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The aim of the present study was to characterize DLBCL of BCR distributions, and to screen specific BCR features as potential indicators related to DLBCL prognosis. A total of 13 patients with DLBCL and 5 healthy individuals were enrolled in the present study. In total, two high throughput sequencing methods, BCR repertoire and WES, were used to reveal BCR distributions and gene mutations, respectively. A series of comprehensive analyses were conducted to identify potential complementarity determining region 3 (CDR3) indicators of DLBCL and to analyze their association with clinical characteristics. The relatively higher BCR diversity in patients with DLBCL was associated with shorter progression-free survival (PFS). In addition, the top 10 differential CDR3s were screened as potential DLBCL indicators, and among these, four CDR3s [immunoglobulin heavy chain IGHV1-8/IGHJ6, IGHV3-74/IGHJ6, IGHV4-39/IGHJ3 and IGHV4-34/IGHJ4] exhibited a close relationship with PFS in patients with DLBCL. In further analysis, IGHV4-34/IGHJ4 and IGHV4-39/IGHJ3 were identified to be directly related to tumor metastasis, and IGHV4-39/IGHJ4 was related to hepatitis B virus (HBV) infection. WES data were used to analyze the gene mutations in patients with DLBCL, indicating that two metastasis pathways (‘focal adhesion’ and ‘ECM-receptor interaction’) were involved in characteristic changes of BCR. In conclusion, BCR diversity and usage of 10 specific CDR3s were identified as potential DLBCL indicators in the present study. Clinically, inflammation and HBV infection were the two main factors associated with changes of BCR characteristics. In terms of the molecular mechanism, BCR distributions were closely associated with metastasis-related pathways.

Most cancers are caused by somatic mutations. Some common mutations in the same cancer type can form a "signature" to specifically predict the prognosis or to distinguish it from other cancers. In this study, 710 somatic cell mutations were identified in 142 cases, including digestive, lung and urogenital cancers, and the digestive cancers were further divided into liver, stomach, intestinal, esophageal and cardia cancer. The above mutations were located in 166 genes. In addition, a group of high-frequency mutation genes with specific characteristics were screened to form predictive signatures for each cancer. Verification using TCGA suggested that the signatures could predict the stages, progression-free survival, and overall survival of digestive, intestinal, and liver cancers (P 0.05). The validation cases further confirmed the predictive role of digestive and liver cancers signatures in diagnosis and prognosis. Overall, this study established predictive signatures for different cancer systems and their subtypes. These findings enable a better understanding in cancer genome, and contribute to the personalized diagnosis and treatment.

Pyroptosis and related gasdermin family proteins are important in tumorigenesis of colorectal cancer (CRC). However, the prognosis roles of pyroptosis-related genes (PRGs) and its relation to infiltrates of immune cell in the pathogenesis of CRC remain unclear. By using this investigation, we set up a prognosis gene pattern on the basis of 13 PRGs(AIM2, CASP1, CASP5, CASP6, CASP8, CASP9, ELANE, GPX4, GSDMD, NLRP7, NOD2, PJVK and PRKACA) for CRC patients. A comprehensive bioinformatics analysis was followed to be performed. With good AUC prediction value of ROC curves, the group with high hazard firstly had poor survival prognosis than the group with low hazard. Secondly, we found that PRGs were significantly related to inflammation-associated genes and immune-associated genes in CRC. Then we identified a correlation of PRGs with immune infiltrations in CRC. For instance, abundances of NK cells resting and neutrophils were more in the low hazard group than in the high hazard group. As a whole, this work indicated that PRGs served a critical function in generating heterogeneity of the tumor microenvironment (TME) in CRC. This prognosis PRGs model may give a starting point for early diagnosis and medication use of CRC.

Liver cancer is a common malignant tumor worldwide, which is a serious threat to the health of people. We try to investigate some mutations and clinical indicators as candidate markers for the development of liver cancer through targeted region capture technology combined with next-generation sequencing. We collected peripheral blood and liver cancer tissue samples from 32 liver patients concurrently. The SeqCap EZ Prime Choice Probe was used to perform the targeted enrichment; this probe captures 1,000 known cancer-associated genes. We calculated the tumor mutation burden (TMB) for each patient. The high-frequency mutations and these relative genes were identified. Eventually, survival analysis was performed based on the mutations and clinical indicators. In 32 liver patients, a total of 29 high-frequency mutations were investigated. They were located in 25 genes, which were enriched in 9 cellular components (CCs), 6 molecular functions (MFs), and 21 biological processes (BPs). Among them, EZH2 c.1544A>G and CCND1 c.839A>T had the highest mutation frequency (5/32). In the protein–protein interaction (PPI) network, EZH2-DNMT3A, NOTCH1-CCND1, and ABL1-CCND1 were the top three pairs. The survival analysis showed that there were significant differences in progression-free survival (PFS) and overall survival (OS) between the Karnofsky performance score (KPS) groups. The PFS and OS in the TMB high group were higher than those in the TMB low group. OS and tumor stage had a remarkable relationship. In conclusion, EZH2 c.1544A>G and CCND1 c.839A>T might be potential biomarkers of liver cancer. TMB might be used as a prognosis and survival indicator of liver cancer.

Stomata are leaf pores that regulate gas exchange and water transpiration in response to environmental cues. They also function in innate immunity by limiting pathogen entry through actively closing in so-called stomatal defense. However, roles of stomata in plant disease resistance are not fully elucidated, especially in monocots. Here, we report that non–race specific resistance of the rice abscisic acid-deficient mutant Osaba1 to Xanthomonas oryzae pv. oryzae is due to increased stomatal conductance. Reducing stomatal conductance in the Osaba1 mutant increases its susceptibility to X. oryzae pv. oryzae. Artificial opening of stomata in wild-type plants leads to enhanced resistance to X. oryzae pv. oryzae. The rice mutant es1-1 with constitutively higher stomatal conductance exhibits strong resistance to X. oryzae pv. oryzae. Additionally, Osaba1 and es1-1 are resistant to X. oryzae pv. oryzicola. The data support that open stomata confer postinvasive resistance against bacterial pathogens in rice, and such resistance probably results from decreased leaf water potential. Our findings reveal a novel role of stomata in plant immunity through modulation of leaf water status, which provides physiological insight into the interactions between plant, pathogen, and environment.

Background Identifying gene mutation signatures will enable a better understanding for the occurrence and development of colorectal cancer (CRC), and provide some potential biomarkers for clinical practice. Currently, however, there is still few effective biomarkers for early diagnosis and prognostic judgment in CRC patients. The purpose was to identify novel mutation signatures for the diagnosis and prognosis of CRC. Methods Clinical information of 531 CRC patients and their sequencing data were downloaded from TCGA database (training group), and 53 clinical patients were collected and sequenced with targeted next generation sequencing (NGS) technology (validation group). The relationship between the mutation genes and the diagnosis, pathological type, stage and prognosis of CRC were compared to construct signatures for CRC, and then analyzed their relationship with RNA expression, immunocyte infiltration and tumor microenvironment (TME). Results Mutations of TP53, APC, KRAS, BRAF and ATM covered 97.55% of TCGA population and 83.02% validation patients. Moreover, 57.14% validation samples and 22.06% TCGA samples indicated that patients with mucinous adenocarcinoma tended to have BRAF mutation, but no TP53 mutation. Mutations of TP53, PIK3CA, FAT4, FMN2 and TRRAP had a remarkable difference between I-II and III-IV stage patients (P PIK3CA, LRP1B, FAT4 and ROS1 formed signatures for the prognosis and survival of CRC patients. The mutations of TP53, APC, KRAS, BRAF, ATM, PIK3CA, FAT4, FMN2, TRRAP, LRP1B, and ROS1 formed the signatures for predicting diagnosis and prognosis of CRC. Among them, mutation of TP53, APC, KRAS, BRAF, ATM, PIK3CA, FAT4 and TRRAP significantly reduced their RNA expression level. Stromal score, immune score and ESTIMATE score were lower in patients with TP53, APC, KRAS, PIK3CA mutation compared non-mutation patients. All the 11 gene mutations affected the distributions of immune cells. Conclusion This study constructed gene mutation signatures for the diagnosis, treatment and prognosis in CRC, and proved that their mutations affected RNA expression levels, TME and immunocyte infiltration. Our results put forward further insights into the genotype of CRC.

PurposeThe aim of this study was to screen biomarkers specific to Lynch syndrome (LS) with colorectal cancer (CRC) or endometrial cancer (EC) to explore the mechanisms by which LS develops into CRC and EC and their differences.MethodsDifferentially expressed or differentially methylated genes and differential mutations were identified in 10 LS, 50 CRC, and 50 EC patients from TCGA, and genes overlapping between LS and CRC or EC (named SGs-LCs and SGs-LEs, respectively) were identified. Afterward, we annotated the enriched GO terms and pathways and constructed a protein–protein interaction (PPI) network. Finally, samples from 10 clinical cases with MSI-H/MSS CRC and EC were collected to verify the mutations and their correlations with five LS pathogenic genes in the SGs-LCs and SGs-LEs.ResultsA total of 494 SGs-LCs and 104 SGs-LEs were identified and enriched in 106 and 14 GO terms, respectively. There were great differences in the gene count and enriched terms between SGs-LCs and SGs-LEs. In the PPI network,SST,GCG,SNAP25, andNPYhad the highest degree of connection among the SGs-LCs, andKIF20AandNUF2had the highest degree of connection among the SGs-LE. In the SGs-LCs and SGs-LEs, the genes whose expression levels affected the survival of LS, CRC or EC patients were quite different.ConclusionsCOL11A1was found to be mutated in MSS CRC patients, similar to the mutations ofMSH6.SST,GCG,SNAP25, andNPYmay be biomarkers for the development of LS into CRC, andKIF20AandNUF2may be markers of LS developing into EC.

Background The purpose of this study was to construct a new typing model for diffuse large B-cell lymphoma (DLBCL) patients based on the B-cell receptor (BCR) and explore its potential molecular mechanism. Methods BCR repertoire sequencing and whole-exome sequencing were performed on formalin-fixed paraffin-embedded samples from 12 DLBCL patients. Subsequently, a typing model was built with cluster analysis, and prognostic indicators between the two groups were compared to verify the typing model. Then, mutation and bioinformatics analyses were conducted to investigate the potential biomarkers of prognostic differences between the two groups. Results Based on BCR sequencing data, we divided patients into two clusters (cluster 1 and cluster 2); this classification differed from the traditional typing method (GCB and non-GCB), in which cluster 1 included some non-GCB patients. The progression-free survival (PFS), overall survival (OS), metastasis and Shannon diversity index of IGH V-J and survival after chemotherapy were significantly different (P FTSJ3, MAGED2, and ODF3L2 were the specific mutated genes in all patients in cluster 2, and these genes could be considered critical to the different prognoses of the two clusters of DLBCL patients. Conclusion We constructed a new typing model of DLBCL based on BCR repertoire sequencing that can better predict the survival time after chemotherapy. FTSJ3, MAGED2, and ODF3L2 may represent key genes for the difference in prognosis between the two clusters.

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High expression of PD-L1 marks the poor prognosis of pancreatic ductal adenocarcinomas (PDAC). However, the regulatory mechanism of PD-L1 remains elusive. We recently reported that cancer Forkhead box protein 3 (Cancer-FOXP3 or C-FOXP3) promoted immune evasion of PDAC by recruiting Treg cells into PDAC via upregulation of CCL5. In this study, we confirmed that PD-L1 was overexpressed in PDAC samples from two independent cohorts of patients with radical resection. Moreover, C-FOXP3 was colocalized and correlated with the expression of PD-L1 in tumor cells at the mRNA and protein levels, and this finding was confirmed by the The Cancer Genome Atlas (TCGA) database. Chromatin immunoprecipitation (ChIP) revealed that C-FOXP3 directly bound to the promoter region of PD-L1 in pancreatic cancer cells. Furthermore, overexpression of C-FOXP3 activated the luciferase reporter gene under the control of the PD-L1 promoter. However, mutation of the binding motif-a completely reversed the luciferase activity. In addition, C-FOXP3-induced upregulation of PD-L1 effectively inhibited the activity of CD8+ T cells. Based on our recent finding that the CCL-5 antibody achieved a better response to PDAC models with high C-FOXP3 levels, we further demonstrated that the PD-L1 antibody strengthened the antitumor effect of CCL-5 blockade in xenograft and orthotopic mouse models with high C-FOXP3 levels. In conclusion, C-FOXP3 directly activates PD-L1 and represents a core transcription factor that mediates the immune escape of PDAC. Combined blockade of PD-L1 and CCL-5 may provide an effective therapy for patients with PDAC that have high C-FOXP3 levels.

8059 Background: Lymphatic system cancer is characterized by high heterogeneity in histology and clinical manifestations, B-cell antigen receptor (BCR) plays a vital role in anti-tumor immune responses. This study aimed to compare the BCR repertoire and identify some specific immune markers for different pathological lymphomas. Methods: 5 pathological types of non-Hodgkin's lymphoma (T-LBL/ALL, PTCL-NOS, B-MCL, B-FL, DLBCL) were collected, with reactive lymph node (RLN) hyperplasia as control. All patients were tested by high-throughput immunohistochemical sequencing (HTS-IR) to analyze the correlation between B-cell immunohistochemistry and clinical indicators, and constructed new strategy typing and overall survival (OS) predicted models for lymphomas. Results: The BCR repertoire had the highest diversity in RLN, followed by T-LBL/ALL, PTCL-NOS, DLBCL, B-MCL and B-FL. The diversity of BCR repertoire and similarity of B cell antigens were higher in B-MCL and B-FL patients. Similar to RLN, T-LBL/ALL and PTCL-NOS had broad and diverse V-J pairs, and rare in B-MCL, B-FL and DLBCL. RLN patients were with the highest average number of amino acids, followed by T-LBL/ALL, DLBCL, PTCL-NOS, B-MCL and B-FL. The expressed amino acid sequencing of ARDLIALDY, ARRPGSFDY, ARDIAGWGAVAGLLGRAYYGMDV, and ARDGPYGGNSVEYFQH were markedly different among 5 groups. Patients tended to recurrence expressed ASLDSSPSGFC, ARGMTTVTTAPNY, ARVPLYDDQNINDV and AGGVGGYDWGSYYFDY (P = 0.01605, 0.02869, and 0.01569), and prone to metastasis with expressions of ARVKEFYGILTGYDY, AHSIIGSSWYNWFDP and VRDGGWQSNNWLGFDV (P = 0.04259, 0.0450 and 0.0481). For all patients, 18 (7 negative, 11 positive) and 12 (10 negative, 2 positive) IGH V-J pairs were respectively associated with lymphoma recurrence and metastasis. The top 3 most significant pairs were IGHV7-4-1_IGHJ4, IGHV3-53_IGHJ5 and IGHV3-7_IGHJ5 bound up with recurrence (P = 0.0019, 0.0020 and 0.0021), and IGHV3-74_IGHJ1, IGHV1-69_IGHJ3 and IGHV1-2_IGHJ1 related to metastasis (P = 0.0022, 0.010 and 0.019). The accuracy of typing model in training and test sets was 78.125% (25/32) and 60% (6/10), respectively. The OS model can predict long (≥ 24 months) or short ( < 24 months) OS. Conclusions: Our study identified new biomarkers, constructed novel lymphoma typing model and OS predicted model based on B cell repertoire. It provides a comprehensive understanding of immune response, and contributes to the diagnosis and prognosis of non-Hodgkin's lymphoma.

e16113 Background: Like most cancers, early diagnosis of colorectal cancer (CRC) contributes to a good prognosis. Recently, some studies indicated that mutation gene signatures have good performances in predicting the treatment and prognosis in CRC patients. However, incomplete understanding of the genotype is not conducive to proper treatment. The objective was to give further genetic insights into CRC and provide more comprehensive references for clinical practice. Methods: In the training group, the whole genome sequencing (WGS), clinical, and demographic data of 531 patients were downloaded from The Cancer Genome Atlas (TCGA). The validation group contained 53 patients, which were collected in Tianjin Medical University Cancer Institute and Hospital (Tianjin, China) from April 2014 to November 2018. The fresh tissues collected within 24 hours after operation were used to extract genomic DNA, and then sequenced with targeted next-generation sequencing (NGS) technology to examine somatic mutations and analyze relevant gene concerning clinical indicators. The correlation between gene variation distribution and various indexes such as cancer species, stage, total survival period, sex, age and race were evaluated. Results: A total of 44 mutant genes with mutation frequencies above 5% were found in both TCGA cases and validated cases. Mutations of TP53, APC, KRAS, BRAF and ATM covered 97.55% of TCGA population and 83.02% validation patients, which were proved to be associated with the development of CRC and could be used as a diagnostic signature. Importantly, mutations of TP53, PIK3CA, FAT4, FMN2 and TRRAP had a remarkable difference between early (I/II) and advanced (III/IV) stage patients (P < 0.0001). Besides, we also confirmed that PIK3CA, LRP1B, FAT4 and ROS1 were a mutated gene signature for the prognosis, LRP1B portended to a higher of recurrence and shorter progression-free survival (PFS); mutation of FAT4 portended to a lower of recurrence and longer PFS, which could predict the recurrence and survival of CRC patients. Conclusions: We have indicated gene mutation signatures related to the diagnosis, pathological classification, staging and prognosis in CRC, which provides further insights into the study of CRC genotype. It is helpful to further improve the personalized diagnosis and treatment.

e21652 Background: EGFR-TKI application is directed by the situation of mutations of epidermal growth factor receptor, in addition, the mutations of ALK, ROS-1,C-met, BRAF, Her-2, RET,NTRK1, PI3K, MEKI have potential effects on guiding treatment. This study aimed to investigate the uncommon and insensitive mutations and their effect on prognosis in non-small cell lung cancer (NSCLC) patients treated with EGFR-TKI. Methods: 21 NSCLC patients treated with EGFR-TKIs were enrolled, 9 with Gefitinib, 5 with Osimertinib and 7 with Icotinib. All patients were detected with targeted NGS 1000 gene panel. Afterwards, the EGFR sensitive mutation, uncommon mutation and mutations accompanied EGFR T790M were analyzed. Furthermore, the correlations of gene mutation with metastasis, OS and PFS were analyzed. SPSS 21.0 was used for statistical analysis, t test for continuous variables and χ2 test for classified variables. Results: In all the patients, EGFR p.[T790M] and p.[L858R] mutations were detected in 4.76% and 23.81% of the patients, respectively. In the 5 patients treated with Osimertinib, one had EGFR p.[T790M] mutation and curative effects was stable disease (SD). The mutations accompanied EGFR T790M included SMARCA4 p.[K1390Q] (2.23%), DNMT3A p.[F755S] (2.17%), MTOR p.[Y1450*] (2.15%), TPMT p.[L182R] (1.84%), etc. For the other four patients treated with Osimertinib, two of them were partial response (PR) and two were SD, accompanied with mutations such as EGFR p.[L858R] (55.00%), ARID1A p.[P16del] (2.83%), TP53 p.[R248W] (21.73%) and AR p.[Q60L] (4.24%). In addition, among all the mutations, 18 uncommon mutations were detected, in which ATM (23.26%), AR (23.26%) and MTOR (37.21%) were markedly associated with OS. Besides, the mutation frequency of 67 genes, such as ABCG2, AURKA, EPHA3 and ATR were correlated with OS. The mutation frequency of FGFR1 was significantly different between the patients with and without metastasis. The mutations of 22 genes, for instance ABCG2 p.[P21L], FAT1 p.[L398F], GNAS p.[F226V] and KMT2C p.[N2842Ilefs], were correlated with PFS during the treatment. Conclusions: EGFR-TKI has similar curative effects in EGFR p.[T790M] negative patients. Gene mutations, including uncommon mutations and EGFR insensitive mutations, can significantly affect the prognosis in NSCLC patients treated with EGFR-TKIs.

MicroRNAs (miRNAs) are endogenous non-coding small RNAs that silence genes through mRNA degradation or translational inhibition. The phytohormone abscisic acid (ABA) is essential for plant development and adaptation to abiotic and biotic stresses. In Arabidopsis, miRNAs are implicated in ABA functions. However, ABA-responsive miRNAs have not been systematically studied in rice. Here high throughput sequencing of small RNAs revealed that 107 miRNAs were differentially expressed in the rice ABA deficient mutant, Osaba1. Of these, 13 were confirmed by stem-loop RT-PCR. Among them, miR1425-5P, miR169a, miR169n, miR390-5P, miR397a and miR397b were up-regulated, but miR162b reduced in expression in Osaba1. The targets of these 13 miRNAs were predicted and validated by gene expression profiling. Interestingly, the expression levels of these miRNAs and their targets were regulated by ABA. Cleavage sites were detected on 7 of the miRNA targets by 5'-Rapid Amplification of cDNA Ends (5'-RACE). Finally, miR162b and its target OsTRE1 were shown to affect rice resistance to drought stress, suggesting that miR162b increases resistance to drought by targeting OsTRE1. Our work provides important information for further characterization and functional analysis of ABA-responsive miRNAs in rice.

Plants have established a complicated immune defense system during co-evolution with pathogens. The innate immune system of plants can be generally divided into two levels. One, named PAMP-triggered immunity (PTI), is based on the recognition of pathogen-associated molecular patterns by pattern-recognition receptors, which confers resistance to most pathogenic microbes. The other begins in cytoplasm and mainly relies on recognition of microbial effectors by plant resistance proteins in direct or indirect ways, which then initiates potent defense responses. This process, termed effector-triggered immunity (ETI), is necessary for defense against pathogens that can secret effectors to suppress the first level of immunity. Activation of these two layers of immunity in plant is based on distinguishing and recognition of "self" and "non-self" signals. Recognition of "non-self" signals can activate signal cascades, such as MAPK cascades, which will then induce defense gene expression and corresponding defense responses. In this review, we focused on underlying molecular mechanisms of plant-pathogen interactions and the latest advances of the PTI and ETI signaling network.

Leaf rolling is considered an important agronomic trait in rice (Oryza sativa) breeding. To understand the molecular mechanism controlling leaf rolling, we screened a rice T-DNA insertion population and isolated the outcurved leaf1 (oul1) mutant showing abaxial leaf rolling. The phenotypes were caused by knockout of Rice outermost cell-specific gene5 (Roc5), an ortholog of the Arabidopsis (Arabidopsis thaliana) homeodomain leucine zipper class IV gene GLABRA2. Interestingly, overexpression of Roc5 led to adaxially rolled leaves, whereas cosuppression of Roc5 resulted in abaxial leaf rolling. Bulliform cell number and size increased in oul1 and Roc5 cosuppression plants but were reduced in Roc5-overexpressing lines. The data indicate that Roc5 negatively regulates bulliform cell fate and development. Gene expression profiling, quantitative polymerase chain reaction, and RNA interference (RNAi) analyses revealed that Protodermal Factor Like (PFL) was probably down-regulated in oul1. The mRNA level of PFL was increased in Roc5-overexpressing lines, and PFL-RNAi transgenic plants exhibit reversely rolling leaves by reason of increases of bulliform cell number and size, indicating that Roc5 may have a conserved function. These are, to our knowledge, the first functional data for a gene encoding a homeodomain leucine zipper class IV transcriptional factor in rice that modulates leaf rolling.

CdS:Cu thin films were prepared using a vacuum co-evaporation technique. The Hall measurements indicate that the conductivity characteristic of CdS thin films transformed from highly compensated in as-grown or weakly annealed materials to p-type conductive in strongly annealed materials. X-ray diffraction spectra show that as-deposited thin films were the hexagonal phase of CdS except the presence of copper for high Cu doping and the diffraction peaks of Cu disappeared after annealing. From the X-ray photoelectron spectroscopy we found the ionization of Cu atoms and the formation of an acceptor level. In situ dark conductivity in vacuum as-deposited CdS:Cu was performed in the temperature range between 27 and 250 °C. An abnormal temperature dependence of conductivity was observed in medium and heavily Cu-doped films. The formation of a p-type material at a certain temperature was also studied by the hot probe measurements, which indicates a complex compensation process in the Cu-doped CdS films.