TSLP, BIRC5, S100B, MDK, and S100P—and three protect genes RARRES3, BLNK, and ACO1. Kaplan‐Meier survival curve showed that patients classified as low‐risk according to optimum cut‐off risk score had better prognosis than those identified within the high‐risk group. ROC analysis indicated that the identified prognostic signature had excellent diagnostic efficiency for predicting 3‐ and 5‐years relapse‐free survival (RFS). Multivariate Cox regression analysis proved that the prognostic signature is independent of other clinical parameters. Stratification analysis demonstrated that the prognostic signature can be used to predict the RFS of BC patients within the same clinical subgroup. We also developed a nomogram to predict the RFS of patients. The calibration plots exhibited outstanding performance. The validation sets  were used to external validation. More convincingly, the real time RT‐PCR results of clinical samples demonstrated that danger genes were significantly upregulated in BC samples, whereas protect genes were downregulated. In conclusion, we developed and validated an immune‐related prognostic signature, which exhibited excellent diagnostic efficiency in predicting the recurrence of BC, and will help to make personalized treatment decisions for patients at different risk score.Breast cancer (BC) is the most common cancer among women worldwide and is the second leading cause of cancer‐related deaths in women. Increasing evidence has validated the vital role of the immune system in BC development and recurrence. In this study, we identified an immune‐related prognostic signature of BRCA that could help delineate risk scores of poor outcome for each patient. This prognostic signature comprised information on five danger genes— This study developed and validated an immune‐related prognostic signature, which exhibited excellent diagnostic efficiency in predicting the recurrence of BC, and will help to make personalized treatment decisions for patients at different risk score. The 2018 GLOBOCAN report revealed that approximately 2.1 million women worldwide were diagnosed with BC in 2018, accounting for one‐fourth of all cancer cases among women.Increasing evidence has shown that the immune system plays a critical role in BC recurrence.In recent years, "omics" technology has developed rapidly. Microarray analysis and whole‐genome sequencing have granted the possibility to explore the genomic characteristics of high‐risk cancers.22.1GSE42568 dataset was used as the training set, while GSE21653, GSE20711, and GSE88770 were used for validation. GPL570 [HG‐U133_Plus_2] Affymetrix Human Genome U133 Plus 2.0 Array was used for gene annotation. We downloaded the original expression profile, and used the robust multi‐array average (RMA) algorithm to perform background correction and quantile normalization. Briefly, for multiple probes corresponding to one gene symbol an average value was considered; single probes corresponding to multiple gene symbols were deleted. After removing incomplete prognostic cases, a total of 121 cases were included in the training set  and 435 cases were included in the validation set . Clinical information, such as T‐stage, ER status, lymph nodes, grade, and recurrence‐free survival were extracted from the dataset for further analysis.IRGs expression data and clinical information were obtained from the GEO database. 2.2t test method to evaluate the P‐value of each gene to determine whether the gene is differentially expressed in tumor and adjacent samples. The Benjamini‐Hochberg method was used to calculate the False Discovery Rate (FDR) to prevent false positive probability in multiple comparisons. Fold change (FC) was used to represent the ratio of gene expression levels between tumor and adjacent samples. Considering FDR <0.05 and | logFC |> 1 as inclusion criteria, the “limma” R package was used to screen differentially expressed genes (DEGs) in the training set. Download all immune genes through the ImmPort website online, and select the immune genes from DEGs(https://www.immport.org/shared/genelists). Then download tumor‐related transcription factors from the Cistrome Cancer website and screen out differentially expressed transcription factors (http://cistrome.org/Cistrome Cancer/).We used the 2.3P‐value < .05 was used as the inclusion criteria to identify candidate IRGs. Hazard ratio (HR) was used to identify IRGs into protect or danger, with HR <1 being considered as protect genes and HR >1 as danger genes. Lasso penalized COX analysis and stepwise multivariate Cox analysis were used to further narrow down the list of significant IRGs. Finally, we developed an immune‐related prognostic signatures (8 genes) based on the following risk score formula:i is the univariate Cox regression coefficient of gene i. The patients were classified as high‐ or low‐risk according to the optimum cut‐off risk score. We used GSE21653, GSE20711, and GSE88770 to validate the accuracy of the identified immune‐related prognostic signature.Univariate Cox proportional hazard regression model was used to screen IRGs associated with RFS in the training set. 2.4GSE42568, GSE21653, and GSE20711 datasets. GSE88770 was not included in further analysis due to lack of clinical information (T_stage). Calibration plots were used to evaluate the diagnostic efficiency of nomogram.In this study, we used "rms" R package to construct a nomogram including clinical information  and immune‐related signature in 2.5BRCA and paired adjacent tissue samples were taken from patients undergoing breast surgery at Zhongnan Hospital of Wuhan University. All specimens were collected after obtaining informed consent by the patients. The samples were immediately stored in liquid nitrogen for further experiments. The Ethics Committee of Zhongnan Hospital  approved the use of these samples for total RNA isolation and quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR) analysis. This study included 40 pairs of samples.2.6−ΔΔCt values were normalized to its levels. The primer sequences for qPCR used in this study are shown in Supplementary Table RNeasy plus mini kits  and 2 × SYBR Master Mix  were used to extract total RNA according to the protocol provided by the manufacturer. qRT‐PCR was conducted in triplicate. GAPDH was used as internal control, and the 22.7The GSEA software (GSEA version 4.0.3) was used to develpoed a gene set enrichment analysis (GSEA) in training and validation sets. The samples were divided into high‐risk and low‐risk groups based on the cut‐off risk score. The c2.cp.kegg.v6.2.symbols.gmt gene set was chosen as the reference gene set. FDR is the adjusted P‐value after multiple hypothesis testing, FDR <25% (Benjamini‐Hochberg) was used as cutoff for significant gene sets. The most significant Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were screened.2.8P‐value < .05 (log‐rank tests) was considered statistically significant. Multivariate Cox regression analysis and stratification analysis were used to determine whether the immune‐related signature was an independent prognostic factor. The "survivalROC" R package was used for time‐dependent receiver operating characteristic (ROC) analysis, and the prognostic performance was verified by comparing the area under the ROC curve (AUC). All statistical tests were performed using R software (version 3.6.1).In this study, we used Kaplan‐Meier plots to evaluate the differences in patients with low‐risk and high‐risk group, and used log‐rank tests to assess the statistical significance. 33.1GSE42568 as training set, which included 17 normal samples and 104 tumor samples. Using FDR <0.05 (Benjamini‐Hochberg) andlogFC>1 inclusion criteria, we identified 1561 DEGs, among which 134 were immune‐related genes and 33 were genes coding for TFs. Volcano plot and heatmap of the training set were shown in Supplementary Figure We used GEO BC dataset 3.2TSLP, BIRC5, S100B, MDK, and S100P—and three protective genes—RARRES3, BLNK, and ACO1. This short list of eight genes was used to develop an immune‐related gene prognostic signature. Figure We first performed a univariate Cox regression analysis to identify RFS‐related prognostic genes in training set. According to the optimal cut‐off risk score, patients were divided into two groups: high‐risk and low‐risk. We identified 19 immune‐related prognostic genes, which were further evaluated by lasso‐penalized Cox regression and stepwise multivariate analysis . Kaplan‐Meier curves confirmed that low‐risk group had better RFS than high‐risk group. Moreover the AUC values were 0.536, 0.606, 0.596, 0.633, and 0.633 in GSE21653 dataset; 0.585, 0.593, 0.639, 0.676, and 0.690 in GSE20711 dataset; 0.728, 0.758, 0.791, 0.798, and 0.700 in GSE88770 dataset, for 1‐, 2‐, 3‐, 4‐, and 5‐year RFS, respectively  The calibration curves indicated that the nomogram had excellent performance  andlogFC>1 were used as the inclusion criteria to screen for differentially expressed genes, a total of 134 IRGs and 33 TFs were identified. Univariate Cox regression analysis was used to determine the association between differentially expressed IRG levels and RFS in training set. Lasso‐penalized Cox regression and stepwise multivariate Cox analysis were performed next to narrow the list of IRGs. Finally, an immune‐related gene prognostic signature was developed (8 genes). We also constructed an interaction network of IRGs and TFs. The Kaplan‐Meier survival curve revealed that low‐risk patients had better prognosis than those identified within the high‐risk group. Moreover ROC and multiple Cox regression analysis indicated that the immune‐related prognosis signature has good diagnostic efficiency to identify relapse‐free events, representing an independent risk factor for BC. Stratification analysis further demonstrated that the prognostic signature can be used to predict the RFS of BC patients within the same clinical subgroup. We also developed a nomogram that integrates clinical features and the IRGs signature, which calibration plots indicated that had excellent performance, to support the clinical assessment of BC patients. To further validate the prognostic signature, we conducted additional qRT‐PCR analysis, which showed that the protect genes were upregulated in adjacent tissues, while the risk genes were up‐regulated in the BC samples. In summary, this study developed and validated an immune‐related prognostic signature that may provide guidance for the future diagnosis of BC recurrence.The workflow of immune‐related gene expression and clinical information preprocessing, gene signature generation, and verification are displayed in Figure The biological functions of the identified immune‐related prognostic genes have been previously reported. Thymic stromal lymphopoietin (TSLP) is a cytokine associated with type 2 immunity and is also associated with the progression of various cancers, including BC, pancreatic cancer, gastric cancer, cervical cancer, and myeloma.Several previous studies have reported prognostic signatures for BC.The authors claim no conflict of interest.ZT and JT wrote this article. GW revised this article. XL, QY, and YW conducted all statistical analyses.Fig S1Click here for additional data file.Fig S2Click here for additional data file.Table S1Click here for additional data file.


Dear editor,et al., et al., Making precise changes in the genomes of organisms is challenging for most genome editing tools. Recently, a search‐and‐replace method, also known as prime editing, was developed that can introduce user‐defined sequence into a target site without requiring double‐stranded breaks (DSBs) or repair templates   gene as a target. A pegRNA containing 13 nt PBS and 16 nt RT template and a nick sgRNA 84 nt downstream of the site of the pegRNA‐induced nick were designed for ALS to introduce an S627N mutation, which makes rice plants resistant to imidazolinone herbicides (Figure To test whether the prime editor Sp‐PE3 can edit rice endogenous genes, we first chose the acetolactate synthase (s Figure . We founs Figure ,l. Amongs Figure . These rs Figure .et al., The SaCas9 is a multiple‐turnover enzyme that releases the cleaved DNA products faster than SpCas9 does (Yourik et al., et al., et al., et al., et al., et al., et al., et al., In summary, the prime editing tool could precisely edit the transgenic reporter and endogenous gene in rice. During the preparation and review of our manuscript, several other prime editing systems in rice and wheat were reported (Li The authors declare no conflict of interests.K.H., W.J., X.T. performed the experiments and analysed the data. K.H. wrote the manuscript. J.‐K. Z. supervised the project and edited the manuscript.


Inflorescence explants of date palm proved to be a promising tool for micropropagation of elite cultivars or rare males and females as organogenesis and somatic embryogenesis could be achieved. These plant materials are abundantly available every year and can be used as cheap and potent explants. Nevertheless, many difficulties could be faced in this protocol according to selection of the spathe size and age, media components, growth regulators, etc. The aim of this study was to determine the influence of various cytokinins on direct organs induction of three date palm cultivars  from immature inflorescence. An additional objective of this study was to investigate the effect of cytokinins and auxins on growth and development of Medjool cultivar.N-phenyl-N′-1,2,3-thidiazol-5-yl urea) (TDZ). TDZ alone or in combination with BA proved to be superior for direct organogenesis in all three cultivars so that another combination of TDZ with BA was conducted. Results showed that moderate concentration of BA, with TDZ, gave superior response. Medjool cultivar response surpassed other two cultivars that made the possibility to conduct some growth regulators treatments on its multiplication and regeneration. TDZ at 0.5 + BA at 1.0 mg/l without activated charcoal seemed to enhance multiplication rate. Medium containing 0.5 mg/l of both naphthaleneacetic acid and indole butyric acid in addition to 1.0 mg/l indole acetic acid appeared to be more suitable for rooting stage of Medjool shootlets.Various combinations of cytokinins were investigated on three date palm inflorescences as N6-(2-isopentenyl) adenine (2iP), kinetin, benzyleadenine (BA), and thidiazuron (In this study, we created an innovation sequence of growth regulators included in nutrient media for date palm direct organogenesis from inflorescence. Organogenesis has been accelerated from immature inflorescence explants and developed to healthy plantlets which acclimatized in greenhouse. Phoenix dactylifera L.) is a dioecious, perennial monocot plant species that belongs to family Arecaceae. It is one of the oldest fruit crops spreadly cultivated in North Africa and Middle East countries and one of the most economically important plants in arid and hot regions. Offshoots are traditionally used for vegetative propagation of date palms. However, tissue culture is the most used technology method to provide large-scale propagation of healthy true-to-type plants. For decades, shoot tip explants have been used for various micropropagation protocols of date palm on the research and commercial levels together [Date palm  has recently emerged as a highly efficient bioregulator of plant morphogenesis in the tissue culture technique. Application of TDZ induces diverse responses ranging from induction of callus to embryogenesis or organogenesis. TDZ is believed to act as an auxin in some cases and as a cytokinin in others. Nevertheless, it has a different structure from either auxins or purine-based cytokinins. TDZ can influence some physiological and biochemical reactions in plant cells. Various reports indicated that TDZ may act through modulation of the endogenous plant growth regulators, modification in cell membranes, energy levels, nutrient uptake, or nutrient assimilation [In fact, the cytokinin type and concentration are very important factors that should be studied for organogenesis technique. Thidiazuron and benzyladenine are the most preferably used cytokinins in the regeneration systems in some fruit trees and woody plants . Thidiazmilation .The aim of this current study was to determine the role of various cytokinins especially thidiazuron on induction of direct shoot organs of three date palm cultivars  from immature inflorescence. An additional objective of this study was to investigate the effect of cytokinins and auxins on growth and regeneration of Medjool cultivar.The present study was carried out through 2018–2020 at the Tissue Culture Lab. of New Valley Regional Centre, Academy of Scientific Research and Technology, Egypt.Phoenix dactylifera L.) cvs. Medjool, Barhee, and Selmi were removed from adult female trees grown at a respected farm in Cairo-Alexandria road, Egypt. Immature inflorescence spathes were collected in late February 2018. The average spathe length was 10–15 cm. Spathes were rinsed under running tap water and liquid soup for half an hour. Surface sterilization in laminar air flow is performed by using 20% of commercial Clorox (5.25% sodium hypochlorite) for 5 min and then rinsed with sterilized distilled water three times. After that, the spathes’ protective sheath was removed, and the spikelets were isolated and sterilized by immersion in 0.1% mercuric chloride (HgCl2) solution for 5 min  to the highest (5) of the three cultivars. In addition, percentage of response  and average number of organs appeared were recorded.Due to higher percentage of the vegetative buds formation of Medjool cv., the following stages were done with this cultivar.At this stage, clusters of vegetative buds (2–3 buds) were cultured on the following culture media which are dispensed on small jars (200 ml) at a rate of 35 ml/ jar to multiply and form new shoots. The culture media contained ¾ MS, 100 mg/l glutamine, 100 mg/l arginine, 5.0 mg/l thiamine HCL, 1.0 mg/l biotin, 80 mg/l adenine sulfate, 0.25 mg/l NAA, and 40 g/l sucrose solidified with 6.0 g/l agar. Cytokinins were added as follows: TDZ at 0.5 mg/l alone and combined with 0.5 mg/l BA, 1.0 mg/l BA, and 2.0 mg/l BA. Previous culture media were used with or without addition of 0.5 g/l activated charcoal. Cultured jars of Medjool cv. were maintained at 27 ± 2 °C and light intensity of 1500 lux, for three subcultures . After that, the number of new shootlets (buds) was calculated.All shootlets from the previous treatments were transferred to ¾ MS + 0.5 mg/l kinetin + 1.0 mg/l indole acetic acid (IAA) for two subcultures . All cultures were incubated at 27 ± 2 °C and light intensity of 2000 lux in order to transfer to rooting media , or 0.5 mg/l NAA + 0.5 mg/l IBA. Culture media were dispensed into test tubes (2.5 in diameter and 25 cm in length) at a rate of 25 ml and capped with polyethylene closures. After 6 weeks, rooted plantlets were transferred to liquid culture media of the same components except sucrose which was reduced to 15 g/l and maintained 4 weeks as a pre-acclimatization treatment. Both solid and liquid culture media were incubated at light intensity of 4000 lux. Root number, root length, plantlet length, hairy roots, and root thickness degrees  were reHealthy rooted plantlets were transferred to greenhouse. Acclimatization process was started by removing the plantlets from test tubes, rinsing them with tap water, immersing them in fungicide solution 0.2% (w/v) for 10 min and then transplanting them in plastic pots (5 cm in diameter and 18 cm in length) filled with moistened mixture of peat moss: perlite 2:1. The plantlets were covered with polyethylene bags for 6 weeks to raise the relative humidity around the plantlets. Bags were removed gradually, and plantlets were irrigated with 1/10 strength of MS inorganic medium.Each treatment of different experiments contained three replicates, and each replicate contained five jars and every jar cultured with five explants. Before analysis, percentage data were normalized by arcsine-transformation. Data obtained were subjected to the analysis of variances of randomized complete design as recommended by Snedecor and Cochran .Immature inflorescence explants of three cultivars of date palm  were cultured in this investigation into media containing MS salts supplemented with 0.5 mg/l NAA, 1.0 mg/l NOA, 40 mg/l adenine sulfate, and 40 g/l sucrose. Various cytokinins were added to see their effect on direct organogenesis of the three cultivars.Data from Table Furthermore, data in Table Data in Table Data in Table According to data in Table Data in Table The same trend could be observed in Table Previous resulted organ cultures of Medjool cultivar were cultured on media containing 3/4 MS salts, 80 mg/l adenine sulfate, 0.25 mg/l NAA, 0.5 mg/l TDZ and various concentrations of BA to study the multiplication rate. Data in Table Data in Table Direct organogenesis presented in the current study was proliferated from immature inflorescence explants of three date palm cultivars . Immature inflorescence explants are a promising alternative source of explants as they provide a quick and safe method for micropropagation of date palms . Direct −1 TDZ combined with auxin. Moreover, benzyladenine incorporation in TDZ-medium enhanced explant response percentage and organ numbers. It was reported that the maximum response on medium supplemented with 1.0 mg/l BA and 0.5 mg/l TDZ was observed, producing an average of 18.2 buds per culture after 24 weeks from date palm culture [Urginea altissima [Passiflora miniata [Aloe vera [Arnebia euchroma [TDZ proved to be crucial for organogenesis of the three cultivars under investigation, and this result is in harmony with Hassan et al.  as they  culture . TDZ at  miniata  and at 1loe vera  and Arneeuchroma .Many researchers tried to understand the role of action of TDZ in plant. For instance, Dey et al.  perceiveResults show that incorporating BA with TDZ and other growth regulators in the media encourage proliferation as new shoots were accelerated in multiplication stage. The increase in shoot proliferation might be due to the physiological role of BA which is considered to be the most widely used cytokinin in the micropropagation industry due to its effectiveness and affordability . It acceIt could be observed from our results that the presence of NAA and IBA with IAA  in rooting media achieved the highest significant root number, root thickness, and hairy root formation on Medjool shootlets compared with IAA alone. The presence of NAA, in the rooting medium, increased root number of date palm somatic embryos. Either NAA or IBA at 0.4, 0.6 or 0.8 mg/l with 60 g/l sucrose gave the best root thickness. It was reported that the effect of NAA is on the main root length while IBA on the lateral root length . Al-MayaIn this study, we created innovation sequences of growth regulators included in nutrient medium for date palm direct organogenesis from inflorescences. Organogenesis has been accelerated from immature inflorescence explants of three cultivars. TDZ and BA are crucial cytokinins to be included into induction and multiplication media of date palm. Medjool proved to be a promising cultivar for micropropagation and biotechnology field, and its shoots were developed to healthy plantlets which acclimatized in greenhouse.


Lung cancer serum tumor markers including carcinoembryonic antigen (CEA), cytokeratin 19 fragment (CYFRA21‐1), and carbohydrate antigen 125(CA125) as prognostic predictors is controversial. Therefore, this study aimed to evaluate the association between these markers and the survival of patients with postoperative stage III‐N2 non‐small cell lung cancer (NSCLC).We enrolled 1011 patients with pathologically confirmed stage III‐N2 NSCLC who underwent resection and whose pretreatment serum tumor marker levels were available. Patients were categorized according to their serum levels into low‐, medium‐, and high‐risk groups. Overall survival (OS), progression‐free survival (PFS), local regional relapse‐free survival (LRFS), and distant metastasis‐free survival (DMFS) were calculated from the date of resection. Their association with each serum tumor marker was assessed using the log‐rank test.P = 0.000). In propensity score matching analysis, the model also achieved prognostic significance for all four survival classifications (P = 0.001–0.004) among the three risk groups.Abnormal CEA levels were associated with worse five‐year OS, PFS and DMFS; abnormal CYFRA21‐1 levels were associated with worse five‐year OS and LRFS; and abnormal CA125 levels were associated with worse five‐year OS, PFS, LRFS and DMFS. Among the risk groups, there were significant differences in five‐year OS, PFS, LRFS and DMFS (The combined model achieved prognostic significance for all survival outcome types. The serum tumor markers tested are useful prognostic predictors for postoperative NSCLC patients but not for all survival outcomes. The combination of the three indices is more reliable in predicting all four of the survival outcomes.Serum CEA, CYFRA21‐1, and CA125 levels can be used as prognostic factors of postoperative N2 non‐small cell lung cancer patients but not for all survival outcomes, suggesting that combinative detection of all three indices would be more reliable.Our model utilizes available technology, with conventional cutoff values, inexpensive costs, and simple mathematics methods and, thus, can be feasibly employed by clinicians or oncologists. From a large number of patients' analysis, we achieved a neat model to be able to predict the OS, PFS, LRFS and DMFS of postoperative III‐N2 NSCLC. Non‐small cell lung cancer (NSCLC) accounts for 85% of lung cancers.Studies have used circulating tumor cells and cell‐free tumor DNA and RNA as a basis for predicting patient survival and recurrence, but their utilization is limited owing to the testing complexity and high costs.Serum tumor markers for lung cancer including carcinoembryonic antigen (CEA), cytokeratin 19 fragment (CYFRA 21‐1), carbohydrate antigen 125 (CA 125), squamous cell carcinoma antigen (SCC), neuron specific enolase (NSE), and progastrin releasing peptide (ProGRP) are widely used for diagnosis, but their role as prognostic predictors is still debated. Among them, SCC, NSE, and ProGRP are strongly associated with certain histological types while the other three are not.This study conformed to the provisions of the Declaration of Helsinki, and it was approved by the ethics committee of the National Cancer Center, China, in 2016 . Informed patient consent was obtained before treatments.Patient eligibility criteria were as follows: patients ≥ 18 years with pathologically confirmed operable stage III‐N2 NSCLC (according to the American Joint Committee of Cancer seventh staging system), underwent R0 resection in our institution, available serum CEA, CYFRA 21‐1, and CA 125 pretreatment data, full medical documents, and follow‐up records. The exclusion criteria were as follows: pregnancy, breastfeeding, other malignancies except nonmelanoma skin cancer or cervical carcinoma in situ, clinical evidence of infection, and autoimmune disease. Data on demographics, pathological subtypes, postoperative treatments, and survival were collected.Serum CEA, CYFRA 21‐1, and CA 125 were tested using immunoelectrochemistry. The records were taken from the database. The normal ranges of CEA, CYFRA 21‐1, and CA 125 were 0.0–5.0 ng/mL, 0.0–3.3 ng/mL, and 0.0–35.0 U/mL, respectively.All patients underwent lobectomy or pneumonectomy. Mediastinal lymphadenectomy or systematic mediastinal lymph node sampling were also performed. Postoperative chemoradiotherapy was administered according to published recommendations following National Comprehensive Cancer Network guidelines.Overall survival (OS), progression‐free survival (PFS), local regional relapse‐free‐survival (LRFS), and distant metastasis‐free survival (DMFS) were observed as outcomes. Survival values were calculated from the time of resection. OS was calculated as the time to death. PFS was calculated as the time to documented clinical progression or death. LRFS was calculated as the time to local progression, defined as primary tumor or regional lymph node recurrence. DMFS was calculated as the time to distant metastasis.We built a prognosis model using the three serum tumor markers. Each abnormal marker was given one point. According to the scores, patients were divided into three groups: low‐risk (0 points), medium‐risk (1 point), and high‐risk (2–3 points).t‐test. If the chi‐squared test or t‐test showed differences in patients' clinical parameters or treatments, propensity score matching (PSM) was used (ratio 1:1:1) to decrease the bias. A P‐value of <0.05 was considered statistically significant. All statistical analyses were conducted using the SPSS statistical software package version 24.0 .Survival values were calculated using the Kaplan‐Meier method. The cutoff value of each serum marker was the upper limit of its normal value . Univariate analysis was performed using the log‐rank test to evaluate the association between each single marker and the survival outcomes. Multivariate analysis was performed using the Cox regression model; variables included sex, age, pathology, Karnofsky Performance Status (KPS), T stage, CEA, CYFRA 21‐1, CA 125, and postoperative treatments. Differences of characteristics and treatments among the three risk groups were evaluated using the chi‐squared test. Continuous variables are described as means; they were compared using Student's From March 2003 to September 2015, 1011 patients fulfilled the enrollment criteria, of which 653 were male. The median age was 57 (range: 25–80) years. Overall, 666 patients were diagnosed with adenocarcinoma, and 275 patients were diagnosed with squamous cell carcinoma. The median follow‐up time was 29.21 (range: 0.56–154.18) months. The details are presented in Table The low‐risk group consisted of 301 patients who received 0 points; 428 medium‐risk patients received one point; and of the 282 high‐risk patients, 211 received two points and 71 received three points. The low‐risk group had younger patients, more female patients, earlier T stages, more nonsquamous carcinomas, and a higher proportion of postoperative chemotherapy and radiotherapy compared with those in the medium‐ and high‐risk groups. After PSM, 226 patients were assigned to each group; age, sex, KPS scores, T stages, postoperative chemotherapy proportions, and postoperative radiotherapy portions were balanced among the three groups. The details are presented in Table P = 0.025), PFS , and DMFS  than patients with abnormal CEA levels. The five‐year LRFS was higher in the normal CEA group but without significant difference   Fig a–d.P = 0.001) and LRFS  than patients with abnormal levels. Five‐year PFS  and DMFS  were similar in patients with normal and abnormal CYFRA 21‐1 levels , PFS , LRFS , and DMFS (32.3% vs. 21.0% P = 0.000)  Fig a–d than P = 0.003) but not with five‐year OS (P = 0.171), PFS (P = 0.052), or LRFS (P = 0.408). CYFRA 21‐1 was associated with five‐year OS (P = 0.025) and LRFS (P = 0.023) but not with five‐year PFS (P = 0.165) or DMFS (P = 0.214). CA 125 was associated with all survival classifications .In multivariate analysis, CEA was associated with five‐year DMFS (P = 0.000). The corresponding results for PFS were 32.3%, 25.0%, and 18.0%, respectively (P = 0.000). This model also showed significant association with five‐year LRFS  and five‐year DMFS . The survival curves are plotted in Fig Among the 1011 patients, the five‐year OS for patients in the low‐, medium‐, and high‐risk groups were 63.9%, 48.0%, and 38.9%, respectively , the model achieved prognostic significance in all four survival classifications. Respectively, five‐year OS: 65.5% vs. 49.8% vs. 41.4%, Serum tumor markers are routinely examined for diagnosis and follow‐up. Although there are newer markers, classic serum tumor markers are very reliable, easily determined, economical, and highly reproducible. Relationships between elevated tumor marker serum levels and prognoses have been proposed, but these reports enrolled patients at different stages and with heterogeneous therapeutic regimens. Few studies have supplied information about locally advanced stages after surgery.CEA has been the most extensively studied tumor marker in lung cancer. However, definite conclusions have not been reported. Perioperative measurement of serum CEA concentration can yield information valuable for predicting poor survival; the pathological stages of these patients has been reported to be very heterogeneous (from IA to IV), which could induce bias.CYFRA 21‐1 is a useful tumor marker for NSCLC, and several studies have concluded that it is a significant prognostic determinant.Compared with CEA and CYFRA 21‐1, there have been fewer studies on the relationship between CA 125 and survival. In operable NSCLC, serum CA 125 could be used as a tool to predict survival.CYFRA 21‐1 has been shown to be more sensitive than CEA in predicting OS;et al. in PFS and OS between patients with high‐CEA/normal‐CYFRA 21‐1 and those with normal‐CEA/high‐CYFRA 21‐1.Previous studies have used multiple tumor markers for modeling NSCLC prognosis. Patients with normal markers may have better PFS and OS than those with one or two high markers; but there were no differences reported in the study by Sone Inevitably, imbalances existed among the risk groups, especially with regard to sex, T stage, pathology, and radiation. To neutralize the effects of these imbalances, we set up a PSM analysis. In PSM analysis, this model still worked well in predicting all four of the survival types. The results of our study agrees with another small trial that analyzed the prognostic value of CEA, CYFRA 21‐1, and CA 125 and showed that abnormal elevations of the three tumor markers worsened the prognosis.Expression levels of CEA, CYFRA 21‐1, and CA 125 have been shown to be linked with disease stage.Some studies have indicated that CEA is a specific tumor marker of adenocarcinoma, which accounts for a large portion of nonsquamous cell cancer.Cutoff values are another concern. Previous studies usually defined their own cutoff values after receiver operating characteristic analysis. CEA cutoff values were 2.5–50 ng/mLThe advantages of this study were multifold. It had a large sample size, and is the first study to investigate the prognostic prediction of N2 NSCLC patients after resection using serum tumor markers. Moreover, the prognosis model also proved to be useful and sensitive after multivariate PSM analysis. Previous models were useful for just one endpoint; this combined model was predictive for four classes: survival, recurrence, progression, and metastasis. The model in our study also utilized available technology, conventional cutoff values, and simple mathematics methods and was economical.Our study had certain limitations. Other NSCLC tumor markers such as NSE and SCC were not analyzed, which might affect survival in certain pathological types such as neuroendocrine NSCLC and squamous carcinoma. We did not investigate the baseline trends and the values after treatment, which might also be helpful in predicting outcomes and has been evaluated in other NSCLC stages.In conclusion, the serum levels of CEA, CYFRA 21‐1, and CA 125 are prognostic predictors of stage N2 patients who have received R0 resection. The three‐marker combination model accurately predicts multiple prognostic outcomes , which might be helpful when deciding on future adjuvant therapy.Yirui Zhai, Zhouguang Hui, Yu Men, Yang Luo, Yushun Gao, Jingjing Kang, Xin Sun, and Jianyang Wang each declare that they have no competing interests.Figure S1 Survival of patients with different CEA levels: (a) overall survival, (b) progression‐free survival, (c) local regional relapse‐free survival, (d) distant metastasis‐free survivalClick here for additional data file.Figure S2 Survival of patients with different CYFRA 21‐1 levels: (a) overall survival, (b) progression‐free survival, (c) local regional relapse‐free survival, (d) distant metastasis‐free survival.Click here for additional data file.Figure S3 Survival of patients with different CA 125 levels: (a) overall survival, (b) progression‐free survival, (c) local regional relapse‐free survival, (d) distant metastasis‐free survival.Click here for additional data file.


X‐chromosome short tandem repeats (X‐STRs) with unique sex‐linkage inheritance models play a complementary role in forensic science. Guizhou is a multiethnic province located in southwest China and some genetic evidence focusing on X‐STRs for various minorities was reported. However, population data of Guizhou Tujia are scarce.A total of 507 Guizhou Tujia individuals were profiled using the AGCU X‐19 STR kit. Allele frequencies and forensic parameters were calculated. Additionally, population genetic relationships between Guizhou Tujia and other 19 populations were explored.A total of 257 alleles with the allele frequencies ranged from 0.0013 to 0.6098 were found. The combined power of discrimination in males and females and mean exclusion chances in all case scenarios were all greater than 0.99999. Population comparisons showed Guizhou Tujia had a homogeneity with all Han populations from different administrative regions, and other ethnic populations residing in Guizhou, while had obviously genetic heterogeneity with the Altaic family populations except Xibe.Nineteen X‐STRs can afford a reliable and informative database of Guizhou Tujia population for human identification and paternity testing, especially in complex biological relations. The genetic relationships of Chinese are significantly influenced by the geographic position and ethnolinguistic origin. 19 X‐STRs can afford a reliable and informative database of Guizhou Tujia population for human identification and paternity testing, especially in complex biological relations. The genetic relationships of Chinese populations are significantly influenced by the geographic position and ethnolinguistic origin. Tujia has its own language which belongs to the Tibeto‐Burman language branch of the Sino‐Tibetan language family but no script. Based on the Chinese historical sources, Tujia people are the main descendants of the Ba people, an ancient tribe in southwest China formed and named between the Xia and the Shang Dynasty. In the early period, the Ba people took Enshi as the center and lived in western Hubei. With the power increasing, the Ba people gradually expanded to the whole Wuling mountain areas, southward to the Guizhou and Hunan province, eastward to Sichuan. After the Ba was defeated by the Qin, its posterity survived and renamed “Tu” in the 1206 A.D. With the stability of the chieftain system, the Tujia people residing in Hunan, Hubei, Sichuan, and Guizhou province integrated and gradually consolidated. Since 1956, Tujia has become an officially recognized independent ethnic group (https://en.wikipedia.org/wiki/Tujia‐people). Guizhou, located in southwest China, is demographically one of China's most diverse provinces with a unique geographical environment and historical mass migration. Approximately 4.1% of the Tujia people in China have settled in Guizhou, and becoming the fifth chiefly ethnic group in the province (https://en.wikipedia.org/wiki/Guizhou).Tujia, one of the most ancient minorities in China, mainly live in the Wuling Mountains boarding Guizhou, Hunan, and Hubei Provinces, and Chongqing Municipality. According to the sixth national population census of the Chinese government, Tujia was the seventh largest minority in 55 Chinese minorities with a population of about 8 million people  Tujia populations using AGCU X‐19 STRs kit. With these highly polymorphic STRs’ allele frequencies data, we further evaluated the X‐chromosome forensic characteristics of AGCU X‐19 STRs kit in Guizhou Tujia populations and reconstructed the population genetic structure with previously reported populations  unrelated healthy Tujia individuals from Guizhou province (southwest China) were collected in accordance with the informed consent principle. Participants were all indigenous without immigration and interracial marriage at least three generations. This study was approved by the Biomedical Research Ethics Committee of Zunyi Medical University (No. 2014‐1‐044).2.22O (sterile deionized H2O). PCR condition consisted of predegeneration of 2 min at 95℃; 10 cycles of 30 s at 94℃, 1 min at 60℃, 1 min at 65℃, then 20 cycles of 30 s at 94℃, 1 min at 59℃, 1 min at 72℃; final extension of 30 min at 60 ℃, and hold at 4℃. The PCR products were detected and separated on an ABI 3500XL Genetic Analyzer . GeneMapper ID v1.4 software was employed to identify and assign the alleles determined by fragment sizes. Positive control (9947A DNA sample) and negative control (sdH2O) ran in each PCR reaction system.DNA extraction of Tujia individuals was performed from the EDTA anticoagulant tube using salting‐out method . SPSS v26.0 Hansen,  was usedp values of 19 X‐STRs between 20 populations, as well as pairwise Nei's genetic distances based on 19 shared X‐STRs, were calculated by Arlequin v3.5 software . Neighbor joining tree (N‐J tree) and multidimensional scaling plots (MDS) were visualized by Mega v7.0 , which explained that the selected sample could represent Guizhou Tujia population.In the present research, we used the AGCU X‐19 kit to genotype the 19 X‐STRs in 507 volunteers  residing in Guizhou province successfully. The Hardy‐Weinberg equilibrium (HWE) for the 19 X‐STR loci was tested using a Markov Chain with the dememorization steps of 100,000 and forecasted chain length of 1,000,000 testing based on the observed heterozygosity (Ho) and expected heterozygosity (He) in 249 unrelated healthy Tujia females Table  Nei & R. The valp > 0.05) in all 19 loci were detected between males and females , power of discrimination in males (PDm) and females (PDf), paternity exclusion chance (MEC) in the Trios  and Duos (MEC_Desmarais_Duos) of 19 X‐STRs based on pool frequencies are performed in Table 3.2p > 0.05/171 = 0.0003) in the females. In the male individuals, there were significant differences in 25 pairs, but only five pairs  still deviated from LD after Bonferroni correction (p > 0.05/171 = 0.0003).The linkage disequilibrium (LD) for 19 X‐STRs intercomparisons was calculated by permutation test using the EM algorithm  of females Table  and was 19 X‐STRs can be clustered into seven linkage groups (LG): LG1  after Bonferroni correction, which were identified between Guizhou Tujia and Xinjiang Kyrgyz at 12 loci, followed by Xinjiang Uyghur at 10 loci, Xinjiang Mogolian at eight loci, Tibet Tibetan and Xinjiang Kazakh at seven loci, Guangxi Zhuang, Sichuan Tibetan, and Sichuan Yi at six loci, Guizhou Sui at five loci, Guangxi Mulao at four loci, Xinjiang Xibe at three loci, Ningxia Hui at two loci, and Shaanxi Han, Guizhou Han, Guizhou Miao at only one loci. No significant differences were observed between Guizhou Tujia and Xinjiang Han, Southern Han, Sichuan Han, and Guizhou Gelao. Significant differences were observed at DXS10159 locus of Guizhou Tujia and 19 reference populations, while no differences were found at DXS10074, DXS10134, and DXS10164 loci.Locus by locus Fst and corresponding Pairwise Nei's genetic distances among 20 populations were estimated and listed in Table Then, phylogenetic relationships between Guizhou Tujia and other reference populations were explored using N‐J tree and MDS plot. As shown on the N‐J tree Figure , three mThe MDS Figure  showed tFinally, PCA was also constructed with the exception of Xinjiang Mogolian populations, which can be prominently separated from others in the N‐J tree and MDS. As shown in Figure 4Herein, genotypes of 507 unrelated Tujia individuals  from Guizhou were successfully obtained using AGCU X‐19 STRs PCR amplification kit. To explore the capacity of 19 X‐STRs in individual identification and forensic complex paternity testing, a series of forensic parameters were calculated, such as PDm, PDf, and four paternity exclusion chance . MEC_Krüger  in Guizhou and other provinces are needed.5In this study, we first analyzed the genetic polymorphisms of the Guizhou Tujia population based on AGCU X‐19 PCR kit. All loci in this population can be used to establish a reliable and informative database of X chromosome markers for human identification and paternity testing, especially in complex biological relations. Additionally, population comparisons indicate that Guizhou Tujia has genetic homogeneity with populations who reside in geographically adjacent regions and share the same language. Besides, Guizhou Tujia as a Tibeto‐Burman‐speaking population has an intimate relationship with geographically close Guizhou Gelao and the Han populations. Additional studies with other provinces and genetic markers of Tujia populations are needed for further understanding of the genetic structure of the Tujia populations.No potential conflict of interest was reported by the authors.PYC and LMY conceived the idea for the study. LL, HYG, LLY, HZ, and FL performed or supervised laboratory work. LL, LSZ, and LLY analyzed the data. PYC, LL, JY, and MLY wrote and edited the manuscript.Fig S1Click here for additional data file.Table S1‐S10Click here for additional data file.


EGFR‐TKI resistance mechanism through tissue can be really challenging due to risks associated with the procedure. Thus, liquid biopsy, especially circulation tumor DNA (ctDNA) analysis, can be an adequate source for biomarker testing in targeted therapy. Our study was aimed at clinical validation of liquid biopsy next‐generation sequencing (NGS) by comparison with tissue biopsy, and we also investigated clinical utility of ctDNA NGS on the prediction of TKI outcomes.Clinical detection of Using hybrid capture panel NGS, we compared the concordance, sensitivity, and specificity of ctDNA using 39 paired plasma and tissue biopsy, and investigated the association between ctDNA genomic alterations of 147 first‐generation TKI‐relapsed patients and their response to first‐ and third‐generation TKIs.EGFR‐TKI‐relapsed patients, T790M was the most common reason for resistance (40.13%). Compared with T790M‐positive patients, patients only detected with sensitizing mutations (sensi‐mutations) had lower mutant allele frequency (MAF) of sensi‐mutations (P = 0.031). TP53 mutation showed negative impact on TKI treatments. In survival analysis of third‐generation TKI, we found a positive correlation between ratio of T790M sensi‐mutation and PFS (P = 0.018); also, higher MAFs of both sensi‐mutation and T790M were observed in the PR group than the SD + PD group.The concordance for ctDNA and tissue biopsy was 84.62% among all patients, and even higher among late stage patients (88.24%). Among 147 EGFR mutations were associated with third‐generation TKI outcomes. Thus, incorporation of high‐throughput NGS into clinical trials may be crucial to identifying the response to osimertinib, as it provides more comprehensive genomic information.Both ratio of T790M sensi‐mutations and MAFs of High concordance of ctDNA and tissue biopsy was observed. NGS of ctDNA from 147 TKI‐relapsed patients showed that both high ratio of T790M sensitizing mutation (sensi‐mutation) and high MAFs of mutations were all associated with better third generation TKI treatment outcomes.The quantification of both MAFs and T790M sensi‐mutation ratio should be taken into consideration in some clinical situations, and incorporation of high‐throughput NGS into clinical trials may be crucial to identifying the response to osimertinib, as it provides more comprehensive genomic information. EGFR mutations,EGFR tyrosine kinase inhibitors (EGFR‐TKIs). However, most patients harboring EGFR‐activating mutations eventually acquire resistance to the first‐generation TKI treatment after a median response of 10–12 months.EGFR gene.MET amplification, ERBB2 amplification, PIK3CA mutation and BRAF mutations have also been reported to cause resistance.Lung cancer represents the leading cause of cancer‐related deaths worldwide.Unfortunately, clinical detection of resistance mechanism through tissue biopsy can be really challenging due to risks associated with the procedure, particularly for TKI‐relapsed patients. Tissue biopsy can also be incomplete because of spatial and temporal tumor heterogeneity. Liquid biopsy obtained by minimal invasive blood draws are therefore became accessible at almost all clinical situations.In this retrospective study, clinical validation of liquid biopsy was performed by concordance, sensitivity, and specificity of ctDNA hybrid capture panel NGS using 39 paired plasma and tissue biopsy from lung cancer patients. We also investigated the resistance mechanisms of 147 plasma samples from first‐generation TKI‐relapsed patients, and analyzed the ability to predict target therapy outcomes by ctDNA NGS.EGFR‐TKI‐relapsed patients were also enrolled in the study in order to analyze the ability to predict target therapy outcomes by ctDNA NGS.We retrospectively reviewed all the patients who had been diagnosed with NSCLC and undergone ctDNA genetic test using NGS between March 2017 to May 2018 at the Cancer Hospital, Chinese Academy of Medical Sciences . A total of 39 blood samples with paired tissue biopsy samples were enrolled in our study for the comparison between liquid and tissue biopsy. Tissue biopsy samples were all collected at the same time period with blood samples. Another 147 blood samples from first‐generation EGFR‐TKI‐relapsed patients , following the manufacturer's instructions. DNA quantity was determined by Qubit 2.0 Fluorometer .We collected 10 mL of whole blood into EDTA‐containing tubes, and plasma was separated within one hour of collection. Briefly, whole blood at 4°C was centrifuged at 2000 × g for 10 minutes, and the supernatant was transferred to a new 15 mL tube and centrifuged at 16000 × g for another 10 minutes at 4°C. About 4 mL plasma was then collected for DNA extraction. CtDNA was extracted from 4 mL plasma using the Plasma Circulating Nucleic Acid Preparation Kit , following the manufacturer's instructions. DNA quantity was determined by Qubit 2.0 Fluorometer .Targeted NGS were performed using a capture‐based panel that covers 168 cancer‐related genes as previously reported.The response evaluation was assessed according to Response Evaluation Criteria in Solid Tumors (RECIST), version 1.12 test for categorical data, and t‐tests for continuous data. PFS were estimated using the Kaplan‐Meier method, and differences in PFS were assessed by log‐rank test. A two‐sided P‐value <0.05 was considered statistically significant.All statistical analyses were performed using the Statistical Package for the Social Sciences  version 22.0. Differences between groups were compared using the Pearson's χEGFR mutations, three cases harboring KRAS mutations, seven cases harboring ALK rearrangement, one case with ROS1 rearrangement, and one case with RET rearrangement. High concordance of 84.62% were observed between ctDNA and tissue biopsies . Among 30 TKI‐naïve paired samples, four cases were proved to be ctDNA NGS false negative. For the type of biopsy procedure,, 21 patients underwent lung biopsies , six cases obtained tumor cells from malignant pleural effusion, and eight cases underwent cervical lymph node biopsies. Liver, hip, and brain biopsies were used for the other three cases, respectively.Among these 39 patients, five were early stage patients (I–II stage) and 34 were late stage patients (III–IV stage) Table . If we oEGFR‐TKI‐relapsed patients are summarized in Table EGFR mutation type was L858R , followed by exon19 deletions (19del) . Other clinical characteristics such as metastasis status are listed in Table The clinicopathologic characteristics of 147 EGFR, MET, ERBB2, TP53, PIK3CA, PTEN, RB1, SMAD4, CTNNB1, CDKN2A, and MTOR, whereas 27 patients (18.36%) were negative in the NGS test  of patients; and 29.93% (44/147) of patients were negative for EGFR mutations  received EGFR‐TKI after chemotherapy (one missing data). A total of 36.36% (16/44) of those patients had brain metastases when liquid biopsy was performed. No difference in brain metastases ratio were observed between the 44 patients and others with EGFR mutations. However, bone metastases were more common in patients detected with EGFR mutations than those without any EGFR mutations . No other significant difference of clinical characteristics were observed between patients who were negative for EGFR mutations and others. We also found 2.04% (3/147) of patients developed alternative pathway activations (including MET amplification and ERBB2 amplification) as resistance mechanisms. A total of 133 cases have been tested for TP53 status, and 45.86% (61/133) of them detected with TP53 mutations. TP53 mutations were more common observed with a concurrent 19del mutation than L858R  including 0/147 incP‐value . Compared with cases only detected with sensitizing mutations, the T790M‐positive group seemed to have higher ctDNA quantity for NGS library construction . No other significant association was observed between other clinical characteristics and T790M MAFs. No significant association was observed between clinical characteristics and sensi‐mutation MAFs.Among 59 T790M‐positive cases, the association of clinical characteristics and MAFs of T790M were also analyzed (one missing data). A total of 35.59% (21/59) of patients underwent surgeries before TKI treatment. When ctDNA NGS was performed, 50.85% (30/59) had bone metastases, 37.29% (22/59) had brain metastases. The majority of patients  had extrapulmonary metastases (M1b), 18.64% (11/59) were M1a, while only three cases (5.08%) did not have metastasis. We found that the MAF of T790M were significantly higher in M1b cases than others (M0 and M1a)  and higher T790M MAF   of patients progressed from third‐generation TKI treatment. A total of 41.30% (19/46) of these patients were treated with second‐line therapy, and 43.48% (20/46) of patients received osimertinib as third‐line treatment . Six patients received more than two lines of treatment before osimertinib, with one missing data. ORR and DCR for third‐generation TKI were 55.32% and 97.87%, respectively. Patients harboring 19del had higher ORR than those harboring L858R , consistent with previous studies.et al. reported that prevalence of T790M was significantly higher in 19del than in the L858R subgroup .et al. the incidence of T790M were similar between the 19del and L858R group (55.56% vs. 53.85%)et al. proved that the incidence of T790M in 19del was slightly lower than in L858R after gefitinib/erlotinib treatment (73.08% vs. 84.62%), but T790M was more likely to coexist with 19del than L858R after afatinib treatment (38.24% vs. 14.29%).We found that T790M was more likely to coexist with 19del than with L858R , and patients with T790M sensi‐mutation >0.22 had better PFS than others.et al. Thus, the cutoff ratio in our study was slightly higher than the study by Del Re et al. As we revealed that the ratio of T790M/sensitizing mutation varied in different patients because of intratumor heterogeneity, the most possible reason for the negative impact of low T790M ratio on third‐generation TKI PFS is that clones lacking T790M are likely to have other resistance mechanisms and respond poorly to third‐generation TKI treatment.In survival analysis of third‐generation TKI, the median PFS was 9.17 months. ORR and DCR for third‐generation TKI were 55.32% and 97.87%, respectively, which was consistent with the previous report.Our study was limited by a few factors. First, our work was a retrospective study and only included first‐generation TKI‐relapsed blood samples. Baseline blood samples collected before first‐generation TKI treatment may be more effective to predict first‐generation TKI outcomes. Second, the number of T790M‐positive patients received osimertinib was relatively small, and not all progressed from third‐generation TKI treatment. Future studies are still needed to confirm the results with a larger population.TP53 mutation showed negative impact on target therapy outcomes. We also found that in third‐generation TKI treatment, both high ratio of T790M sensi‐mutations and high MAFs of EGFR mutations were associated with better outcomes of third‐generation TKI treatment. Therefore, the quantification of both MAFs and T790M sensi‐mutation ratio should be taken into consideration in some clinical situations, and incorporation of high‐throughput NGS into clinical trials may be crucial in identifying the response to osimertinib, as it provides more comprehensive genomic information, such as mutation types and the MAF.In summary, our study indicated that high‐throughput NGS based platforms are competent for the detection of ctDNA mutations to guide the target therapy, especially when tissue biopsy was not available. However, it should always be noted that for those patients harboring low MAF sensitizing mutation without T790M in ctDNA NGS, it is possible that their T790M mutation is under LOD, and can still benefit from third‐generation TKI treatment. In survival analysis, No authors report any conflict of interest.Figure S1 Kaplan‐Meier estimates of first‐generation TKI progression‐free survival of 147 cases. (a) Survival curves of all 147 cases; (b) survival curves according to sensitizing mutation types; and (c) survival curves according to TP53 status.Figure S2 Kaplan‐Meier estimates of third‐generation TKI progression‐free survival of 46 patients. (a) Survival curves according to sensitizing mutation types; and (b) survival curves according to TP53 status.Figure S3 Receiver operating characteristic (ROC) curve, point a: ratio (T790M sensitizing mutation) = 0.4.Figure S4 The association between TP53 status and other genetic alterations. (a) TP53 status and EGFR sensitizing mutations. (b) TP53 status and other cancer‐related gene mutations detected by next‐generation sequencing (NGS).Click here for additional data file.


This study analyzed the optical coherence tomography angiography (OCTA) macular parameters in primary angle-closure glaucoma (PACG) patients after acute primary angle closure (APAC) episodes. Thirty-three patients with 33 APAC eyes and 33 primary angle closure suspect (PACS) eyes and 33 age-matched normal subjects (controls) were enrolled. Macular vessel density (VD) in central, inner, outer and full regions and foveal avascular zone (FAZ) parameters  were compared between APAC, PACS, and control eyes. For resolved APAC eyes, the VD in each macular region was significantly lower than that in control eyes, with less central and inner macular VD than PACS eyes. The central macular VD was significantly lower in PACS eyes than in controls. There was no difference in FAZ area and perimeter between APAC, PACS, and control eyes. FAZ circularity was highest in control eyes, followed by PACS eyes, and lowest in APAC eyes. The AUC, sensitivity and specificity of FAZ circularity were 0.944, 93.9% and 84.8%, respectively, in APAC eyes and 0.881, 84.8% and 81.8%, respectively, in PACS eyes. Therefore, FAZ circularity had the best discrimination capability for detecting both APAC and PACS eyes. Macular assessment with OCTA could provide an accurate early-stage diagnostic tool for PACG. Though it is an important cause of blindness globally, the exact pathogenic mechanisms of PACG are not clear. Recently, some studies have indicated that the retinal vascular alterations are related to the development of this disease4.Primary angle-closure glaucoma (PACG) is a notable type of glaucoma with high incidence in the population worldwide7. A number of studies substantiated a significant decrease in peripapillary and macular vessel density (VD) in primary open angle glaucoma (POAG), and normal tension glaucoma (NTG) eyes compared to normal controls10. Some investigations11 focused on PACG patients and demonstrated that peripapillary VD was lower in acute PACG eyes than in unaffected eyes after an acute primary angle closure attack13. In addition, PACG eyes with well-controlled intraocular pressure (IOP) had higher peripapillary VD than eyes with IOP that was not well controlled14.At first reported in 2012, optical coherence tomography angiography (OCTA) is a safer technique to quantitatively assess retinal vasculature than conventional angiography methods16. Regarding anatomy, the peripapillary area is supported by a double-layered capillary support system  and ganglion cell layer (GCL)), while the vascular supply of the macula is only a single-layered parafoveal capillary arcade. Physiological evidence also supports that the macula is more sensitive to hypoxia and ischaemia because it consumes more oxygen than other tissues in the body17. Subsequently, macular VD and foveal avascular zone (FAZ) metrics were regarded as potential biomarkers in the clinical evaluation of early glaucoma in recent studies20. However, few researches have examined macular vasculature in PACG. Zhu et al.14 demonstrated decreased macular circulation in PACG with glaucomatous optic neuropathy and visual field (VF) defects, but no literature has explored macular VD and FAZ metrics using OCTA in APAC eyes after a recent acute episode. The purpose of our study was to investigate the potential macular vascular differences with OCTA by comparing macular VD and FAZ parameters between APAC eyes after the first acute attack, fellow eyes confirmed to be affected with primary angle closure suspect (PACS) and heathy normal control eyes.Some studies reported macular damage in early-stage glaucoma, such as macular ganglion cell-inner plexiform layer loss, and these attracted a significant amount of research to understand the importance of the macula in glaucomaA total of 33 unilateral adult APAC patients (33 APAC eyes and 33 PACS eyes) who were diagnosed as acute angle-closure glaucoma patients were enrolled in the study, and 33 age-matched healthy subjects were enrolled as controls, with 33 eyes randomly selected, from June 2018 to December 2019 at the Department of Ophthalmology of Xiangya Hospital. All research methods follow the Helsinki Declaration. After approval by the Medical Ethics Committee of Xiangya Hospital, all the subjects signed an informed consent form according to the principle of ethics.21: (1) elevated IOP > 21 mmHg with Goldmann applanation tonometry; (2) at least 3 of the following signs: conjunctival hyperaemia, corneal epithelial edema, enlarged pupil, direct disappearance of light reflex, and the disappearance of the shallow anterior chamber or anterior chamber; (3) at least two of the following symptoms: severe eye pain, a sharp decline in vision, ipsilateral migraine, nausea, vomiting, elevated body temperature, and pulse acceleration.; and(4) at least 2 quadrants of the chamber angle were closed on gonioscopic examination. A PACS eye was defined by the presence of the following22: (1) presence of iridotrabecular contact more than 180° without peripheral anterior synechiae (PAS) on gonioscopy; (2) the absence of a glaucomatous optic nerve and visual field damage; (3) no history or signs of a previous AAC attack; and (4) an IOP of < 21 mmHg without medication. Exclusion criteria for APCG and PACS patients were as follows: (1) secondary glaucoma, such as lens-derived glaucoma, neovascular glaucoma, iridocyclitis secondary glaucoma; (2) POAG, chronic angle-closure glaucoma, congenital or developmental glaucoma; (3) retinal or optic nerve disease; (4) dioptre spherical degree > 6.00–6.00 D and/or astigmatism > 3.00–3.00 D; (5) previous history of endoscopic or laser treatment; and (6) history of hypertension and diabetes.Eyes that suffered the first attack and were resolved by treatment with medicine were designated as APAC eyes, and the contralateral unaffected eyes were designated as PACS eyes. During hospitalization the APAC eyes underwent trabeculectomy, and the PACS eyes were treated with laser peripheral iridotomy. An APAC eye was defined by the presence of the followingThe control subjects were excluded if they met the following criteria: (1) ophthalmic diseases such as glaucoma, fundus diseases, corneal diseases; (2) history of ocular trauma and intraocular surgery, history of hypertension and diabetes, family history of glaucoma; (3) retinal or optic nerve diseases or visual field defect; (4) dioptre spherical power > 6.00 –6.00 D and / or astigmatism > 3.00–3.00 D (5) IOP was higher than 21 mmHg; and (6) a cup-to-disc ratio ≥ 0.5 or cup to disc asymmetry ≥ 0.2.We used anti-glaucoma medicine and intravenous acetazolamide or mannitol to reduce IOP for all APAC eyes after admission. A series of ophthalmologic examinations were performed in APAC and PACS eyes after the first acute attack had been fully resolved by medicine. Subsequently, the APAC eyes underwent trabeculectomy, and the PACS eyes were treated with laser peripheral iridotomy during hospitalization.23.The complete ophthalmologic examinations included vision examination, slit lamp examination, gonioscopic examination, IOP measurement (Goldmann applanation tonometry), anterior chamber depth (ACD) and axial length (AL) measurements, field visual measurements, and OCT and OCTA examinations. We used OCT  to measure RNFL thickness and used a Humphrey Field Analyser  for field visual inspection. Measurements of IOP, systolic blood pressure (SBP), and diastolic blood pressure (DBP) were performed while the patients was in a sitting position for 20 min before the patient underwent the OCTA examination. The mean arterial pressure (MAP) was calculated by the following formula: MAP = DBP + (SBP − DBP)/3, the ocular perfusion pressure (OPP) calculation formula was OPP = 2 (MAP − IOP)/324.All subjects underwent SD-OCTA examination using the OCTA system . OCTA obtained a 6 × 6 mm volumetric macular superficial retinal vessel image that consisted of vessels from the layer of the inner limiting membrane (ILM) to the inner plexiform layer (IPL). Several factors, including glaukomaflecken and cataract, can affect VD measurements in the macula. Therefore, poor-quality scans with a signal strength index (SSI) values less than 7 were excluded from this study. The system software was used to automatically divide the image into 3 regions, including a 1 mm diameter central disc and an inner and outer ring with diameters of 3 mm and 6 mm, respectively  . The FAZ circularity index is a measure of the shape of the FAZ, where 1 indicates a circular shape and 0 indicates an irregular shape. By observing the parameters of the FAZ, we can understand the damage that the disease to inflicts upon the capillaries in the macular arch area. The FAZ circularity index calculation method was based on the area and perimeter of the FAZ, and the calculation formula is as followsP < 0.0167 was considered statistically significant. The discrimination capability of OCTA parameters were evaluated with the area under the receiver operating characteristic curve (AUROC), and P < 0.05 was considered statistically significant. Multiple linear regression analyses were conducted to evaluate the relationship between OCTA parameters and other clinical features in the APAC and PACS eyes. To further evaluate these co-factors for macular VD, we established regression models for predicting macular VD based on the clinical features.Statistical analysis was performed with SPSS version 20.0 . The normal distribution of the data was tested by the Kolmogorov–Smirnov test. Therefore, the data are presented as the mean ± standard deviation (range) of the Gaussian distribution value and the median  of the non-Gaussian distribution. The comparison between two different groups was analysed with the LSD-t test with Bonferroni correction for Gaussian distribution values and Tamhane’s T2 test with Bonferroni correction for non-Gaussian distribution values, and There were 33 PACG patients, including those who had both eyes as APAC eyes and PACS eyes, and 33 age-matched normal subjects in this study.P > 0.0167). Compared with control eyes, APAC eyes and PACS eyes all had shorter AL (P < 0.001); there was no difference in AL between APAC and PACS eyes. The ACD of APAC eyes (1.74 (1.49–1.86) mm) was significantly lower than that of PACS (1.90 (1.80–2.00) mm) (P < 0.001) and control eyes (2.86 (2.63–2.99) mm) (P < 0.001); the ACD of PACS eyes was significantly lower than that of control eyes (P < 0.001). The VFof APAC group (− 8.92 (− 11.81 to − 7.14) dB) was significantly lower than that of PACS group (− 3.85 (− 5.34 to − 2.31) dB) (P < 0.001) and control group (− 3.47 (− 3.92 to − 2.89) dB) (P < 0.001). For APAC eyes, the average IOP at attacking was 52.00 (42.00–58.00) mmHg, and the duration of IOP exposure was 2.35 ± 1.50 days. There was no significant difference in IOP at attack and VF between PACS eyes and control eyes (P > 0.0167). The SBP values of APAC and PACS eyes were significantly higher than that of control eyes (P = 0.001), but the DBP values of APAC and PACS eyes were significantly lower than that of control eyes (P = 0.003).Table P ≤ 0.015), though there was still no significant change in the outer and full macular regions (P ≥ 0.025). In APAC eyes, VLD and VPD values were significantly reduced in all macular regions compared with control eyes, with statistically significant differences (P ≤ 0.001). VLD and VPD values in the central macular region of PACS eyes were significantly lower than those of control eyes (P ≤ 0.004), while the differences in other regions were not statistically significant (P ≥ 0.032). For the FAZ index, there was no difference in the FAZ area and perimeter between the three groups (P ≥ 0.018). The FAZ circularity index was highest in control eyes (0.76 ± 0.07), followed by PACS eyes (0.61 ± 0.11), and lowest in APAC eyes (0.52 ± 0.12) (P ≤ 0.007)  to evaluate the accuracy of detection Fig. . The AUCP = 0.032; r = 0.473, P = 0.005); the central VLD was positively correlated with the VF and SBP . The outer and full VPD were positively correlated with the RNFL thickness significantly ; the FAZ area and circularity index were positively correlated with the RNFL thickness significantly  , DBP , and OPP  significantly; the inner VPD was positively correlated with OPP . The FAZ area and perimeter index were positively correlated with DBP  and OPP  significantly  and SBP (P = 0.037). The determination coefficient R2 = 0.265 indicated that 26.5% of the central VLD variation may have been attributed to the joint variations in VF and SBP.Additionally, we used multivariate stepwise regression analyses to further evaluate the co-factors for macular VD Table . For theP = 0.039), DBP (P = 0.713), and OPP (P = 0.342). The determination coefficient R2 = 0.182 indicated that 18.2% of the outer VPD variation may have been attributed to joint variations in RNFL thickness, DBP and OPP. Equation (3) shows that we could predict changes in the full VPD by looking at changes in RNFL thickness (P = 0.051), DBP (P = 0.831), and OPP (P = 0.250). The determination coefficient R2 = 0.179 indicated that 17.9% of the full VPD variation may have been attributed to joint variations in RNFL thickness, DBP and OPP. Although RNFL thickness, DBP and OPP had combined effects on the outer and full VPD, RNFL thickness had a larger impact on VPD variations than DBP and OPP in PACS eyes.For the PACS group, Eq. (2) shows that we could predict the outer VPD by looking at changes in the RNFL thickness (P = 0.871) and OPP (P = 0.014). The determination coefficient R2 = 0.317 indicated that 31.7% of the FAZ area variation may have been attributed to joint variations in DBP and OPP. Equation (5) shows that we could predict changes in the FAZ perimeter by looking at changes in DBP (P = 0.256) and OPP (P = 0.001). The determination coefficient R2 = 0.409 indicated that 40.9% of the FAZ perimeter variation may have been attributed to joint variations in DBP and OPP. These two multi-variate regression models presented that OPP was a more important factor than DBP for FAZ area and perimeter measurements in PACS eyes.Equation (4) shows that we could predict changes in the FAZ area by looking at changes in DBP  and OCTA was not approved for glaucoma39. The FAZ circularity index was applied to measure the shape relative to a circle. The index indicates an irregular shape when close to 0, and a circular shape when close to 119. Geometrically, it is possible for different shapes to have the same area and perimeter according to Heron’s formula. While on the aspect of anatomy, there is only a single layer of vascular support in the FAZ, and the subtle change in the vascular arcade may render an irregular outline of the FAZ. The FAZ circularity index may be more sensitive than the FAZ area and perimeter in detecting subtle changes in the vascular arcade, and it is therefore plausible that we can detect a decreased circularity index in APAC and PACS eyes. Additionally, Choi et al.40 reported POAG eyes with an increased FAZ perimeter and decreased FAZ circularity index compared with control eyes, which is similar to our finding. It may suggest that there are similar pathophysiological processes in advanced POAG and PACG; accordingly we will explore the differences in FAZ parameters in different types of glaucoma in the following research. Our investigations showed that the FAZ circularity index was positively correlated with RNFL thickness in an APAC eye. Hence, we hypothesize that the reduction in RNFL thickness may be relate to microcirculatory alterations in macula. Although we can’t know whether the microcirculatory or structural alterations appear first during the development of PACG, the FAZ metric and macular VD could help us to understand the pathologic and physiological processes in PACG better.The FAZ is a central round 400 μm area devoid of retinal capillaries in the fovea, whose morphology relates to many pathologic conditionsIn the current study we examined the diagnostic accuracy of all 11 parameters with AUROC. By corollary, macular VD has great diagnostic value. However, our analysis demonstrated that the FAZ circularity index was the best parameter for discriminating between PACG and control eyes (AUC 0.944), and between APAC and control eyes (AUC 0.881), with both demonstrating better AUC, sensitivity and specificity. The diagnostic efficacy of macular VD in distinguishing an APAC or PACS eye from a normal eye was significantly worse than that of the FAZ circularity index. In addition, we found that central and inner VLD and VPD had better diagnostic ability than outer and full VLD and VPD in distinguishing PACS eyes from normal eyes. All these data suggested the subtle loss of capillaries of the parafoveal vascular arcade might render an irregular outline of the FAZ prior to the decrease in macular VD outside the fovea at the early stage of PACG. Therefore, the fovea, parafoveal macular VD and FAZ circularity index may not only be an additional diagnostic approach for PACG eyes after an attack but may also help in the recognition and management of PACS patients without a history of attack.41. Thirdly, all the APAC eyes were examined after fully resolving with medications, but we could not distinguish the influence of medications.Our study has some limitations. Firstly, this is a retrospective study with a relatively small sample of 33 subjects. Secondly, we did not examine the deep retinal and choroidal vasculature of our subjects, as those with PACG eyes had decreased VDIn conclusion, the changes in macular VD and FAZ parameters in APAC and PACS eyes were described compared to those of healthy control eyes using OCTA when IOP was controlled after an acute attack. APAC eyes had a progressive reduction in macular VD and FAZ circularity index, while PACS eyes presented a dropout of VD in the central macular region and FAZ circularity index. Besides, the loss of VD in the central and inner macular regions and the lower FAZ circularity index were confirmed in eyes with APAC compared with PACS eyes. Non-invasive macular assessment with OCTA could provide an accurate diagnostic tool for PACG, and the FAZ circularity index may be a potential biomarker in detecting vascular damage at an early stage in PACG eyes.


In rodents, neurotensin contributes to high fat diet induced obesity by facilitation of intestinal fat absorption. The effect of oral lipid load on plasma proneurotensin and relationship with plasma triglycerides in humans is unknown.To investigate the acute effects of an oral lipid load on proneurotensin and plasma triglycerides and their interrelationships in healthy individuals.Twenty-two healthy subjects were given 150 mL of full milk cream (54 g fat) and 59 mL of pure olive oil (54 g fat) in the fasted state at two different occasions separated by at least 1 week in random order. Venous blood was drawn at fasted before 0 h (h) and at 1 h, 2 h and 4 h after ingestion. Post-ingested values of proneurotensin and plasma triglycerides were compared with fasting levels and post ingestion Area Under the Curve (AUC) of proneurotensin was correlated with that of plasma triglycerides.(P < 0.001) and at 3 h for triglycerides of 0.60 (0.43–0.78) mmol/L (P < 0.001). Similarly, plasma proneurotensin and plasma triglycerides increased after ingestion of olive oil with maximum increase of proneurotensin at 3 h of 62 (46–78) pmol/L (P < 0.001) and plasma triglycerides at 3 h of 0.32 (0.18–0.45) mmol/L (P < 0.001). The post lipid load AUC for proneurotensin correlated significantly with the AUC for plasma triglycerides both after cream  and olive oil , respectively.An immediate rise of plasma proneurotensin and plasma triglycerides were observed after ingestion of cream with maximum increase at 2 h for proneurotensin [mean ] of 22 (12–31) pmol/L Proneurotensin increases after an oral lipid load of both cream and olive oil and the rise of post-ingestion plasma triglycerides is significantly related to the rise of post-ingestion proneurotensin. The global increase in the incidence and prevalence of obesity is occurring in parallel with the burden of cardiovascular disease (CVD) and metabolic diseases such as type 2 diabetes, hypertension and dyslipidemia. Large prospective and observational studies have already confirmed the adverse effects of obesity and dyslipidemia on CVD . HoweverLevels of plasma triglycerides, both in the fasted and the non-fasted state, are higher in diabetes patients with insulin resistance than in diabetes patients without insulin resistance , and thiNeurotensin (NT) is a 13-amino acid peptide expressed primarily in the brain and neuroendocrine cells of the small intestine. It is assumed that the absolute majority of the NT that is measurable in blood comes from the periphery, i.e. from the small intestine, where it is stimulated by dietary fat intake .In the brain, NT acts as a neurotransmitter and regulates different functions like temperature regulation, nociception, pituitary hormone secretion and dopaminergic transmission . In the Research in recent years have shown that elevated fasting level of proneurotensin, i.e. a peptide derived from the NT precursor hormone that is release in parallel with the mature peptide , predict2) was calculated as weight (kg) divided by height (m) squared. Subjects were classified according to BMI using WHO classification: normal weight: BMI < 25 kg/m2; overweight: BMI ≥ 25 kg/m2 and < 30 kg/m2; and obese: BMI ≥ 30 kg/m2.In this study, 22 healthy young subjects (10 men and 12 women) without any previous medications and diseases volunteered to participate. Trained research nurses conducted the standard physical examinations. It involved the measurement of blood pressure, weight, height, waist circumference, as well as collection of blood samples. Blood pressure was measured in the supine position after 5 min rest with the arm at the level of heart. The mean of the two measurements, taken 1 min apart, was calculated. Height was measured to the nearest cm. Weight is measured to the nearest kg in subjects wearing light clothes, without shoes. BMI  by using a one-step sandwich immunoassay based on a chemiluminescence label and coated-tube technique . The limit of detection of proneurotensin precursor fragment was 1.9 pmol/L , 15. ChoThe inter assay CV for proneurotensin was 6.2% at 48 pmol/L and 4.1% at 191 pmol/L.Participants received milk cream and olive oil at two separate occasion and all routine plasma laboratory analyses were performed at the University Hospital’s central clinical laboratory by using certified methods.After over-night fast (since 22:00 evening before the test day), all 22 subjects underwent ingestion of 150 mL of milk cream (54 g fat) and 59 mL of pure olive oil (54 g fat) at two different occasions in random order, with the two oral lipid loads separated by 1 week. Blood samples were taken for measurement of plasma triglycerides, proneurotensin and blood glucose in the fasted state and after every hour for 4 h.p-values were less than 0.05.Clinical characteristics at the first visit were presented as means ± standard deviations (SD) for continuous variables and as numbers and percentage for dichotomous variables. The individual hourly plasma concentration values of proneurotensin, triglycerides and glucose after the respective oral lipid load was compared to the fasted value  using paired t-test and the delta values were presented as means and 95% confidence intervals. The change at the maximal post-lipid ingestion concentration of proneurotensin and triglycerides versus the baseline concentration was calculated. Post lipid ingestion areas under the Curve (AUC) of plasma proneurotensin and triglycerides were calculated and correlated the AUCs of proneurotensin and triglycerides using Pearson correlation analysis. Differences were considered statistically significant when two-tailed (P < 0.001) after which it declined. Triglycerides also rose significantly after 1 h and reached its maximum after 3 h [0.60 (0.43–0.78)] mmol/L (P < 0.001) before declining. Similarly, after ingestion of olive oil, proneurotensin rose significantly after 1 h and reached its maximum values after 3 h [62 (46–78) pmol/L] (P < 0.001). Triglycerides rose significantly after 2 h and reached its maximum after 3 h 0.32 (0.18–0.45) mmol/L] (P < 0.001). After cream, the AUC for proneurotensin and triglycerides are 49 ± 74 pmol/L x h and 1.5 ± 0.95 mmol/L x h respectively. After olive oil, the AUC for proneurotensin and triglycerides are 196 ± 115 pmol/L x h and 0.71 ± 0.77 mmol/L x h respectively. The AUC of both proneurotensin and triglycerides differed significantly between cream and olive oil (Table The baseline (first visit) clinical characteristics of the study subjects are shown in Table r = 0.49, P = 0.021) and olive oil   nor after olive oil .In contrast to proneurotensin and triglycerides, plasma glucose concentrations significantly decreased at all-time points after lipid ingestion (after both cream and olive oil) as compared to the fasted plasma glucose levels . HoweverHere it is shown for the first time that there is a relationship between the degree of post-lipid ingestion rise in plasma concentration of proneurotensin and triglycerides in humans, as demonstrated by significant correlation between the post lipid load AUC of proneurotensin and the corresponding AUC of triglycerides after two different forms of lipid loads, i.e. cream and olive oil. This finding extends evidence of a pivotal role of NT in intestinal fat absorption from prior animal studies , to suggPreviously, high levels of proneurotensin have been shown to predict obesity, diabetes mellitus and CVD, as well as shows relationship with NAFLD , 14, 19.Of note, most of the previous epidemiological studies have examined the associations between fasting level of proneurotensin and cardiometabolic diseases , 19, 20,The most obvious intervention to reduce postprandial proneurotensin levels is to reduce its trigger, i.e. fat intake. From a pharmacological point of view, the intestinal lipase inhibitor orlistat is of particular interest, which helps in weight reduction by decreasing levels of tumor necrosis factor alpha and interleukin 6  and may Another finding was that glucose significantly decreased after fat ingestion. One explanation for this finding is that neurotensin acts synergistically with glucagon-like peptide-1 (GLP1) and peptide YY (PYY) in the distal small intestines to decrease palatable food intake and to inhibit gastric emptying, thus leading to lower glucose concentration . As a seStrengths of the study include the controlled timing and amounts of ingested fat as well as inclusion of healthy study participants with no medications that would potentially bias lipid metabolism. Limitations are that the exact mechanisms by which post-lipid ingestion rise in proneurotensin affects plasma postprandial triglyceride concentrations cannot be explained and no control diets (i.e. protein or carbohydrate) were included, however, the suggested mechanism emanates from the previous animal experimental studies. The study included a rather small number of normal subjects to see the physiological interplay between fat, proneurotensin and triglycerides. There is no prior study to base the assumption of size of the effect estimate on and therefore power calculation was not done. Finally, ELISA (Enzyme-Linked ImmunoSorbent Assay) was not performed to compare the mature hormone (neurotensin) and triglycerides in this study, instead the more stable proneurotensin was used as a surrogate.In conclusion, proneurotensin increases after an oral lipid load of both cream and olive oil and the degree of rise of post ingestion triglycerides is significantly related to the rise of post ingestion plasma proneurotensin. Findings of this study can be of importance for future treatment strategies for metabolic diseases and CVD through neurotensin related mechanisms.Additional file 1: Table S1. Changes in plasma glucose concentration at all-time points both after cream and after olive oil ingestion.