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Background: Esophageal cancer is a tumor type with high invasiveness and low prognosis. As immunotherapy has been shown to improve the prognosis of esophageal cancer patients, we were interested in the establishment of an immune-associated gene prognostic index to effectively predict the prognosis of patients. Methods: To establish the immune-related gene prognostic index of esophageal cancer (EC), we screened 363 upregulated and 83 downregulated immune-related genes that were differentially expressed in EC compared to normal tissues. By multivariate Cox regression and weighted gene coexpression network analysis (WGCNA), we built a prognostic model based on eight immune-related genes (IRGs). We confirmed the prognostic model in both TCGA and GEO cohorts and found that the low-risk group had better overall survival than the high-risk group. Results: In this study, we identified 363 upregulated IRGs and 83 downregulated IRGs. Next, we found a prognostic model that was constructed with eight IRGs (OSM, CEACAM8, HSPA6, HSP90AB1, PCSK2, PLXNA1, TRIB2, and HMGB3) by multivariate Cox regression analysis and WGCNA. According to the Kaplan–Meier survival analysis results, the model we constructed can predict the prognosis of patients with esophageal cancer. This result can be verified by the Gene Expression Omnibus (GEO). Patients were divided into two groups with different outcomes. IRGPI-low patients had better overall survival than IRGPI-high patients. Conclusion: Our findings indicated the potential value of the IRGPI risk model for predicting the prognosis of EC patients [ABSTRACT FROM AUTHOR]

The tumor microenvironment is composed of tumor cells, stroma cells, immune cells, blood vessels, and other associated non-cancerous cells. Gene expression measurements on tumor samples are an average over cells in the microenvironment. However, research questions often seek answers about tumor cells rather than the surrounding non-tumor tissue. Previous studies have suggested that the tumor purity (TP)—the proportion of tumor cells in a solid tumor sample—has a confounding effect on differential expression (DE) analysis of high vs. low survival groups. We investigate three ways incorporating the TP information in the two statistical methods used for analyzing gene expression data, namely, differential network (DN) analysis and DE analysis. Analysis 1 ignores the TP information completely, Analysis 2 uses a truncated sample by removing the low TP samples, and Analysis 3 uses TP as a covariate in the underlying statistical models. We use three gene expression data sets related to three different cancers from the Cancer Genome Atlas (TCGA) for our investigation. The networks from Analysis 2 have greater amount of differential connectivity in the two networks than that from Analysis 1 in all three cancer datasets. Similarly, Analysis 1 identified more differentially expressed genes than Analysis 2. Results of DN and DE analyses using Analysis 3 were mostly consistent with those of Analysis 1 across three cancers. However, Analysis 3 identified additional cancer-related genes in both DN and DE analyses. Our findings suggest that using TP as a covariate in a linear model is appropriate for DE analysis, but a more robust model is needed for DN analysis. However, because true DN or DE patterns are not known for the empirical datasets, simulated datasets can be used to study the statistical properties of these methods in future studies.

The Pan-Cancer Atlas consists of original sequencing data from various sources, provides the opportunity to perform systematic studies on the commonalities and differences between diverse cancers. The analysis for the pan-cancer dataset could help researchers to identify the key factors that could trigger cancer. In this paper, we present a novel pan-cancer classification method, referred to MI_DenseNetCAM, to identify a set of genes that can differentiate all tumor types accurately. First, the Mutual Information (MI) was utilized to eliminate noise and redundancy from the pan-cancer datasets. Then, the gene data was further converted to 2D images. Next, the DenseNet model was adopted as a classifier and the Guided Grad-CAM algorithm was applied to identify the key genes. Extensive experimental results on the public RNA-seq data sets with 33 different tumor types show that our method outperforms the other state-of-the-art classification methods. Moreover, gene analysis further demonstrated that the genes selected by our method were related to the corresponding tumor types.

The tumor microenvironment is composed of tumor cells, stroma cells, immune cells, blood vessels, and other associated non-cancerous cells. Gene expression measurements on tumor samples are an average over cells in the microenvironment. However, research questions often seek answers about tumor cells rather than the surrounding non-tumor tissue. Previous studies have suggested that the tumor purity (TP)—the proportion of tumor cells in a solid tumor sample—has a confounding effect on differential expression (DE) analysis of high vs. low survival groups. We investigate three ways incorporating the TP information in the two statistical methods used for analyzing gene expression data, namely, differential network (DN) analysis and DE analysis. Analysis 1 ignores the TP information completely, Analysis 2 uses a truncated sample by removing the low TP samples, and Analysis 3 uses TP as a covariate in the underlying statistical models. We use three gene expression data sets related to three different cancers from the Cancer Genome Atlas (TCGA) for our investigation. The networks from Analysis 2 have greater amount of differential connectivity in the two networks than that from Analysis 1 in all three cancer datasets. Similarly, Analysis 1 identified more differentially expressed genes than Analysis 2. Results of DN and DE analyses using Analysis 3 were mostly consistent with those of Analysis 1 across three cancers. However, Analysis 3 identified additional cancer-related genes in both DN and DE analyses. Our findings suggest that using TP as a covariate in a linear model is appropriate for DE analysis, but a more robust model is needed for DN analysis. However, because true DN or DE patterns are not known for the empirical datasets, simulated datasets can be used to study the statistical properties of these methods in future studies. [ABSTRACT FROM AUTHOR]

The Pan-Cancer Atlas consists of original sequencing data from various sources, provides the opportunity to perform systematic studies on the commonalities and differences between diverse cancers. The analysis for the pan-cancer dataset could help researchers to identify the key factors that could trigger cancer. In this paper, we present a novel pan-cancer classification method, referred to MI_DenseNetCAM, to identify a set of genes that can differentiate all tumor types accurately. First, the Mutual Information (MI) was utilized to eliminate noise and redundancy from the pan-cancer datasets. Then, the gene data was further converted to 2D images. Next, the DenseNet model was adopted as a classifier and the Guided Grad-CAM algorithm was applied to identify the key genes. Extensive experimental results on the public RNA-seq data sets with 33 different tumor types show that our method outperforms the other state-of-the-art classification methods. Moreover, gene analysis further demonstrated that the genes selected by our method were related to the corresponding tumor types. [ABSTRACT FROM AUTHOR]

Next-generation sequencing has emerged as an essential technology for the quantitative analysis of gene expression. In medical research, RNA sequencing (RNA-seq) data are commonly used to identify which type of disease a patient has. Because of the discrete nature of RNA-seq data, the existing statistical methods that have been developed for microarray data cannot be directly applied to RNA-seq data. Existing statistical methods usually model RNA-seq data by a discrete distribution, such as the Poisson, the negative binomial, or the mixture distribution with a point mass at zero and a Poisson distribution to further allow for data with an excess of zeros. Consequently, analytic tools corresponding to the above three discrete distributions have been developed: Poisson linear discriminant analysis (PLDA), negative binomial linear discriminant analysis (NBLDA), and zero-inflated Poisson logistic discriminant analysis (ZIPLDA). However, it is unclear what the real distributions would be for these classifications when applied to a new and real dataset. Considering that count datasets are frequently characterized by excess zeros and overdispersion, this paper extends the existing distribution to a mixture distribution with a point mass at zero and a negative binomial distribution and proposes a zero-inflated negative binomial logistic discriminant analysis (ZINBLDA) for classification. More importantly, we compare the above four classification methods from the perspective of model parameters, as an understanding of parameters is necessary for selecting the optimal method for RNA-seq data. Furthermore, we determine that the above four methods could transform into each other in some cases. Using simulation studies, we compare and evaluate the performance of these classification methods in a wide range of settings, and we also present a decision tree model created to help us select the optimal classifier for a new RNA-seq dataset. The results of the two real datasets coincide with the theory and simulation analysis results. The methods used in this work are implemented in the open-scource R scripts, with a source code freely available at https://github.com/FocusPaka/ZINBLDA.

Next-generation sequencing has emerged as an essential technology for the quantitative analysis of gene expression. In medical research, RNA sequencing (RNA-seq) data are commonly used to identify which type of disease a patient has. Because of the discrete nature of RNA-seq data, the existing statistical methods that have been developed for microarray data cannot be directly applied to RNA-seq data. Existing statistical methods usually model RNA-seq data by a discrete distribution, such as the Poisson, the negative binomial, or the mixture distribution with a point mass at zero and a Poisson distribution to further allow for data with an excess of zeros. Consequently, analytic tools corresponding to the above three discrete distributions have been developed: Poisson linear discriminant analysis (PLDA), negative binomial linear discriminant analysis (NBLDA), and zero-inflated Poisson logistic discriminant analysis (ZIPLDA). However, it is unclear what the real distributions would be for these classifications when applied to a new and real dataset. Considering that count datasets are frequently characterized by excess zeros and overdispersion, this paper extends the existing distribution to a mixture distribution with a point mass at zero and a negative binomial distribution and proposes a zero-inflated negative binomial logistic discriminant analysis (ZINBLDA) for classification. More importantly, we compare the above four classification methods from the perspective of model parameters, as an understanding of parameters is necessary for selecting the optimal method for RNA-seq data. Furthermore, we determine that the above four methods could transform into each other in some cases. Using simulation studies, we compare and evaluate the performance of these classification methods in a wide range of settings, and we also present a decision tree model created to help us select the optimal classifier for a new RNA-seq dataset. The results of the two real datasets coincide with the theory and simulation analysis results. The methods used in this work are implemented in the open-scource R scripts, with a source code freely available at https://github.com/FocusPaka/ZINBLDA. [ABSTRACT FROM AUTHOR]

In the past five years, RNA-Seq has become a powerful tool in transcriptome analysis even though computational methods dedicated to the analysis of high-throughput sequencing data are yet to be standardized. It is, however, now commonly accepted that the choice of a normalization procedure is an important step in such a process, for example in differential gene expression analysis. The present article highlights the similarities between three normalization methods: TMM from edgeR R package, RLE from DESeq2 R package, and MRN. Both TMM and DESeq2 are widely used for differential gene expression analysis. This paper introduces properties that show when these three methods will give exactly the same results. These properties are proven mathematically and illustrated by performing in silico calculations on a given RNA-Seq data set.

In the past 5 years, RNA-Seq has become a powerful tool in transcriptome analysis even though computational methods dedicated to the analysis of high-throughput sequencing data are yet to be standardized. It is, however, now commonly accepted that the choice of a normalization procedure is an important step in such a process, for example in differential gene expression analysis. The present article highlights the similarities between three normalization methods: TMM from edgeR R package, RLE from DESeq2 R package, and MRN. Both TMM and DESeq2 are widely used for differential gene expression analysis. This paper introduces properties that show when these three methods will give exactly the same results. These properties are proven mathematically and illustrated by performing in silico calculations on a given RNA-Seq data set. [ABSTRACT FROM AUTHOR]