Potentially functional genetic variants of the notch signaling pathway genes predict survival of Chinese patients with esophageal squamous cell carcinoma.

BACKGROUND: The Notch signaling pathway is involved in the progression of esophageal squamous cell carcinoma (ESCC), although the roles of single nucleotide polymorphisms (SNPs) of the Notch signaling pathway genes in the process remain unknown. METHODS: The present study included 1009 patients with histopathologically diagnosed ESCC at Fudan University Shanghai Cancer Center. Two-stage multivariate Cox proportional hazards regression analysis was used to estimate associations between 13,248 SNPs in 103 Notch signaling pathway genes and overall survival of the patients. RESULTS: We found that overall survival of the patients was significantly associated with genotypes of HDAC9 rs1729318 (AT+TT vs. AA: hazard ratio = 1.44, 95% confidence interval = 1.16-1.80, pcombined  = 0.001) and HDAC9 rs1339555498 (GT + TT vs. GG: hazard ratio = 1.38, 95% confidence interval = 1.10-1.74, pcombined = 0.005). Further receiver operator characteristic (ROC) curve analysis indicated that the model with both available clinical factors and these two SNPs improved the area under the ROC curve compared to the model with clinical factors only (1-year: 0.66 vs. 0.64, p = 0.034). Additional expression quantitative trait loci analysis showed that the rs1729318 T variant genotypes were associated with increased mRNA expression levels of HDAC9 in normal esophageal muscular tissue (p = 0.003). CONCLUSIONS: The results suggest that these two potential functional SNPs on HDAC9 may serve as biomarkers for predicting survival of ESCC patients. However, further studies are needed to confirm these findings.

Potentially functional variants in nucleotide excision repair pathway genes predict platinum treatment response of Chinese ovarian cancer patients.

Acquired platinum resistance impedes successful treatment of epithelial ovarian cancer (EOC), and this resistance may be associated with inherited DNA damage-repair response. In the present study, we performed a two-phase analysis to assess associations between 8191 single-nucleotide polymorphisms within 127 genes of nucleotide excision repair pathway from a genome-wide association study dataset and platinum treatment response in 803 Han Chinese EOC patients. As a result, we identified that platinum-based chemotherapeutic response was associated with two potentially functional variants MNAT1 rs2284704 T>C [TC + CC versus TT, adjusted odds ratio (OR) = 0.89, 95% confidence interval (CI) = 0.83-0.95 and P = 0.0005] and HUS1B rs61748571 A>G (AG + GG versus AA, OR = 1.10, 95% CI = 1.03-1.18 and P = 0.005). Compared with the prediction model for clinical factors only, models incorporating HUS1B rs61748571 [area under the curve (AUC) 0.652 versus 0.672, P = 0.026] and the number of unfavorable genotypes (AUC 0.652 versus 0.668, P = 0.040) demonstrated a significant increase in the AUC. Further expression quantitative trait loci analysis suggested that MNAT1 rs2284704 T>C significantly influenced mRNA expression levels of MNAT1 (P = 0.003). These results indicated that MNAT1 rs2284704 T>C and HUS1B rs61748571 A>G may serve as potential biomarkers for predicting platinum treatment response of Chinese EOC patients, once validated by further functional studies.

Functional genetic variants of RUVBL1 predict overall survival of Chinese patients with epithelial ovarian cancer.

To date, the 5-year overall survival of epithelial ovarian cancer (EOC) remains poor. Because studies suggest that RUVBL1 may be a chemotherapeutic target for the treatment of cancer, in this study, therefore, we investigated the role of potentially functional single nucleotide polymorphisms (SNPs) of RUVBL1 in the survival of Chinese patients with EOC, and we subsequently performed functional prediction and validation of the identified significant SNPs. We found that RUVBL1 rs1057156 A>G and RUVBL1 rs149652370 A>G were associated with survival of EOC patients in the multivariate Cox proportional hazards regression analysis. Specifically, the RUVBL1 rs149652370 AG genotype was associated with a shorter progression-free survival ([adjusted hazards ratio (HR)] = 3.32, 95% confidence interval (CI) = 1.76-6.25 and P = 2.01E-04), compared with the AA genotype. The RUVBL1 rs1057156 AG (only nine had GG) genotype was also associated with a poor overall survival (adjusted HR = 1.73, 95% CI = 1.19-2.52, P = 0.004), compared with the AA genotype. Further experiments showed that the RUVBL1 rs1057156 A>G change lowered its binding affinity to microRNA-4294 and led to upregulation of the RUVBL1 expression. We further found that overexpression of RUVBL1 promoted cell proliferation and metastatic potential. Overall, RUVBL1 enhanced EOC cell proliferation, invasion and migration presumably by stimulating the process of glycolysis. Thus, this study provides evidence that functional variants of RUVBL1 may regulate its gene expression, a possible mechanism affecting survival of EOC patients and that RUVBL1 may be a potential chemotherapeutic target for the treatment of EOC patients.

microRNA-messenger RNA regulatory network of esophageal squamous cell carcinoma and the identification of miR-1 as a biomarker of patient survival.

Emerging evidence indicates that microRNAs (miRNAs) play an important role in tumor carcinogenesis and progression by targeting gene expression. The goal of this study was to comprehensively analyze the vital functional miRNAs and their target genes in esophageal squamous cell carcinoma (ESCC) and to explore the clinical significance and mechanisms of miR-1 in ESCC. First, the miRNA and messenger RNA (mRNA) expression profiles of ESCC were determined with microarray technology. Using an integrated analysis of miRNAs and their target genes with multistep bioinformatics methods, the miRNA-mRNA regulatory network in ESCC was constructed. Next, miR-1 expression in 292 ESCC patients and its relationship with clinicopathological features and prognosis were detected by in situ hybridization. Furthermore, the biological functions of miR-1 were determined with in vitro and in vivo functional experiments. Finally, real-time quantitative reverse transcription polymerase chain reaction, Western blot analysis, and luciferase reporter assays were performed to verify the target genes of miR-1. In this study, 67 miRNAs and 2992 genes were significantly differentially expressed in ESCC tissues compared with their expression in adjacent normal tissues, and an miRNA-mRNA regulatory network comprising 59 miRNAs and 162 target mRNAs was identified. Low miR-1 expression was correlated with pathological T stage, lymph node metastasis, vessel invasion, and poor clinical outcome. miR-1 suppressed ESCC cell proliferation and invasion and promoted ESCC cell apoptosis. Fibronectin 1 (FN1) was verified as a direct target of miR-1. Taken together, the present results suggest that miR-1 may be a valuable prognostic predictor for ESCC, and the miR-1/FN1 axis may be a therapeutic target.

Functional variant of MTOR rs2536 and survival of Chinese gastric cancer patients.

We previously reported that some single nucleotide polymorphisms (SNPs) of candidate genes involved in the MTOR complex1 (MTORC1) were associated with risk of gastric cancer (GCa). In the present study, we further evaluated associations of eight potentially functional SNPs of MTOR, MLST8 and RPTOR with survival of 1002 GCa patients and also investigated molecular mechanisms underlying such associations. Specifically, we found that the MTOR rs2536 C allele at the microRNA binding site was independently associated with a 26% reduction of death risk (HR = 0.74, 95% CI = 0.57-0.96, p = 0.022). The results remained noteworthy with a prior false positive probability of 0.1. Genotype-phenotype correlation analysis in 144 patients' adjacent normal gastric tissue samples revealed that the MTOR expression levels were lower in rs2536 TC/CC carriers than that in wild-type TT carriers (p = 0.043). Dual luciferase assays revealed that the rs2536 C allele had a higher binding affinity to microRNA-150, leading to a decreased transcriptional activity of MTOR, compared to the rs2536 T allele. Further functional analysis revealed that MTOR knockdown by small interference RNA impaired proliferation, migration, and invasion ability in GCa cell lines. In conclusion, The MTOR rs2536 T > C change may be a biomarker for survival of Chinese GCa patients, likely by modulating microRNA-induced gene expression silencing. Additional studies are needed to validate our findings.

Variants in Notch signalling pathway genes, PSEN1 and MAML2, predict overall survival in Chinese patients with epithelial ovarian cancer.

To identify genetic variants in Notch signalling pathway genes that may predict survival of Han Chinese patients with epithelial ovarian cancer (EOC), we analysed a total of 1273 single nucleotide polymorphisms (SNPs) within 75 Notch genes in 480 patients from a published EOC genomewide association study (GWAS). We found that PSEN1 rs165934 and MAML2 rs76032516 were associated with overall survival (OS) of patients by multivariate Cox proportional hazards regression analysis. Specifically, the PSEN1 rs165934 AA genotype was associated with a poorer survival (adjusted hazards ratio [adjHR] = 1.41, 95% CI = 1.07-1.84, and P = .014), compared with the CC + CA genotype, while MAML2 rs76032516 AA + AC genotypes were associated with a poorer survival (adjHR = 1.58, 95% CI = 1.16-2.14, P = .004), compared with the CC genotype. The combined analysis of these two SNPs revealed that the death risk increased as the number of unfavourable genotypes increased in a dose-dependent manner (Ptrend < .001). Additionally, the expression quantitative trait loci analysis revealed that the SNP rs165932 in the rs165934 LD block (r2 = .946) was associated with expression levels of PSEN1, which might be responsible for the observed association with SNP rs165934. The associations of PSEN1 rs165934 and MAML2 rs76032516 of the Notch signalling pathway genes with OS in Chinese EOC patients are novel findings, which need to be validated in other large and independent studies.

An ERCC4 regulatory variant predicts grade-3 or -4 toxicities in patients with advanced non-small cell lung cancer treated by platinum-based therapy.

Platinum-based chemotherapy (PBC) in combination with the 3rd generation drugs is the first-line treatment for patients with advanced non-small cell lung cancer (NSCLC); however, the efficacy is severely hampered by grade 3-4 toxicities. Nucleotide excision repair (NER) pathway is the main mechanism of removing platinum-induced DNA adducts that contribute to the toxicity and outcome of PBC. We analyzed data from 710 Chinese NSCLC patients treated with PBC and assessed the associations of 25 potentially functional single nucleotide polymorphisms (SNPs) in nine NER core genes with overall, gastrointestinal and hematologic toxicities. Through a two-phase study, we found that ERCC4 rs1799798 was significantly associated with overall and gastrointestinal toxicities [all patients: GA/AA vs. GG, odds ratio (OR)adj =1.61 and 2.35, 95% confidence interval (CI)=1.11-2.33 and 1.25-4.41, and Padj =0.012 and 0.008, respectively]. Our prediction model for the overall toxicity incorporating rs1799798 demonstrated a significant increase in the area under the curve (AUC) value, compared to that for clinical factors only (all patients: AUC = 0.61 vs. 0.59, 95% CI = 0.57-0.65 vs. 0.55-0.63, P = 0.010). Furthermore, the ERCC4 rs1799798 A allele was associated with lower ERCC4 mRNA expression levels according to the expression quantitative trait loci (eQTL) analysis. Our study provided some new clue in future development of biomarkers for assessing toxicity and outcomes of platinum drugs in lung cancer treatment.

Genetic variants of JNK and p38α pathways and risk of non-small cell lung cancer in an Eastern Chinese population.

The JNK and p38α pathways play an important role in carcinogenesis. Therefore, we hypothesize that single nucleotide polymorphisms (SNPs) of genes involved in these pathways are associated with risk of lung cancer. We first selected and genotyped 11 independent SNPs of the JNK and p38α pathway-related genes in a discovery set of 1,002 non-small cell lung cancer (NSCLC) cases and 1,025 cancer-free controls of Eastern Chinese. Then, we validated those significant SNPs in a replication set of 1,333 NSCLC cases and 1,339 cancer-free controls of Eastern Chinese. Multifactor dimensionality reduction (MDR) and classification and regression tree (CART) analyses were used to identify interactions between significant SNPs and other covariates. In both discovery and replication as well as their pooled analysis, carriers of GADD45G rs8252T variant genotypes had a significantly lower risk of NSCLC (adjusted OR = 0.81 and 0.79, 95% CI = 0.72-0.92 and 0.64-0.99 and p = 0.001 and 0.040 for dominant and recessive genetic models, respectively) and carriers of MAP2K7 rs3679T variant genotypes had an increased risk of NSCLC (adjusted OR = 1.19 and 1.29, 95% CI = 1.05-1.34 and 1.09-1.54 and p = 0.005 and 0.004 for dominant and recessive genetic models, respectively). Furthermore, rs8252 variant CT/TT carriers showed significantly higher levels of GADD45G mRNA expression than CC carriers in the target tissues. We observed some evidence of interactions between rs8252 genotypes and sex in NSCLC risk. These results indicate that GADD45G rs8252 and MAP2K7 rs3679 SNPs may be susceptibility biomarkers for NSCLC in Eastern Chinese populations.

The spectrum of BRCA mutations and characteristics of BRCA-associated breast cancers in China: Screening of 2,991 patients and 1,043 controls by next-generation sequencing.

To characterize the prevalence of BRCA mutations and characteristics of BRCA carriers in China and to update the clinical recommendations for BRCA testing, we conducted a wide screen for BRCA mutations using next-generation sequencing (NGS). A total of 4,034 Chinese subjects were screened for germline BRCA1/2 mutations, including 2,991 breast cancer patients and 1,043 healthy individuals from the community enrolled as controls. We developed an NGS-based approach to perform BRCA1/2 screening. BRCA mutations were identified in 9.1% (232/2,560) of cases with at least one risk factor, in 3.5% (15/431) of sporadic patients and in 0.38% (4/1,043) of healthy controls. The mutation frequency ranged from 8.9 to 15.2% in cohorts with a single risk factor to 16.6-100% in groups with multiple risk factors. We identified 70 novel BRCA mutations. A high frequency of BRCA1 c.5470_5477del was detected, accounting for 13.9% (16/115) of the BRCA1 mutations detected in our study. Clinical characteristics such as family history, invasive carcinoma, negative human epidermal growth factor receptor 2 (HER2), high Ki67 index, lymph node status, and high tumour grade were closely related to BRCA mutations. BRCA2 carriers had poorer disease-free survival among HER2- or hormone receptor-positive patients (hazard ratio = 1.892; 95% confidence interval: 1.132-3.161; p = 0.013). This study shows that BRCA mutation carriers could be frequently identified among breast cancer patients with multiple risk factors. Importantly, we established an NGS-based pipeline for BRCA1/2 testing in clinical practice and strongly suggest that breast cancer patients of premier- and moderate-grade risks receive BRCA1/2 mutations testing in China.

Plasma microRNA-based signatures to predict 3-year postoperative recurrence risk for stage II and III gastric cancer.

Our aim was to identify plasma microRNA (miRNA)-based signatures to predict 3-year postoperative recurrence risk for patients with stage II and III gastric cancer (GC), so as to provide insights for individualized adjuvant therapy. Plasma miRNA expression was investigated in three phases, involving 407 patients recruited from three centers. ABI miRNA microarray and TaqMan Low Density Array were adopted in the discovery phase to identify potential miRNAs. Quantitative reverse-transcriptase polymerase chain reaction was used to assess the expression of selected miRNAs. Logistic regression models were constructed in the training set (n = 170) and validated in the validation set (n = 169). Receiver operating characteristic analyses, survival analyses and subgroup analyses were further used to assess the accuracy of the models. We identified a 7 miRNA classifier and 7miR + pathological factors index that provided high predictive accuracy of GC recurrence (area under the curve = 0.725 and 0.841 in the training set; and 0.627 and 0.771 in the validation set). High-risk patients defined by the signatures had significantly shorter disease-free survival and overall survival than low-risk patients. The 7 miRNA classifier is an independent prognostic factor, and could add predictive value to traditional prognostic factors. Subgroup analyses revealed the satisfactory performance persisted regardless of stage, and the two models both displayed high accuracy in stage IIA patients. In conclusion, identified microRNA signature may potentially provide some additional benefit for prediction of disease recurrence in patients with stage II and III GC.

Functional genetic variants of XRCC4 and ERCC1 predict survival of gastric cancer patients treated with chemotherapy by regulating the gene expression.

DNA repair protects genomic integrity and may modulate chemotherapy efficacy. Few large-scale studies have evaluated predictive roles of genetic variants of DNA repair genes in survival of Chinese gastric cancer (GCa) patients treated with chemotherapy. Here, we assessed the roles of 35 single nucleotide polymorphisms (SNPs) in DNA repair genes in survival of 1002 GCa patients, of whom 694 received chemotherapy and 308 did not. Among patients receiving chemotherapy, the ERCC1 rs2298881A allele was associated with a better survival [hazards ratio (HR) = 0.82, 95% confidence interval (CI) = 0.69-0.98; P = 0.03], whereas two XRCC4 SNPs were associated with a worse survival (HR = 1.26, 95% CI = 1.03-1.54 for the rs10040363G allele, P = 0.02; and HR = 1.30, 95% CI = 1.06-1.59 for the rs2075685T allele, P = 0.01). These three SNPs were unique survival predictors for patients treated with chemotherapy (P < 0.05 for all) but not for patients without chemotherapy (P > 0.05 for all), suggesting that they modulated chemotherapy efficacy. Patients who received chemotherapy and had haplotypes with at least one death-risk allele in XRCC4 had a poor survival, and the trend for an increase in the number of death-risk alleles adversely affecting the survival was also observed in an allelic dose-dependent manner (Ptrend = 0.001). Further functional analysis revealed that the death-risk alleles up-regulated the gene expression, leading to a worse survival as suggested by our meta-analysis pooling both mRNA microarray data from the GEO database and published data (ERCC1: HR = 1.31 [1.08-1.58]; P = 0.006). These functional genetic variants may independently or jointly affect survival in chemotherapy-treated GCa patients by modulating the gene expression in the tumors.

Interleukin-11 Receptor Is a Candidate Target for Ligand-Directed Therapy in Lung Cancer: Analysis of Clinical Samples and BMTP-11 Preclinical Activity.

We previously isolated an IL-11-mimic motif (CGRRAGGSC) that binds to IL-11 receptor (IL-11R) in vitro and accumulates in IL-11R-expressing tumors in vivo. This synthetic peptide ligand was used as a tumor-targeting moiety in the rational design of BMTP-11, which is a drug candidate in clinical trials. Here, we investigated the specificity and accessibility of IL-11R as a target and the efficacy of BMTP-11 as a ligand-targeted drug in lung cancer. We observed high IL-11R expression levels in a large cohort of patients (n = 368). In matching surgical specimens (i.e., paired tumors and nonmalignant tissues), the cytoplasmic levels of IL-11R in tumor areas were significantly higher than in nonmalignant tissues (n = 36; P = 0.003). Notably, marked overexpression of IL-11R was observed in both tumor epithelial and vascular endothelial cell membranes (n = 301; P < 0.0001). BMTP-11 induced in vitro cell death in a representative panel of human lung cancer cell lines. BMTP-11 treatment attenuated the growth of subcutaneous xenografts and reduced the number of pulmonary tumors after tail vein injection of human lung cancer cells in mice. Our findings validate BMTP-11 as a pharmacologic candidate drug in preclinical models of lung cancer and patient-derived tumors. Moreover, the high expression level in patients with non-small cell lung cancer is a promising feature for potential translational applications.

FGF-1/-3/FGFR4 signaling in cancer-associated fibroblasts promotes tumor progression in colon cancer through Erk and MMP-7.

Cancer-associated fibroblasts (CAFs), as the activated fibroblasts in the tumor stroma, are important modifiers of tumour progression. In the present study, we observed that azoxymethane and dextran sodium sulfate treatments induced increasingly severe colorectal mucosal inflammation and the intratumoural accumulation of CAFs. Fibroblast growth factor (FGF)-1 and FGF-3 were detected in infiltrating cells, and FGFR4, the specific receptor for FGF-1 and FGF-3, was detected in colon cancer tissues. The phosphorylation of FGFR4 enhanced the production of metalloproteinase (MMP)-7 and mitogen-activated protein kinase kinase (Mek)/extracellular signal-regulated kinase (Erk), which was accompanied by excessive vessel generation and cell proliferation. Moreover, we separated CAFs, pericarcinoma fibroblasts (PFs), and normal fibroblasts (NFs) from human colon tissue specimens to characterize the function of CAFs. We observed that CAFs secrete more FGF-1/-3 than NFs and PFs and promote cancer cell growth and angiogenesis through the activation of FGFR4, which is followed by the activation of Mek/Erk and the modulation of MMP-7 expression. The administration of FGF-1/-3-neutralizing antibodies or the treatment of cells with FGFR4 siRNA or the FGFR4 inhibitor PD173074 markedly suppressed colon cancer cell proliferation and neovascularization. These observations suggest a crucial role for CAFs and FGF signaling in the initiation and progression of colorectal cancer. The inhibition of the FGF signaling pathway may be a useful strategy for the treatment of colon cancer.

Genetic variations in the mTOR gene contribute toward gastric adenocarcinoma susceptibility in an Eastern Chinese population.

BACKGROUND AND AIM: Genetic variants in the mammalian target of rapamycin (mTOR) gene have become an interesting topic for the study of genetic susceptibility to cancer, but their associations with the risk of gastric cancer have not been fully investigated. MATERIALS AND METHODS: In a hospital-based case-control study of 1002 gastric cancer patients and 1003 cancer-free controls, we genotyped four potentially functional single nucleotide polymorphisms (SNPs) (rs1034528G>C, rs17036508T>C, rs3806317A>G, and rs2295080T>G) of mTOR and assessed their associations with the risk of gastric cancer using univariate and multivariate logistic regression analyses. We also used the multifactorial dimension reduction analysis to explore possible interactions and the false-positive report probabilities to assess significant findings. RESULTS: We found that rs1034528 CG/CC and rs3806317 GA/GG variant genotypes were associated with an increased risk of gastric cancer under a dominant model (adjusted odds ratio=1.27 and 1.22, respectively). In the combined analysis of all four SNPs under investigation, patients with 3-4 risk genotypes of mTOR had a significantly increased risk of gastric cancer (adjusted odds ratio=1.46, 95% confidence interval=1.19-1.79) compared with those with 0-2 risk genotypes. Stratified analysis indicated that this risk was more pronounced in subgroups of men, never-smokers, never-drinkers, and clinical stages III+IV. The multifactorial dimension reduction analysis suggested some evidence of interactions between the combined genotypes and other risk factors for gastric cancer. CONCLUSION: These findings suggest that potentially functional SNPs of mTOR may individually or collectively contribute to the risk of gastric cancer. Larger studies with diverse ethnic populations are warranted to validate our findings.

Polymorphisms in the kinesin-like factor 1 B gene and risk of epithelial ovarian cancer in Eastern Chinese women.

The kinesin-like factor 1 B (KIF1B) gene plays an important role in the process of apoptosis and the transformation and progression of malignant cells. Genetic variations in KIF1B may contribute to risk of epithelial ovarian cancer (EOC). In this study of 1,324 EOC patients and 1,386 cancer-free female controls, we investigated associations between two potentially functional single nucleotide polymorphisms in KIF1B and EOC risk by the conditional logistic regression analysis. General linear regression model was used to evaluate the correlation between the number of variant alleles and KIF1B mRNA expression levels. We found that the rs17401966 variant AG/GG genotypes were significantly associated with a decreased risk of EOC (adjusted odds ratio (OR) = 0.81, 95 % confidence interval (CI) = 0.68-0.97), compared with the AA genotype, but no associations were observed for rs1002076. Women who carried both rs17401966 AG/GG and rs1002076 AG/AA genotypes of KIF1B had a 0.82-fold decreased risk (adjusted 95 % CI = 0.69-0.97), compared with others. Additionally, there was no evidence of possible interactions between about-mentioned co-variants. Further genotype-phenotype correlation analysis indicated that the number of rs17401966 variant G allele was significantly associated with KIF1B mRNA expression levels (P for GLM = 0.003 and 0.001 in all and Chinese subjects, respectively), with GG carriers having the lowest level of KIF1B mRNA expression. Taken together, the rs17401966 polymorphism likely regulates KIF1B mRNA expression and thus may be associated with EOC risk in Eastern Chinese women. Larger, independent studies are warranted to validate our findings.

Frequent copy number variations of PI3K/AKT pathway and aberrant protein expressions of PI3K subunits are associated with inferior survival in diffuse large B cell lymphoma.

BACKGROUND: It has been reported that the PI3K/AKT signaling pathway is activated in diffuse large B-cell lymphoma (DLBCL), PI3K constitutive activation plays a crucial role in PI3K/AKT pathway. However, the copy number variations (CNVs) of PI3K subunits on gene level remain unknown in DLBCL. Therefore, the aim of the study is to investigate the CNV of PI3K subunits and their relationship with clinicopathological features exploring the possible mechanism underlying of PI3K activation in DLBCL. METHODS: CNV of 12 genes in the PI3K/AKT pathway was detected by NanoString nCounter in 60 de novo DLBCLs and 10 reactive hyperplasia specimens as controls. Meanwhile, immunohistochemistry (IHC) was performed to examine the expression of p110α, p110ß, p110γ, p110δ, and pAKT on DLBCL tissue microarrays. RESULTS: All PI3K and AKT subunits, except for PIK3R1, had various CNVs in the form of copy number amplifications and copy number losses. Their rates were in the range of 8.3-20.0%. Of them PIK3CA and PIK3CB gene CNVs were significantly associated with decreased overall survival (P = 0.029 and P = 0.019, respectively). IHC showed that the frequency of strong positive expression of p110α, p110ß, p110γ, and p110δ were 26.7%, 25.0%, 18.3%, and 25.0% respectively, and they were found to be associated with decreased survival (P = 0.022, P = 0.015, P = 0.015, and P = 0.008, respectively). Expression of p110α was not only significantly associated with CNVs of PIK3CA (P = 0.002) but also positively correlated with strong positive expression of pAKT (P = 0.026). CONCLUSIONS: CNV of PIK3CA is highly associated with aberrant p110α protein expression and subsequent activation of PI3K/AKT pathway. CNVs of PIK3CA and PIK3CB, and aberrant protein expression of p110 isoforms are of great important value for predicting inferior prognosis in DLBCL. Frequent CNVs of PI3K/AKT subunits may play an important role in the tumorigenesis of DLBCL.

Number of negative lymph nodes is associated with survival in thoracic esophageal squamous cell carcinoma patients undergoing three-field lymphadenectomy.

BACKGROUND: The number of negative lymph nodes (NLNs) can be used for predicting clinical outcomes for patients with esophageal carcinoma as it is believed to reflect the extent of lymphadenectomy. However, when patients are treated with the same surgical procedure, its prognostic value is not clear. METHODS: We reviewed the records of 332 patients with thoracic esophageal squamous cell carcinoma (ESCC) who underwent three-field lymphadenectomy (3FLND) and had at least 15 lymph nodes removed. We used Kaplan-Meier estimates to compute overall survival (OS), the log-rank tests to assess the equality of survival rates, and Cox regression analyses to evaluate the association between survival and NLN count after adjusting for potential confounders. RESULTS: At a median follow-up interval of 36 months, the median OS was 47 months and the 5-year survival rate was 47.0 %. NLN count was independently associated with OS, and higher numbers of NLNs were linked to better OS (hazard ratio [HR] 0.970; 95 % confidence interval [CI] 0.955-0.986); the effect did not change after we stratified patients into node-negative (HR 0.966; 95 % CI 0.933-1.000) and node-positive (HR 0.973; 95 % CI 0.955-0.991) groups. CONCLUSION: The NLN count is an important independent prognostic factor for patients with thoracic ESCC treated with 3FLND.

Homology Identification and Cross-Contamination Analysis: A Method for Evaluating the Quality of Biological Samples Stored in a Biobank Using the Advanta Sample ID Genotyping Panel.

Biological samples are important resources for scientific research. These samples are stored in biobanks over years until needed, and some of them can never be retrieved if they are improperly stored, causing them to be wasted. Thus, they are priceless, and they should be used correctly and effectively. Sample quality substantially affects biomedical research results. However, sample misidentification or mix-up is common. It is necessary to establish quality standards for sample identification. In this study, we used the Advanta Sample ID genotyping panel to detect homology identification and cross-contamination. We compared the single-nucleotide polymorphism (SNP) typing results of two different samples and calculated the similarity score of homologous sample pairs and nonhomologous sample pairs. Through analysis, we obtained a similarity score cutoff point of 0.8620, which was an effective way to distinguish homology and nonhomology. Cross-contamination was detected in two sets of mixtures (STD8:STD6 and jj3:1-P) mixed at a series of special ratios. Sensitivity was dependent on the sample characteristics and mixing ratios. Finally, we assessed the effect of sample degradation degree on SNP genotyping and found that degraded samples with a minimal DNA integrity number of 1.9 had complete genotyping results. On the whole, this study shows that the Sample ID panel is reliable for homology identification and cross-contamination analysis. Moreover, this technology has promising further applications in biological sample quality control.

Microsatellite instability in mismatch repair proficient colorectal cancer: clinical features and underlying molecular mechanisms.

BACKGROUND: Both defects in mismatch repair (dMMR) and high microsatellite instability (MSI-H) have been recognised as crucial biomarkers that guide treatment strategies and disease management in colorectal cancer (CRC). As MMR and MSI tests are being widely conducted, an increasing number of MSI-H tumours have been identified in CRCs with mismatch repair proficiency (pMMR). The objective of this study was to assess the clinical features of patients with pMMR/MSI-H CRC and elucidate the underlying molecular mechanism in these cases. METHODS: From January 2015 to December 2018, 1684 cases of pMMR and 401 dMMR CRCs were enrolled. Of those patients, 93 pMMR/MSI-H were identified. The clinical phenotypes and prognosis were analysed. Frozen and paraffin-embedded tissue were available in 35 patients with pMMR/MSI-H, for which comprehensive genomic and transcriptomic analyses were performed. FINDINGS: In comparison to pMMR/MSS CRCs, pMMR/MSI-H CRCs exhibited significantly less tumour progression and better long-term prognosis. The pMMR/MSI-H cohorts displayed a higher presence of CD8+ T cells and NK cells when compared to the pMMR/MSS group. Mutational signature analysis revealed that nearly all samples exhibited deficiencies in MMR genes, and we also identified deleterious mutations in MSH3-K383fs. INTERPRETATION: This study revealed pMMR/MSI-H CRC as a distinct subgroup within CRC, which manifests diverse clinicopathological features and long-term prognostic outcomes. Distinct features in the tumour immune-microenvironment were observed in pMMR/MSI-H CRCs. Pathogenic deleterious mutations in MSH3-K383fs were frequently detected, suggesting another potential biomarker for identifying MSI-H. FUNDING: This work was supported by the Science and Technology Commission of Shanghai Municipality (20DZ1100101).

Mutation landscape in Chinese nodal diffuse large B-cell lymphoma by targeted next generation sequencing and their relationship with clinicopathological characteristics.

BACKGROUND: Diffuse large B-cell lymphoma (DLBCL), an aggressive and heterogenic malignant entity, is still a challenging clinical problem, since around one-third of patients are not cured with primary treatment. Next-generation sequencing (NGS) technologies have revealed common genetic mutations in DLBCL. We devised an NGS multi-gene panel to discover genetic features of Chinese nodal DLBCL patients and provide reference information for panel-based NGS detection in clinical laboratories. METHODS: A panel of 116 DLBCL genes was designed based on the literature and related databases. We analyzed 96 Chinese nodal DLBCL biopsy specimens through targeted sequencing. RESULTS: The most frequently mutated genes were KMT2D (30%), PIM1 (26%), SOCS1 (24%), MYD88 (21%), BTG1 (20%), HIST1H1E (18%), CD79B (18%), SPEN (17%), and KMT2C (16%). SPEN (17%) and DDX3X (6%) mutations were highly prevalent in our study than in Western studies. Thirty-three patients (34%) were assigned as genetic classification by the LymphGen algorithm, including 12 cases MCD, five BN2, seven EZB, seven ST2, and two EZB/ST2 complex. MYD88 L265P mutation, TP53 and BCL2 pathogenic mutations were unfavorable prognostic biomarkers in DLBCL. CONCLUSIONS: This study presents the mutation landscape in Chinese nodal DLBCL, highlights the genetic heterogeneity of DLBCL and shows the role of panel-based NGS to prediction of prognosis and potential molecular targeted therapy in DLBCL. More precise genetic classification needs further investigations.