equation can be based on the availability of resources for measuring the anthropometry in the particular setting.


ESPL1, SPAG5, MKI67, PLK1, and PGR. EarlyR has been validated in multiple cohorts profiled using microarrays. This study sought to verify the prognostic features of EarlyR in a case-cohort sample from BIG 1–98, a randomized clinical trial of ER+ postmenopausal breast cancer patients treated with adjuvant endocrine therapy (letrozole or tamoxifen).EarlyR gene signature in estrogen receptor–positive (ER+) breast cancer is computed from the expression values of Expression of EarlyR gene signature was estimated by Illumina cDNA-mediated Annealing, Selection, and Ligation assay of RNA from formalin-fixed, paraffin-embedded primary breast cancer tissues in a case-cohort subset of ER+ women  from BIG 1–98. EarlyR score and prespecified risk strata  were “blindly” computed. Analysis endpoints included distant recurrence–free interval and breast cancer–free interval at 8 years after randomization. Hazard ratios (HRs) and test statistics were estimated with weighted analysis methods.The distribution of the EarlyR risk groups was 67% low, 19% intermediate, and 14% high risk in this ER+ cohort. EarlyR was prognostic for distant recurrence–free interval; EarlyR high-risk patients had statistically increased risk of distant recurrence within 8 years  compared with EarlyR low-risk patients. EarlyR was also prognostic of breast cancer–free interval .This study confirmed the prognostic significance of EarlyR using RNA from formalin-fixed, paraffin-embedded tissues from a case-cohort sample of BIG 1–98. EarlyR identifies a set of high-risk patients with relatively poor prognosis who may be considered for additional treatment. Further studies will focus on analyzing the predictive value of EarlyR signature. Selection of an effective treatment regimen for a primary breast cancer patient must take into account the molecular heterogeneity of the disease. Approximately 80% of primary breast cancers are estrogen receptor–positive (ER+), almost all of which are treated with endocrine therapy. Although prognosis for early-stage ER+ breast cancer patients treated with endocrine therapy alone is considered “good,” at least 20% of patients will suffer a distant recurrence within 10 years. Traditionally, clinical factors such as tumor size, grade, number of positive lymph nodes, and indicators of proliferation such as Ki67 have been used to assess the utility of adjuvant chemotherapy. However, these factors alone fail to define all molecular traits that have a considerable effect on prognosis. There is a considerable clinical need for a molecular assay that can differentiate between the small percentage of ER+ breast cancer patients who have high risk of relapse and may benefit from chemotherapy and the majority who can safely avoid it.Multi-gene signatures such as Oncotype DX Recurrence Score , MammaprESPL1, SPAG5, MKI67, PLK1, and PGR [To address the limitations of the current assays, we developed the EarlyR gene signature based on the expression of In most previous studies of EarlyR, gene expression was measured from frozen tissues collected from patient cohorts of convenience, limiting its clinical applicability. Additionally, these patients may not have been treated with regimens that constitute current standard of care. Validation of the prognostic significance of EarlyR in a contemporary, well-annotated, randomized, controlled clinical trial is needed. The current study aims to assess the prognostic significance of EarlyR using a cohort of patients from the randomized, controlled BIG 1–98 clinical trial of endocrine therapy for postmenopausal women with hormone receptor–positive early breast cancer .Between March 1998 and May 2003, 8010 postmenopausal women with hormone receptor–positive operable invasive breast cancer were randomly assigned to monotherapy with letrozole or tamoxifen for 5 years, a sequential strategy of letrozole for 2 years followed by tamoxifen for 3 years, or the reverse sequence . At the protocol-specified update 12 years after trial commencement, and with a median follow-up of 8.1 years, 1022 distant recurrences were observed  carried out retrospective tissue collection in accordance with institutional guidelines and national laws. Formalin-fixed, paraffin-embedded (FFPE) primary breast cancer tissue blocks were assessed for availability of invasive tumor material for translational research under the approval of the IBCSG Biological Protocols Working Group. All material processing and assaying was done without knowledge of patients’ treatment assignments or outcomes. Appropriate permissions from the University of Notre Dame and Indiana University institutional review boards were also obtained for use of the samples and data.A case-cohort sampling design was used to select 1218 FFPE tissue samples  for whole-genome profiling using the DASL protocol on the Illumina HT-12 v4 microarray as follows. All breast cancer recurrence cases  with available RNA were included, and nonrecurrence cases were sampled according to four stratification factors, resulting in 48 strata (classes). There were differences in the proportion of nonrecurrence patients sampled by the four stratification factors:  lymph noThe EarlyR cohort  at Florida Scripps. Gene expression values were cubic spline normalized with no background correction using BeadStudio software (Illumina). The natural logarithms of microarray probe expression values were used in this analysis. Expression values for probes other than the five used to compute EarlyR were not available for this project. Access to these data may be requested through the IBCSG data-sharing process.ESPL1 (ILMN_1742145), MKI67 (ILMN_1734827), SPAG5 (ILMN_2141259), PLK1 (ILMN_1736176), and PGR (ILMN_1811014). The EarlyR score (0–100) was computed for each sample as described in the Expression values for the EarlyR panel genes were obtained from the Illumina DASL assay data for the study cohort using the same Illumina probes used to compute EarlyR in the METABRIC cohort , specifiTo investigate the prognostic feature of EarlyR with disease outcomes, the analysis endpoints included DRFI, defined as time from randomization to breast cancer recurrence at a distant site, and BCFI, defined as time from randomization to first invasive breast cancer recurrence at a local, regional, or distant site or invasive contralateral breast cancer. Both endpoints were censored at the minimum of last disease follow-up, death or 8 years since diagnosis, in an attempt to focus on the assessment of “early” prognostic features of EarlyR. Eight years was chosen as a threshold based on a prior publication showing that the prognostic utility of current genomic signatures for ER+ breast cancer deteriorates after 8 years .Sampling weights were computed as described above. Weighted analysis methods  were used to adjust estimates of recurrence-free interval test statistics to obtain unbiased analyses and to give consistent estimates of prognosis in the full ER+ cohort. In the weighted analyses, contributions to Kaplan-Meier estimators and tests were weighted proportional to the inverses of the sampling fractions; special methods were used to compute variances . WeighteStatistical power was assessed based on the overall ER+ cohort (N = 1174) and the endpoint of BCFI, the endpoint that guided the case-cohort sampling. Due to the case-cohort design, an adjusted standard error estimate for the coefficient of interest  was obtained using the weighed Cox regression model. The standard error was subsequently used to assess statistical power to detect a difference (hazard ratio) comparing two adjacent EarlyR groups based on a trend test. Assuming a two-sided significance level of .05, there was a 90% power to detect a hazard ratio of 1.33 for comparing two adjacent EarlyR risk strata.P values less than .05 in the overall EarlyR cohort were considered statistically significant.Results were presented in accordance with REporting recommendations for tumour MARKer prognostic studies criteria . All staUnweighted distributions of clinical features of the EarlyR cohort (N = 1174) were consistent with those of patients enrolled in the BIG 1–98 intention-to-treat population, when considering the case-cohort sampling design , EarlyR score  classifiPtrend = .008) within 8 years of randomization; specifically, with weighted HR = 1.73 (95% CI = 1.14 to 2.64) comparing EarlyR-High vs EarlyR-Low or HR = 1.35 (95% CI = 0.91 to 2.0) comparing EarlyR-Int vs EarlyR-Low. The percent free of distant recurrence at 8 years was estimated as 91% (95% CI = 89% to 92%) for EarlyR-Low and 84% (95% CI = 80% to 88%) for EarlyR-High. The continuous EarlyR score was also statistically significantly prognostic. Specifically, for a 10-unit increase of EarlyR risk score, we observed an HR = 1.07 (95% CI = 1.03 to 1.12) for DRFI. In a multivariable model adjusting for age, LN status, tumor size, grade, and Ki-67 labeling index, we observed an HR = 1.03 (95% CI = 0.99 to 1.08) for EarlyR risk score.The EarlyR stratification was statistically significantly prognostic of DRFI in the overall ER+ cohort . The percent estimated free of breast cancer recurrence at 8 years was 87% (95% CI = 86% to 89%) for EarlyR-Low and 79% (95% CI = 75% to 84%) for EarlyR-High. Also, a continuous EarlyR score is prognostic of BCFI with HR = 1.07 (95% CI = 1.03 to 1.11) for a 10-unit increase in the EarlyR risk score.Similarly, the EarlyR risk stratification was prognostic of BCFI within 8 years .As exploratory analyses, we assessed the early (within 8 years) prognostic value of EarlyR in subsets of this ER+ cohort. The EarlyR risk stratification was shown to be prognostic of DRFI in ER+/HER2− . In this subgroup of patients, the estimated 8-year DRFI was 95.4% (95% CI = 94% to 96.8%) in EarlyR-Low (N = 334) and 88.5% (95% CI = 82.7% to 94.6%) in EarlyR-High (N = 44), as shown in The principal application of prognostic signatures such as OncoIn the BIG 1–98 clinical trial, DRFI was statistically significantly improved with letrozole vs tamoxifen monotherapy . There wClinical trials such as BIG 1-98 have shown the benefit of hormone therapy to treat early-stage ER+ breast cancer in postmenopausal women. Although the majority of early-stage ER+ breast cancer patients remain disease-free when treated with only tamoxifen or an aromatase inhibitor, as many as 20% suffer a distant recurrence within 10 years of diagnosis. Increasingly, multi-gene signatures have proven to be more effective than clinico-pathological traits alone in identifying those ER+ patients who are at a high risk of recurrence and may benefit from cytotoxic chemotherapy in addition to hormone therapy.The EarlyR genomic signature was shown to be prognostic of DRFI and BCFI in the METABRIC ER+ cohort and of distant metastasis–free survival in multiple other cohorts, independent of clinico-pathological features . HoweverIn the current study, we validated the prognostic significance of EarlyR in a case-cohort sample (N = 1174) of BIG 1-98 in which all patients were prescribed endocrine therapy for 5 years. Importantly, mRNA was extracted from FFPE tissues in a single laboratory using standardized protocol and subsequently assayed to measure gene expression.A strength of this study is the independence of the computation of EarlyR score and strata (Buechler lab), blind to clinical data, from gene expression analysis (Avera Cancer Institute) and statistical analysis . Importantly, the thresholds defining the risk strata were prospectively defined, again blind to clinical data, satisfying the criteria for a prospective study using archived samples defined by Simon et al. . The stutype DX Recurrence Score, ROR, and Mammaprint. In this study, prognosis for the EarlyR-Low patients in the overall ER+ postmenopausal breast cancer cohort was excellent, with the estimated 8-year DRFI being 91% (95% CI = 89% to 92%), and in ER+/LN− patients not treated with chemotherapy the estimated 8-year DRFI was 95.4% (95% CI = 94% to 96.8%) and estimated 8-year BCFI was 92.5% (95% CI = 90.8% to 94.3%). This low rate of distant recurrence is comparable with the distant recurrence rates of the low-risk patients identified by Oncotype DX and PAM50 ROR in the translational protocol of the Arimidex, Tamoxifen, Alone, or in Combination (transATAC) cohort of the ATAC trial  of patients with excellent prognosis and comparably few patients (at most 19%) with intermediate risk. Moreover, in the patients identified as low risk by EarlyR, there was less than 5% risk of distant recurrence at 8 years in the ER+/LN− patients not treated with chemotherapy. Additional studies are needed to determine whether patients identified as high risk by EarlyR are likely to benefit from chemotherapy.Novartis contracted with the IBCSG to support the BIG 1–98 trial. IBCSG had responsibility for the trial design; to collect, analyze, and interpret the data; and report the results. This report presents translational research based on collected tumor material , nucleic acid extraction, and gene expression assay . SB is supported by a Susan G. Komen Scholar award (SAC 110004).Affiliations of authors: University of Notre Dame, Notre Dame, IN (SB); Harper Cancer Research Institute, Notre Dame, IN (SB); IBCSG Statistical Center Department of Biostatistics and Computational Biology, Dana Farber Cancer Institute, Boston, MA ; Harvard Medical School, Boston, MA (MMR); Harvard T.H. Chan School of Public Health, Boston, MA (KPG); Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, IN ; Avera Cancer Institute, Department of Molecular and Experimental Medicine, Sioux Falls, SD ; International Breast Cancer Study Group Coordinating Center Pathology Office, Bern, Switzerland (RK); Breast Center, Kantonsspital, St. Gallen, Switzerland (BT); Swiss Group for Clinical Cancer Research SAKK, Berne, Switzerland (BT); Indiana University Melvin and Bren Simon Cancer Center, Indianapolis, IN (SSB); University of Milan, IEO European Institute of Oncology IRCCS, Milan, Italy (GV).All authors read the manuscript before submission. SAB, YGP, and SSB are holders of the NotreDame/Indiana University patent for the gene signature.We are indebted to the women, physicians, nurses, and data managers who participated in the BIG 1–98 clinical trial; to the many pathologists who submitted tumor blocks; to the BIG 1–98 Collaborative Group and BIG 1–98 Steering Committee; to Novartis for supporting the execution of the clinical trial and the collection of tumor blocks; and to the IBCSG for the design and coordination of the trial and the IBCSG Central Pathology Office for tumor block collection and processing.pkz051_Supplementary_DataClick here for additional data file.


To determine the predictive CT imaging features for diagnosis in patients with primary pulmonary mucoepidermoid carcinomas (PMECs).CT imaging features of 37 patients with primary PMECs, 76 with squamous cell carcinomas (SCCs) and 78 with adenocarcinomas were retrospectively reviewed. The difference of CT features among the PMECs, SCCs and adenocarcinomas was analyzed using univariate analysis, followed by multinomial logistic regression and receiver operating characteristic (ROC) curve analysis.P < 0.05). Only lesion location, shape, margin and degree of enhancement remained independent factors in multinomial logistic regression analysis. ROC curve analysis showed that the area under curve of the obtained multinomial logistic regression model was 0.805 (95%CI: 0.704–0.906).CT imaging features including tumor size, location, margin, shape, necrosis and degree of enhancement were significant different among the PMECs, SCCs and adenocarcinomas, as determined by univariate analysis (The prediction model derived from location, margin, shape and degree of enhancement can be used for preoperative diagnosis of PMECs. Primary pulmonary mucoepidermoid carcinoma (PMEC) is a rare malignant neoplasm of the lung and accounts for approximately 0.1–0.2% of all lung malignancies , arisingIn this study, we retrospectively reviewed the CT findings of PMECs, SCCs and adenocarcinomas in order to identify the independent predictive radiological features for the diagnosis of PMECs.Between July 2012 and March 2019, 37 patients with pathologically proven PMECs were retrospectively reviewed. In addition, 76 patients with SCCs and 78 patients with adenocarcinomas from September 2018 to July 2019 were included in order to analyze the characteristic CT features of PMECs. Patients were included if they were pathologically confirmed, and chest CT examination before treatment. Patients with the same tumor in any other organ or have previous history of the same tumor were excluded. This study was approved by the Institutional Review Board of every participating center, and patient informed consent was waived for this type of review.Clinical data including age, sex, and clinical presentation were reviewed. The main symptoms of three diseases included cough, phlegm, pectoralgia, fever and asthma.All patients had chest CT imaging before treatment. CT imaging was performed in 88 patients using a 64-slice spiral CT , and in the remaining 103 patients using a 64-slice spiral CT . The main imaging parameters were 120 kVp, 150–300 mA of automatic adjustment, a pitch of 0.982. Axial and coronal multiplanar reconstructions (MPR) images with 2–5 mm thick were obtained with lung or mediastinum kernels. After acquisition of unenhanced images, contrast-enhanced images were obtained after a bolus intravenous injection of 1.5 ml/kg contrast media , with a rate of 3 ml/s.CT data were reviewed on PACS system for all patients by two experienced radiologists in consensus . CT imaging features including primary tumor size, location, margin, shape, calcification, necrosis and degree of enhancement were determined. Tumor size was measured in maximal dimension on the transverse plane. The location of the lesions was classified as central type which is in main bronchi and trachea, hilar type which is in lobar or segment bronchi or the right middle bronchus, and peripheral type which is in small bronchi. The margin of tumor is considered as well-defined or ill-defined. The shape of the lesion was classified as irregular or regular. The regular shape was defined as round/ovoid, and irregular was lobulated. Degrees of enhancement were graded as mild, moderate and marked. Comparing the enhanced and plain CT, degree of enhancement was defined as mild if the increased CT value after enhancement is lower than or equal to 20 HU, as moderate if greater than 20HU but great less than or equal to 40 HU, and as marked if greater than 40 HU. Intra-tumor necrosis was discerned on contrast-enhanced CT images and tumor calcification was identified on unenhanced CT images.2 test or Fisher’s Exact Test, and compare lesion size of three groups by variance analysis. A multinomial logistic regression analysis was subsequently developed to determine the association between the three groups and radiologic variables. Variables with P value less than 0.05 as determined by univariate analysis were further chosen as the independent predictor for diagnosis of PMECs in the multivariate logistic regression analysis. A two-side P value of less than 0.05 was considered statistically significant. Furthermore, receiver operating characteristic (ROC) curve analysis was used to evaluate the obtained multinomial logistic regression model. All statistical tests were performed by using software SPSS .The CT imaging features for statistical analysis were categorized as follows: lesion location , margin (well-defined or ill-defined), shape (irregular or regular), necrosis (presence or absence), calcification (presence or absence), degree of contrast enhancement . Univariate analysis was applied to compare the frequency of these imaging findings among PMECs, SCCs and adenocarcinomas by using χIn cases of PEMCs, there were 15 women (40.5%) and 22 men (59.5%), aged from 10 to 77 years with a mean of 44.95 years ±19.26. In cases of SCCs, there were 7 women (9.2%) and 69 men (90.8%), aged from 36 to 83 years with a mean of 63.11 years±7.72. In cases of adenocarcinomas, there were 31 women (39.7%) and 47 men (60.3%), aged from 37 to 93 years with a mean of 61.83 years±10.28.The main CT characteristics of three groups are summarized in Table P < 0.05) (Table P < 0.05). Meanwhile, the primary tumors in lung with central type (OR = 185.586) or hilar type (OR = 518.164), well-defined margin (OR = 7.283), regular shape (OR = 9.279) are more likely to be PMECs than adenocarcinomas (P < 0.05). Further ROC curve analysis showed that the area under curve (AUC) of the obtained logistic regression model was 0.805 (95% CI: 0.704–0.906)  Table . Multino5) Table . Moreove5) Table . The priOR = 7.28, regularPrimary PMECs are rare and still a challenge for diagnosis. Preoperative identification of patients with PMECs is important for the assessment of clinical outcome and the choice of treatment , 14. HerClinically, PMECs tended to affect young adults , which wPMECs are commonly found in the segmental or lobar bronchi , 17–19. To improve diagnosis, we used multinomial logistic regression analysis to identify the predictive radiological features for the diagnosis of PMECs. In our study, PMECs were central and hilar type in 89.2%, SCCs were central and hilar in 46.1% patients, and adenocarcinomas were central and hilar just in 9% patients. In multinomial logistic regression analysis, location of lung cancer is an independent feature for differential diagnosis. Primary lung cancer with central type of location are more likely to be PMEC than SCC (OR = 177.326) or adenocarcinoma OR = 185.586), and the tumor with hilar type of location is also more likely to be PMEC than SCC (OR = 56.484) or adenocarcinoma (OR = 518.164). PMECs are usually arises from large airway, including the trachea and the main or lobar bronchi . However5.586, anOur study has several limitations. First, as a retrospectively study using CT data from different hospitals over different periods of years, it would inevitably suffer from some degree of selection bias and various CT facilities and scans parameters. However, all patients in our series had slice thicknesses of CT images less than 5 mm, which may be acceptable for imaging analysis. Second, the number of patients in this study was limited because of PMECs are rare and the follow-up period is just 7 years. Third, no state-of-art CT was used in our study, such as dual-source or spectral CT. CT spectral quantitative parameters are valuable in evaluating histological types of lung cancers, and it is maybe even possible to correctly determine the lesion density before contrast enhancement . Future In conclusion, our study results suggest that CT features including location, margin, shape, and degree enhancement are predictors for diagnosis of PMEC. The primary lung tumor with central type or hilar type, well-defined margin, regular shape, and marked enhancement indicates PMEC.


Esophageal squamous cell carcinoma (ESCC) is a leading cause of cancer death worldwide and in China. We know miRNAs influence gene expression in tumorigenesis, but it is unclear how miRNAs affect gene expression or influence survival at the genome-wide level in ESCC.We performed miRNA and mRNA expression arrays in 113 ESCC cases with tumor/normal matched tissues to identify dysregulated miRNAs, to correlate miRNA and mRNA expressions, and to relate miRNA and mRNA expression changes to survival and clinical characteristics.P < 1.92E-04), including several not previously reported in ESCC . Expressions of 16 miRNAs were highly correlated with expressions of 195 genes . Increased expressions of miRNA in tumor tissue for both miR-30e* and miR-124 were associated with increased survival (P < 0.05). Similarly, nine probes in eight of 818 dysregulated genes had RNA expression levels that were nominally associated with survival, including NF1, ASXL1, HSPA4, TGOLN2, BAIAP2, EZH2, CHAF1A, SUPT7L.Thirty-nine miRNAs were identified whose tumor/normal tissue expression ratios showed dysregulation (28 down- and 11 up-regulated by at least two-fold with Our characterization and integrated analysis of genome-wide miRNA and gene expression in ESCC provides insights into the expression of miRNAs and their relation to regulation of RNA targets in ESCC tumorigenesis, and suggest opportunities for the future development of miRs and mRNAs as biomarkers for early detection, diagnosis, and prognosis in ESCC. Esophageal carcinoma occurs worldwide as the sixth leading cause of cancer mortality  and is aGene expression profiling can improve our understanding of molecular alterations during carcinogenesis. Biomarkers of these molecular alterations, in turn, may be useful in diagnosing cancers, particularly early, curable cancers. They may also identify druggable targets for therapy or be useful in predicting prognosis. Regulatory mechanisms underlying gene expression are vital functions in biological processes. The discovery of microRNAs (miRNAs) has revealed a hidden layer of gene regulation that can tie multiple genes together into biological networks. More than 2500 mature human miRNAs have been identified thus far (miRBase assembly version GRCh38)  since thThus far there have been only a few reports of genome-wide analyses of both miRNA and mRNA expression in paired tumor/normal tissues from ESCC patients, but these studies have included only a small number of cases  or very Patients enrolled in the project included consecutive cases of ESCC who presented to.the Surgery Department of the Shanxi Cancer Hospital in Taiyuan, Shanxi Province, PR China, between 1998 and 2003, who had no prior therapy for their cancer, and who underwent surgical resection of their tumor at the time of their hospitalization. After obtaining written informed consent, patients were interviewed to obtain information on demographic and lifestyle cancer risk factors, and family history of cancers. Clinical data were collected at the time of hospitalization (between 1998 and 2003) and cases were followed after surgery for up to 69 months to ascertain vital status (median follow-up 23 months). In total, 113 ESCC cases were evaluated in the present study. All cases were histologically confirmed as ESCC by pathologists at both the Shanxi Cancer Hospital and the National Cancer Institute (NCI). This study was approved by the Institutional Review Boards of the Shanxi Cancer Hospital and the NCI.http://tools.invitrogen.com/content/sfs/manuals/trizol_reagent.pdf). A second method of RNA extraction was by using Allprep RNA/DNA/Protein mini kit from Qiagen, following the manufacturer’s instructions (http://www.qiagen.com/literature/render.aspx?id=2067). For both extraction methods, the quality and quantity of total RNA were determined on the RNA 6000 Labchip/Agilent 2100 Bioanalyzer .Paired esophageal cancer and normal tissue distant to the tumor were collected during surgery. Tissues for RNA analyses were snap frozen in liquid nitrogen and stored at − 130 °C until used. Selection of patients for RNA studies was based solely on the availability of appropriate tissues for RNA testing  tumor, and tissue RNA quality/quantity adequate for testing). Total RNA was extracted by two methods: one was extracted by the Trizol method following the protocol of the manufacturer  for a total of 664 unique human miRNAs. In addition, each card contained one selected endogenous control assay (MammU6) printed four times, 5 endogenous gene probes , and one negative control assay (ath-miR159a). Card A focused on more highly characterized miRNAs, while Card B contained many of the more recently discovered miRNAs along with the miR* sequences.http://www3.appliedbiosystems.com/cms/groups/mcb_support/documents/generaldocuments/cms_042167.pdf. Briefly, three microliter (ul) of total RNA (350–1000 ng) was added to 4.5uL of RT reaction mix, which consisted of 10x Megaplex RT Primers, 100 mM dNTPs with dTTP, 50 U/uL MultiScribe Reverse Transcriptase, 10x RT buffer, 25 mM MgCl2, 20 U/uL RNase Inhibitor, and nuclease-free H2O. The samples were run on a thermal cycler using the following conditions: 40 cycles of 16 °C for 2 min, 42 °C for 1 min, and 50 °C for 1 s. All reactions were completed with a final incubation at 85 °C for 5 min. Six microliters of cDNA generated were mixed with 450uL of 2x TaqMan Universal PCR Master Mix with no AmpErase UNG, and 444uL of nuclease-free H2O. 100uL of the reaction mix was added to each of 8 fill ports on a TaqMan MicroRNA Array. The filled Array was centrifuged twice at 1200 rpm for 1 min, and then sealed with 8 fill ports film. Arrays were run on a 7900HT RT-PCR System with the SDS software and the comparative CT method was used to determine the expression levels of mature miRNAs.The protocol was according to the manufacturer’s manual at http://www.affymetrix.com/support/index.affx).Of the 113 paired ESCC samples, 34 pairs were run on Human U133A chips, 73 pairs on U133A_2 chips, and 6 pairs on U133Plus_2 chips from Affymetrix. Probes were prepared according to the protocol provided by the manufacturer , available from: Procedures included first strand synthesis, second strand synthesis, double-strand cDNA cleanup, in vitro transcription, cRNA purification, and fragmentation. Twenty micrograms of biotinylated cRNA were finally applied to the hybridization arrays of the Affymetrix GeneChip. After hybridization at 45 °C overnight, arrays were developed with phycoerythrin-conjugated streptavidin by using a fluidics station (Genechip Fluidics Station 450) and scanned (Genechip Scanner 3000) to obtain quantitative gene expression levels. Paired tumor and normal tissue specimens from each patient were processed simultaneously during the RNA extractions and hybridizations.http://www.lifetechnologies.com/about-life-technologies.html). The miRNAs that showed signals in tumor only or normal only were dropped from further analysis. In the present study, the data are presented as fold change calculated using the 2 -ΔΔCT method. Results of the real-time PCR data were represented as CT values, where CT was defined as the threshold cycle number of PCRs at which amplified product was first detected. The average CT was calculated for both the target genes and MammU6, and the ΔCT was determined as the mean of the CT values for the target gene minus the mean of the quadruplicate CT values for MammU6. The ΔΔCT represented the difference between the paired tissue samples, as calculated by the formula ΔΔCT = . The N-fold differential expression in the target gene of a tumor sample compared to the normal sample counterpart was expressed as 2 -ΔΔCT.RQ Manager integrated software from the ABI was used to normalize the entire signal generated. Expression levels  were calculated when both tumor and normal sample gave signals in the assays using DataAssist software v2.0  were calculated for each case separately for the 113 normal and 113 tumor tissue samples tested. Over all samples, CVs for MammU6 were determined to be very low – 1.3% for normal tissues and 0.7% for tumor tissues, indicating that technical variation was minimal; thus, reproducibility was excellent for use of MammU6 in our normalization procedure.As our normalization procedure was based on MammU6, our endogenous control, we assessed the technical variation of our normalization procedure by determining the coefficient of variation (CV) of the quadruplicate CAs miRNAs span a wide range of expression levels, median fold changes are a more accurate representation of miRNA expression values and are used throughout our miRNA analysis.http://www.targetscan.org/ by Whitehead Institute for Biomedical Research  to check for conserved miRNA at the 3’UTR for genes affected. We also used the http://mirtarbase.mbc.nctu.edu.tw/index.html database to search miRNA target genes. This database collects data on miRNA-target interactions based on validated experiments.We used http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE44021 and GSE67268 for miRNA at http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE67268All statistical analyses were developed using R packages. MicroRNAs that showed signal in both tumor and normal tissue in at least 50% of cases were included in analyses presented here . Spearman correlations were used to evaluate the association between expressions of miRNA and mRNA. Nearly six million  Spearman correlations and their corresponding P-values were computed. To address the multiple testing problem here we used a Bonferroni corrected P-value cut off of 8.40E-09  to select significant miRNA–target gene pairs. We also explored associations between miRNA and mRNA expression and clinical/pathological variables using Spearman analysis. For all evaluations presented here , we used the miRNA signals (average delta Ct) or mRNA signals (average) for tumor:normal expressed as fold change ratios. For each miRNA or mRNA, we applied the Kaplan-Meier method to visualize differences and the Log-Rank test to statistically compare survival by expression levels divided as high versus low expression.Paired t-tests were used to identify differences in matched tumor/normal samples for mRNA expression. To find miRNAs with significant fold changes, we applied the Wilcoxon method to the fold change data in log10 scale with Bonferroni correction at 0.05, which resulted in a threshold To further explore patterns of expression of miRNAs visually, we performed hierarchical clustering of data from miRNA expression by case. For this clustering, missing values were replaced by the median for each probe, and data were transformed to normalize their distribution. The R function ‘heatmap’ was used to generate the heatmap with the method set to ‘ward’ to calculate the distance used for the hierarchical clustering. We also evaluated the 11 demographic/clinicopathologic variables shown in Supplementary Table We used Cox proportional hazards regression models to evaluate survival as the hazard ratio (HR) for miRNA and gene expression fold change with adjustment of the four clinical variables age, gender, metastasis, and stage. We coded the fold change variables for miRNA and gene expression in two ways. First we assigned a single ordinal variable to represent each of the four quantile intervals . Second, we created indicator variables for each of the four quartiles so that we could compare Q2, Q3, and Q4 separately to Q1 as the reference category.Characteristics of the 113 total ESCC patients evaluated here are summarized . In order to have sufficient numbers of cases with expression data for each miRNA, we required that at least half the patients express an miRNA in both tumor and normal tissue for it to be included in our analysis. This restriction reduced the number of miRNAs we analyzed here from 523 to 260.P-value less than 0.05 after Bonferroni correction  and a P-values > 0.10).Hierarchical clustering was performed to characterize miRNA expression for all tumors and matched normal tissues. Heat maps showed similar patterns when using probe sets that had signals across all 113 samples in either 50% or 90% of the samples, so we report only results for probe sets with signals on at least half the samples. Here, we show that miRNAs (rows) cluster into two main groups with several sub-groups . The 10 most up-regulated genes were MMP1, SPP1, COL11A1, COL1A1, POSTN, MMP12, MAGEA6, MAGEA3, COL1A2, and KRT17; while the 10 most down-regulated genes were CRISP3, CRNN, MAL, TGM3, CLCA4, SCEL, CRCT1, SLURP1, TMPRSS11E, and FLG.Gene expression (mRNA) was profiled on Affymetrix U133A chips and results analyzed with paired t tests. A total of 818 genes showed dysregulated gene expression between tumor and normal tissues, including 422 down-regulated and 396 up-regulated genes  and a n = 22,277 mRNA probes, including all 818 dysregulated genes described above). Expression of 16 miRNAs showed correlation with expression of 195 genes at the P < 8.42E-09 level (Table ACOX2 under expression) and negative (rho = − 0.54 with EZH2 over expression) correlations. Taken together, these results indicate that one miRNA can target multiple genes and execute positive or negative effects on the expression of these genes.Spearman analysis was applied for the correlation analysis between 267 microRNAs and all mRNAs expressed in both tumor and matched normal tissues , tumor grade (grade 1 and 2 vs grade 3 and 4), and tumor stage (stage I and II vs III and IV).P < 0.05; Supplementary Table P < 1.92E-04). Nine miRNAs correlated with the presence of metastasis , seven with higher tumor grade , and 10 with higher tumor stage . These correlations were all moderate in magnitude, ranging from 0.19 to 0.28, and the fold changes observed were similarly modest, except for eight which exceeded twofold differences (six with FC < 0.50 and two with FC > 2.00). No overlapped miRNA was seen in the three categories. Taken together, we found no striking or clear-cut associations between miRNA expression and the clinicopathological features studied here.Twenty-six miRNA expressions were correlated with one of the three clinical phenotypes we evaluated at the level of nominal significance (P < 0.05), including miR-30e*  and miR-124 , P = 0.0459).We analyzed the expression of 39 dysregulated miRNAs with survival using Cox models with adjustment for age, gender, metastasis, and tumor stage  were associated with survival at the nominal significance level (P < 0.05) in Cox models adjusted for age, gender, metastasis, and stage (Supplementary Table NF1 (HR = 0.30); this translated into a median survival improvement of 11.1 months (for Q4 vs Q1). Median survival differences for persons in the highest vs lowest quartiles of gene expression were largest for EZH2 and CHAF1A at − 18.3 and − 20.1 months, respectively.We also investigated associations between the 195 genes (395 probes) that were significantly associated with miR expression (as shown in Table MicroRNAs (miRs) play an important central role in regulating the stability and expression of messenger RNA. To our knowledge, the present study is the largest to date to characterize genome-wide expressions of miRs and mRNAs in matched tumor/normal tissues from ESCC patients and relate those expressions to prognosis.We identified 39 miRs that showed significant dysregulation in ESCC, including 11 up- and 28 down-regulated. Some of these miRNAs have been reported in cancers before, including ESCC . Among the dysregulated miRNAs identified, miR-196b showed the greatest up-regulation (FC 9.3) while miR-375 had the greatest down-regulation (FC 0.02). Over-expression of miR-196b has been previously described in ESCC, in pancreatic and gastric cancers, and in leukemia –26. ThisSLC2A1 (Solute Carrier Family 2 Member 1) (FC 2.40). This gene encodes a major glucose transporter in the mammalian blood-brain barrier. Lazar et al. reported increased expression of this gene in some malignant tumors and suggested a role for glucose-derivative tracers to detect in vivo thyroid cancer metastases by positron-emission tomography scanning [PPL(Periplakin) (FC 0.17) and EVPL (Envoplakin) (FC 0.29). The EVPL gene encodes a member of the plakin family of proteins that form components of desmosomes and the epidermal cornified envelope. This gene is located in the tylosis esophageal cancer locus on chromosome 17q25, and its deletion is associated with both familial and sporadic forms of ESCC [PPL is an important paralog of the EVPL gene and both EVPL and PPL were down-regulated, indicating that miR-203 can regulate expression of more than one gene in ESCC. These results suggest that some miRs may act as tumor suppressors  while others function as oncogenes  in ESCC.Many studies have identified numerous dysregulated miRs in various cancers. However, whether these dysregulated miRs influence gene targets in tumors is unclear. To better understand the associations between expression levels of miRs and gene targets, we performed genome-wide expression of miRs and mRNA using patient-matched tumor and normal tissues. We identified 16 miRs whose expressions correlated with gene expression (after Bonferroni correction), including six miRs whose tumor:normal expression FCs were < 0.50. For example, decreased expression of miR-133a (FC 0.19) correlated with up-regulation of scanning . On the  of ESCC . PPL is EZH2 .We identified three miRs , and miR-650 (FC 0.98) correlated with over-expression of CXCL13 (FC 2.80). It seems clear that there are many factors that can influence gene expression beyond just the effect of miRs , and that widespread dysregulation of cancer-associated miRs is a complex phenomenon. Many of these miRs have been located downstream of major oncogenes and tumor suppressors that act as transcription factors [Although we found 16 miRs whose expression correlated with gene expression, the magnitude of the tumor:normal expression level ratios in 10 of these miRs was in the normal range . For example, miR-155 (FC 1.73) correlated with over-expression of  factors . Thus, pmiR expression has been associated with diagnosis, prognosis, and response to treatment in various cancers. The present study suggests a number of potential miRs that might serve as early detection or diagnostic markers. Essentially all 39 dysregulated miRs shown in Table This study also found evidence that expression of selected individual miRs might serve as prognosis markers in ESCC cases. Expressions of two miRs – miR-124 and miR-30e* – were both associated with survival in ESCC patients. miR-124 was up-regulated in ESCC patients (FC 2.98) and patients with higher expression levels lived longer, while miR-30e* was down-regulated (FC 0.40) but patients with lower expression levels also survived longer.Based on findings from the current study, the most attractive individual miR for future development as a potential biomarker in ESCC appears to be miR-124. This is because miR-124 was markedly elevated in tumor compared to normal tissue (FC 2.98), making it attractive as a potential early detection/diagnosis marker. At the same time, expression of miR-124 was also associated with survival, suggesting that it may serve as a prognosis marker as well.Hierarchical clustering identified collections of ESCC cases whose miR expression profiles grouped them together. Unfortunately, survival experience for patients in these different groupings did not differ.In addition to the miR-30* and miR-124 associations with survival noted above, expression of eight genes (9 different mRNA probes) were also nominally associated with survival. Additional work is needed to establish if the observed associations with survival in ESCC for these mRNAs are real or just false positives. Further, inherent instability in mRNAs makes their practical use as biomarkers very difficult, so, in addition to further validating the use of these mRNAs as prognosis markers, efforts to translate assessment of mRNA expression into gene-specific protein expression assays that can be readily applied in clinical labs  are also needed.Our identification of both miRs and genes whose expressions separately appear to relate to survival in ESCC patients suggests that further exploration of models employing miR and gene markers together might lead to markers or signatures with improved predictive performance. Thus, integrating the data from different sources initially generated to inform on the biologic role of these small molecules might also find clinical relevance as markers for early detection, diagnosis, or prognosis.Using genome-wide platforms in tumor and normal tissues we identified 39 miRs with dysregulated expression in ESCC patients. Combining these miR data with genome-wide RNA expression data on the same patients, we further determined that expression of 16 miRs strongly correlated with RNA expression in 195 genes. We found both miRs and RNAs whose expressions showed suggestive associations with clinical characteristics and survival. Taken together, our findings provide insights into the expression of miRs and their relation to regulation of RNA targets in ESCC tumorigenesis, and suggest opportunities for the future development miRs and mRNAs as biomarkers for early detection, diagnosis, and prognosis in ESCC.Additional file 1: Table S1. 260 miRNAs with tumor & normal tissue expression in ≥50% of cases.Additional file 2: Table S2. Demographic and clinical characteristics of ESCC cases (n = 113).Additional file 3: Figure S1. Heatmap of miRNAs dysregulated in at least half of ESCC cases.Additional file 4: Table S3. Gene/probe expressions correlated with miR-203Additional file 5: Table S4. Correlation of miRNA expression with clinical characteristics.Additional file 6: Table S5. Survival by miR expression for 16 miRs correlated with gene expression  correlated with miRs.Additional file 8: Figure S2. Kaplan-Meier survival plots by quartile of RNA expression for 9 probes correlated with miRNAs (from Supplementary Table


The following salivary biomarkers were determined: total proteins (TP), measured by the Lowry method; oxidative damage, measured by the TBARS method; total antioxidant capacity (TAC); superoxide dismutase (SOD) enzymatic antioxidant activity; and uric acid (UA) non-enzymatic antioxidant activity. Data were analyzed by ANOVA, followed by the Student-Newman-Keuls test, Pearson and Spearman correlation coefficients, and multivariable linear regression (p < 0.05). TP, TAC, SOD enzymatic antioxidant activity, and UA non-enzymatic antioxidant activity increased with caries severity, consequently reducing salivary oxidative damage. It was concluded that higher caries severity increases salivary antioxidant system activity, with consequent reduction in salivary oxidative damage.Oxidative stress biomarkers can be found at detectable concentrations in saliva. These salivary biomarkers reflect specific oxidation pathways associated with caries and periodontitis. Our study evaluated the influence of dental caries severity (assessed using the ICCMS™ criteria) on the levels of oxidative stress biomarkers in saliva from children. Unstimulated saliva samples were collected from patients (from one to three years old) in a day care center in Birigui, SP, Brazil, two hours after fasting. Children were divided into four groups ( Severe Early Childhood Caries (S-ECC) is defined as any sign of dental caries on smooth dental surfaces  occurrinSaliva, which can be collected in a noninvasive and safe way, has been used as a biological fluid to substitute plasma samples in the diagnosis and prognosis of systemic disease, such as sickle cell anemia in children , dementiPhysiological metabolism is the main source of free radicals , and salStudies have reported a positive correlation between salivary protein concentration and salivary total antioxidant capacity (TAC) , 20, 21.Considering the complex interplay described above, the aim of the present study was to determine the influence of caries severity (classified according to the ICCMS™ criteria) on the levels of enzymatic and nonenzymatic antioxidants in saliva of children, as well as oxidative damage in saliva of children. We hypothesized that carious lesion severity influences the salivary antioxidant status; that is, in the early stages of carious lesions oxidative damage markers would be increased, but salivary antioxidant systems would be activated in advanced stages in order to reduce oxidative damage.″ south and longitude 50°20′24″ east at an altitude of 450 meters; it has an annual average temperature of 22.1°C and human development index of 0.780).The Human Ethics Committee of the School of Dentistry, Araçatuba, São Paulo State University , reviewed and approved the research protocol (CAAE 71063417.4.0000.5420). This research involved children aged between one and three years of age, who were participating in educational and preventive oral health programs at the childcare center Dionisia Miragaia Carmine, in the city of Birigui, São Paulo, Brazil . Subsequently, 120 children were randomly enrolled in the study (blocking stratification), comprising 30 caries-free children (Group A) and 90 subjects presenting carious lesions at different stages . Initially, 32 children were selected for the caries-free group (Group A); however, two children were excluded due to noncooperation.The exclusion criteria were children whose parents/guardians did not provide the signed informed consent form and children presenting carious lesions with more than one ICCMS™ classification. Moreover, any alteration in gingival color and morphology, visible biofilm, previous restorations, and dental eruptions associated with exacerbated inflammatory response observed during the clinical examination were also adopted as exclusion criteria. Besides, systemic diseases, chronic or not, and use of medications were also considered as exclusion criteria.n = 30/group) according to the ICCMS™ criteria [An experienced pediatric dentist calibrated in the area examined all the children under natural light, with the aid of a WHO probe and mirror; a gauze was used for drying. Children were divided into four groups  and stored at -80°C until analysis [To minimize the possible influence of recent food/beverage intake, unstimulated saliva was collected between 7:00 a.m. and 8:00 a.m., after fasting for 2 h. Oral hygiene was conducted at home by the parent or guardian, who employed only water and toothbrush without fluoride products. In cases of adverse event, in which saliva collection was difficult , the procedure was interrupted, and it was not attempted again. Saliva samples were collected with the Salivette® cotton swab, which was positioned and maintained in the sublingual space for 5 min. The collected samples were kept on ice. Then, the samples were centrifuged at 5500 × uots 100–0 μL and All biochemical analyses were performed by colorimetric reactions. Samples were placed in 96-well plates, except in the case of SOD analysis  and read in a PowerWave 340 BioTek reader.Salivary flow was determined by the total value of each sample collected (mL) during 5 minutes, and the results were expressed in mL/min.μL saliva aliquots using the method by Lowry et al. in 1951 [Protein was quantified in 20  in 1951 . A standw/v) was added to 125 μL of saliva, which acidified the solution and caused the proteins to precipitate. The mixture was centrifuged , thiobarbituric acid  was added to the supernatant, and the solution was kept in a water bath at 100°C for 15 min. After reaching room temperature, the absorbance was read at 535 nm; a molar absorption coefficient of 1.56 × 105 M−1 cm−1 was employed. Results are expressed in nmol/L and in nmol/L/mg of protein.The oxidative damage was evaluated by the TBARS method, which determines the substances reactive to thiobarbituric acid. TBARS was determined as described by Buege and Aust in 1978 . Trichlo3+ TPTZ), which forms Fe2+ in acidic medium. A 15 μL saliva aliquot was used. Absorbance was determined at 595 nm; a standard ferrous sulfate curve with values ranging from 20 to 260 μmol/L FeSO4 was employed. Results are expressed in μmol/L FeSO4 and in μmol/L FeSO4/mg of protein.Salivary TAC was evaluated by the antioxidant power of iron reduction (FRAP assay), as previously standardized by Benzie and Strain in 1996 . This meμL of saliva by means of the enzymatic method of Trinder. The manufacturer's instructions were followed. Results are expressed as mg/mL and as mg/mL/mg of protein.A commercial kit  was used to determine UA in 20 μL aliquot of saliva was previously diluted . Absorbance was read at 420 nm. Five readings were taken every 1 min. The amount of enzyme that was necessary to reduce pyrogallol autoxidation by 50% was considered as a unit of enzymatic activity. Results are expressed in UE/mL and in UE/mL/mg of protein.On the basis of pyrogallol autoxidation inhibition, SOD activity was determined in saliva by the method of Maklund in 1985 . A 20 μLr between 0.50 and 0.69, 0.70 to 0.89, and >0.90 were considered as moderate, strong, and very strong correlations, respectively [p < 0.05.Data are expressed as mean ± SD (standard deviation). Data showed normal distribution (Shapiro-Wilk's test) and was submitted to ANOVA and Student-Newman-Keuls' posttest. The relationship between the dependent variables  was examined by the Pearson correlation coefficient, and the nonparametric Spearman correlation test was performed for correlations between groups (ICCMS™) and dependent variables . Values of ectively . The mulGender was fairly distributed within each group, with boys corresponding to 46.6% in Groups A and C and 50% in Groups B and D. The age of the children progressively increased from Group A to Group D. The number of erupted teeth also increased progressively from Groups A to C, but there was no difference between Groups C and D. The number of carious lesions was not significant among the Groups B, C, and D. Salivary flow rate was not significantly different among the groups .p < 0.001) in Group D as compared to the other groups. Higher concentrations were observed in Group C than in Groups A and B (p < 0.001), with no significant differences between Groups A and B (p > 0.05) .Total salivary protein concentration was higher ( > 0.05) . There wp < 0.0001) in Group D than in the other groups. Such levels were lower in Group C than in Groups A and B, with no significant differences between Groups A and B (p > 0.05). There was a strong negative correlation between caries severity and salivary MDA values . MDA values and total salivary proteins correlated negatively, but moderately  in Group D than in the other groups. Higher levels were observed in Group C than in Groups A and B (p < 0.001), without significant differences between Groups A and B (p > 0.05). A positive and strong correlation was observed between caries severity (ICCMS™) and salivary TAC . In addition, there was a very strong negative correlation between TAC and salivary MDA  in Group D as compared to the other groups. In Group C, UA concentration was higher than in Groups A and B (p < 0.001), without significant difference between Groups A and B (p > 0.05) (p < 0.0001) in Groups C and D as compared to Groups A and B; however, there were no significant differences between Groups C and D (p > 0.05) . Moreover, UA/mg of protein values and salivary TAC correlated strongly and positively, whilst UA and salivary MDA correlated weakly and negatively  . When sa > 0.05) . There wgatively .p < 0.001) in Group D than in the other groups. In Group C, SOD activity was greater than in Groups A and B (p < 0.001), without significant differences between Groups A and B (p > 0.05) (p < 0.001) in Groups C and D than in the Groups A and B, but there were no differences between Groups A and B (p > 0.05) and C and D (p > 0.05) . SOD activity correlated moderately negatively with salivary oxidative damage (SOD activity was significantly higher ( > 0.05) . When no > 0.05) . There we damage .i.e., without cavitation) were not significantly different from caries-free children. On the other hand, the higher the lesion severity, the higher the concentrations of salivary proteins and oxidative stress biomarkers. Together, these findings may contribute to future clinical and biochemical studies in the area of cariology.The development of clinical diagnostic criteria that consider carious lesions at different stages of tissue compromising has allowed a better comprehension of dental caries as a progressive disease, in addition to the establishment of preventive and therapeutic measures depending on the degree of tissue damage. Considering the lack of studies assessing the relationship between caries severity and salivary oxidative stress in children, the present study assessed the oxidative stress biomarkers in saliva of children presenting carious lesions at different stages, according to the ICCMS™ criteria. The present results showed that the concentrations of total protein and oxidative stress biomarkers in saliva of children with initial carious lesions  resemble the protein concentration previously quantified in saliva of children with S-ECC (grouped without taking caries severity into account) , but areCaries severity was shown to increase the amount of salivary proteins, as these variables were positively correlated. Carious lesions in dentin are associated with mineral and organic matrix degradation by the activity of collagenolytic enzymes, mainly matrix metalloproteinases (MMPs), and high MMP-8 concentrations have been found in saliva of individuals with carious lesions in dentin . In addiμmol/L/mg of protein) is lower than in saliva of caries-free children (0.0039 μmol/L/mg of protein). However, recent studies [μmol/L and 0.71 nmol/mL, respectively) are increased as compared to caries-free patients. Methodological differences may account for the discrepancies between our results and literature data. It is noteworthy that TBARS values were not normalized by the salivary protein concentration in neither of the aforementioned literature studies. Because the presence and progression of dental caries raise the salivary protein concentration, the TBARS concentration values must be normalized by the amount of salivary protein found in each group. Caries severity and salivary TBARS values are strongly and negatively correlated, which reinforces our results. In addition, multivariate linear regression shows that caries severity is the variable associated with salivary oxidative damage. Taken together, our data demonstrate that carious lesion progression reduces salivary oxidative damage [TBARS assay was used as a rapid screening procedure , since i studies  have repe damage .i.e., a high amount of antioxidant contributes to reduce oxidative damage [The mechanisms that reduce salivary oxidative damage have been recently evaluated, and TAC assessment provides information on the balance between oxidant and antioxidant systems, e damage . Salivare damage  and in ce damage  have beee damage . Surprise damage , suggeste damage .TAC reflects the combined effects of antioxidants (mainly nonenzymatic) present in plasma and body fluids . Given ti.e., higher SOD activity in saliva increases NO bioavailability.The contribution of enzymatic antioxidant activity to oxidative damage in saliva of children with caries lesions has also been evaluated. To this end, the activity of SOD, which is the enzymatic antioxidant system that is considered as the body's first line of defense against reactive species, has been investigated . SOD catOn the basis of the results presented herein, we can conclude that caries severity has a direct effect on the activity of antioxidant systems and salivary TAC, which gradually reduces salivary oxidative damage.


The aim of the present study was to investigate the role and underlying mechanisms of FAO in the protective effects of mild hypothermia. We used male mice to establish the experimental models as previously described. In brief, before exposure to in situ ischemia for 1 h and reperfusion for 6 h, mice received pretreatment with mild hypothermia for 2 h and etomoxir (inhibitor of FAO) or leptin (activator of FAO) for 1 h, respectively. Then, tissue and blood samples were collected to evaluate the liver injury, oxidative stress, and changes in hepatic FAO. We found that mild hypothermia significantly reduced the hepatic enzyme levels and the score of hepatic pathological injury, hepatocyte apoptosis, oxidative stress, and mitochondrial injury. In addition, the expression of the rate-limiting enzyme (CPT1a) of hepatic FAO was downregulated almost twofold by IR, while this inhibition could be significantly reversed by mild hypothermia. Experiments with leptin and etomoxir confirmed that activation of FAO could also reduce the hepatic enzyme levels and the score of hepatic pathological injury, hepatocyte apoptosis, oxidative stress, and mitochondrial injury induced by IR, which had the similar effects to mild hypothermia, while inhibition of FAO had negative effects. Furthermore, mild hypothermia and leptin could promote the phosphorylation of JAK2/STAT3 and upregulate the ratio of BCL-2/BAX to suppress hepatocyte apoptosis. Thus, we concluded that FAO played an important role in hepatic IR injury and mild hypothermia attenuated hepatic IR injury mainly via the regulation of JAK2/STAT3-CPT1a-dependent FAO.Hepatic ischemia–reperfusion (IR) injury is a clinical issue that can result in poor outcome and lacks effective therapies at present. Mild hypothermia (32–35°C) is a physiotherapy that has been reported to significantly alleviate IR injury, while its protective effects are attributed to multiple mechanisms, one of which may be the regulation of fatty acid  Hepatic ischemia–reperfusion (IR) injury is a serious clinical problem that can hardly be avoided in certain kinds of surgeries, such as liver transplantation and resection, as well as hemorrhagic shock , 2. The Mild hypothermia (32–35°C)  is a cliAs is well known, hypothermia could lead to a significant reduction in body metabolism; cellular oxygen and glucose requirements decrease by an average of 5–8% for every degree of decrease in temperature , 16, 17.β-oxidation (FAO) in the liver is the primary pathway for the oxidation of fatty acids and also the key metabolic pathway for energy homeostasis [The liver is a key regulatory organ in lipid metabolism; it can take up as well as oxidize fatty acids; it can synthesize, store, and secrete TG in VLDL particles; and it is the main metabolic orsgan that controls whole-body cholesterol and BA metabolism , 22. Moreostasis , 24. Fureostasis . A largeeostasis , 25, 26.eostasis , 28. Zhaeostasis . In vieweostasis , and theBoth mild hypothermia and IR injury have significant effects on lipid metabolism. However, the effects of changes in fatty acid metabolism under mild hypothermia on IR injury have not been addressed. Hence, the concrete effects of hepatic FAO under mild hypothermia on liver IR injury in male mice and the exact signaling pathway associated with these processes will be described in the following sections.n = 5 for each group): (1) normal group (N), with mice only suffering a midline incision to expose the liver; (2) mild hypothermia pretreatment group (MH), with mice only receiving pretreatment with mild hypothermia; (3) IR group (IR), with mice exposed to in situ ischemia for 1 h and reperfusion for 6 h; (4) mild hypothermia pretreatment+IR group (MHP), with mice receiving pretreatment with mild hypothermia for 2 h and then exposure to IR; (5) etomoxir+IR group (EIR), with mice receiving pretreatment with etomoxir for 1 h and then exposure to IR; and (6) leptin+IR group (LIR), with mice receiving pretreatment with leptin for 1 h and then exposure to IR. The animal experiments were similar to those described previously [The experiments were performed with 6- to 8-week-old C57BL/6J male mice purchased from Wuhan Wan Qian Jia Xing Bio-Technology Co., Ltd. . Mice were housed in the Animal Center of Zhongnan Hospital of Wuhan University and kept in a temperature-controlled environment (20-25°C) with a 12/12 h light/dark cycle with free access to food and water. The study was approved by the Ethical Committee of Wuhan University. All animal experiments were carried out in accordance with the Experimental Animal Management Ordinance  and the Guide for the Care and Use of Laboratory Animals , Bethesda, MD, USA). Animals fasted overnight while with access to water before the experiments. Mice were randomly divided into six groups  and aspartate aminotransferase (AST) by automatic analysis in the Zhongnan Hospital of Wuhan University.Ischemic lobes were harvested and fixed in 4% formalin. Samples were embedded in paraffin as previously described . All parApoptosis was assayed by Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining following the manufacturer's instructions. The total hepatocytes and TUNEL-positive cells were detected in three randomly chosen views (100x) for each liver section using a fluorescence microscope. The rate of apoptosis  in each view was calculated with Image-Pro Plus 6.0 .V : V = 1 : 1) for 1 h and (V : V = 3 : 1) for 3 h, then treated with embedding agent overnight, and heated at 70°C overnight. Finally, samples were sliced to 70–90 nm sections in a Reichert ultrathin slicer and then stained with lead citrate solution and uranyl acetate 50% ethanol-saturated solution for 15 min for observation.Fresh ischemic livers were fixed in 2.5% glutaraldehyde and washed with PBS for 15 min three times. Samples were fixed in 1% osmic acid for 1–2 h followed by washing with PBS for 15 min three times again. Next, samples were dehydrated with a graded series of ethanol solutions  for 15 min at each concentration and then treated with ethanol and acetone for 20 min each. Next, samples were treated with a mixture of embedding agent and acetone  and GAPDH . The protein expression was quantified by densitometric analysis using the Image J software.Western blot was performed using whole lysates extracted from livers as previously described ; phospha+/NADH Assay Kit , acetyl coenzyme A (Acetyl CoA) , and malonyl coenzyme A  ; detections were performed following the manufacturers' instructions. The results were measured as ng/ml, μmol/gprot, μg/g, and μg/g.For the evaluation of mitochondrial function and FAO, frozen liver tissue was homogenized with specified buffer that was included in the ELISA kits specific for CPT1 , adenosine triphosphate (ATP) , ADP/ATP Ratio Assay Kit , NADFor the evaluation of oxidative stress, frozen liver tissue was homogenized with specified buffer that was included in the colorimetric assay kits specific for superoxide dismutase (SOD), malondialdehyde (MDA), reactive oxygen species (ROS) , and 4-hydroxynonenal (4-HNE) , which were detected following the manufacturers' instructions. The results were measured as U/mgprot, nmol/mgprot, FI/mg, and ng/g.P < 0.05 was considered statistically significant.Data were analyzed using SPSS 19.0 statistical software for Windows . All results are presented as mean ± SD. Differences between experimental groups were analyzed by one-way analysis of variance (ANOVA); P < 0.05), as ALT and AST levels decreased to 5071.2 ± 776 U/L and 5735.2 ± 1886.3 U/L. Meanwhile, hepatic histopathology changes were assessed. As shown in Figures P < 0.05). Similarly, as shown in Figures P < 0.05). In addition, the hepatic functional and morphological changes and cell apoptosis did not exhibit a significant difference between the normal group and the mild hypothermia pretreatment group (P > 0.05). These results further confirm our previous research findings that mild hypothermia could attenuate IR injury.We monitored the rectal temperature throughout the experiment to ensure the stability of the experimental model; data is shown in +/NADH in liver tissue were measured to evaluate the mitochondrial functional changes. As shown in +/NADH were significantly decreased, and ADP/ATP Ratio was significantly increased after IR injury, while mild hypothermia could improve these changes in the liver. These results were consistent with the mitochondrial morphological changes (P < 0.05). Moreover, mitochondrial injury induced by IR resulted in increased ROS, 4-HNE, and MDA levels and decreased SOD levels, which are biomarkers of lipid peroxidation and oxidative stress. Pretreatment with mild hypothermia could also significantly prevent these changes and thus ameliorates lipid peroxidation and reduces oxidative stress injury (P < 0.05) . These results show that mild hypothermia could reduce IR-induced mitochondrial injury, lipid peroxidation, and oxidative stress.Based on the aforementioned results and our preliminary studies, we performed a proteomic analysis and found that metabolism, especially lipid metabolism, was markedly changed in these animal models see ; this suβ-oxidation cycle, was downregulated nearly twofold as well. Other proteases that participate in FAO were also obviously downregulated. The expression levels of these key enzymes could, however, be preserved by mild hypothermia (P < 0.05). Moreover, the production of ATP was also significantly decreased upon IR injury, whereas these changes could be reversed by pretreatment with mild hypothermia (P < 0.05). However, the production of Acetyl CoA and Malonyl CoA and the expression of HMGCS1, the rate-limiting enzyme of ketogenesis, did not exhibit significant differences between the normal group and the IR group (P > 0.05); they were, however, significantly different from the mild hypothermia pretreatment groups, with or without IR (P < 0.05). These results further highlight the notable effects of mild hypothermia on FAO. Thus, we conclude that mild hypothermia alleviates hepatic IR injury, primarily by regulating FAO.To test whether regulation of FAO was involved in mild hypothermia which attenuates hepatic IR injury, we evaluated the levels of proteases that are involved in the process of FAO and the associated products of FAO in liver tissue, such as ATP, Acetyl CoA, and Malonyl CoA. As shown in Figures P > 0.05). However, the application of leptin could significantly reduce the damage; levels of ALT and AST were reduced to 4065.6 ± 759.7 U/L and 4632.2 ± 1500 U/L, respectively, the score of hepatic pathological injury decreased to 3.8 ± 0.8, and the hepatocyte apoptosis rate was lowered to 13.06% ± 1.92% (P < 0.05). In addition, inhibition of FAO had negative effects but activation of FAO had positive effects on mitochondrial injury and oxidative stress, i.e., less-damaged mitochondrial morphology, decreased ROS, 4-HNE, MDA levels, and ADP/ATP Ratio, and increased SOD and ATP levels, and NAD+/NADH were observed after pretreatment with leptin (P < 0.05); these were consistent with the observed hepatic functional and morphological changes (see P < 0.05), while inhibition of FAO had the opposite effects , which is the key enzyme of glycolysis, and Citrate Synthase (CS) and Isocitrate Dehydrogenase 2 (IDH2), which are the key enzymes of TCA cycle, were downregulated after IR injury, while these changes could be preserved by mild hypothermia pretreatment and leptin. However, these changes were not as significant as those in FAO (see P < 0.05). Moreover, the apoptosis-related proteins BCL2 and BAX showed significant changes; that is, the ratio of BCL2/BAX increased after activation of FAO and pretreatment with mild hypothermia, while it decreased after inhibition of FAO (P < 0.05). These results strongly suggest that activation of the JAK2/STAT3 pathway is closely associated with the protective effects of mild hypothermia against hepatic IR injury. These suggest that the JAK2/STAT3 pathway is of great importance in the protective effects of mild hypothermia against IR injury.To further illustrate the predominant signaling pathway involved in these processes, we measured the activation of the JAK2/STAT3 pathway, which is upstream of CPT1 and downstream of leptin. As shown in Hepatic IR injury is a complex pathological process that can result in serious liver injury and severely threatens the short- and long-term prognosis of the patients who undergo liver surgery , 32, 33.In order to elucidate the underlying mechanisms, we performed a proteomic analysis and found that metabolism exhibited significant changes upon IR injury and mild hypothermia pretreatment, of which, lipid metabolism, especially fatty acid oxidation, was of great significance. As is well known, the liver is one of the most important organs for FAO, and mitochondria are the main site where metabolism occurs. Therefore, we evaluated the mitochondrial morphological and functional changes and the hepatic FAO activity; results showed the IR-induced mitochondrial injury and FAO inhibition were largely reversed by mild hypothermia, thus demonstrating for the first time that FAO is one of the potential mechanisms by which mild hypothermia protects against IR injury. These results are consistent with previous studies, which concluded that metabolic disturbances during ischemia are a necessary precursor for reperfusion-induced inflammation and could also serve as a molecular target for therapeutic intervention .β-oxidation cycle [β-oxidation cycle is a process in which acyl-CoA is shortened, whereby the two carboxy-terminal carbon atoms are released as acetyl-CoA units for each cycle. In short, directly after import, acyl-CoA is metabolized first by an acyl-CoA dehydrogenase, such as very long-chain acyl-CoA dehydrogenase (ACADVL), and then by mitochondrial trifunctional protein (HADHA), which exerts hydratase, long-chain hydroxyacyl-CoA dehydrogenase, and thiolase activity [β-oxidation produces large amounts of acetyl-CoA, flavin adenine dinucleotide (FADH2), and nicotinamide adenine dinucleotide (NADH). Of the processes, acetyl-CoA can enter the citric acid cycle to produce ATP, it can be converted into ketones upon the catalysis of hydroxymethylglutaryl-CoA synthase (HMGCS1), and it can also be converted into malonyl-CoA. Moreover, FADH2 and NADH can be used to produce ATP [FAO plays a pivotal role in energy homoeostasis, and the process is extremely complex. Briefly, after translocation across the plasma membrane, fatty acids are rapidly converted into acyl-CoA by acyl-CoA synthetases such as long-chain-fatty-acid-CoA ligase 1 (ACSL1) , 35, 36.on cycle . The β-oactivity . The produce ATP . The impduce ATP , 39. In in vivo pharmacological interventions of FAO were performed. Etomoxir, a small molecule developed for metabolic and cardiovascular disease that exhibits nanomolar potency toward CPT1 upon enzymatic conversion to the active inhibitor etomoxiryl-CoA, was used to block mitochondrial FAO [To further confirm the above results, rial FAO . At the rial FAO –43. The α pathway [Based on the above results, we investigated the main signaling pathway involved in these processes. Several pathways have been demonstrated to be associated with FAO, including the PPAR pathway , 44, the pathway , and the pathway . These p pathway . Phospho pathway , 48. As in vivo hepatic ischemia for 1 h and reperfusion for 6 h, the time is relatively short, while the time of the models in the studies opposite to us are relative long, which often is more than 24 h. In addition, there are also many studies consistent with our results [in vivo intervention experiments, and we did not verify our findings by in vitro experiments or clinical trials.Importantly, regarding the protective effects of leptin on hepatic IR injury, Lin et al. first found that endogenous leptin fluctuates in hepatic IR injury  and Carb results –56; the The major novel conclusions of the present study were the following. (1) Mild hypothermia effectively attenuates IR injury. (2) One of the mechanisms by which mild hypothermia exerts protective effects against IR injury is the preservation of mitochondrial FAO. (3) Pharmacological interventions of FAO have significant effects on IR injury; that is, activation of FAO can significantly attenuate IR injury, while inhibition of FAO has negative effects. (4) The JAK2/STAT3-CPT1a signaling pathway may play a vital role in these processes. A schematic representation of the underlying mechanisms is presented in