File S1 - A 16-Gene Signature Distinguishes Anaplastic Astrocytoma from Glioblastoma

Supplementary Text: Supplementary Methods and Supplementary Results. Supplementary Tables: Table S1. Primers used for RT-qPCR. Table S2. List of genes selected for expression analysis by PCR array. Table S3. Number of AA and GBM patient samples in training set, test set and three independent cohorts of patient samples (TCGA, GSE1993 and GSE4422). Table S4. Expression of 16 genes in AA (n = 20) and GBM (n = 54) samples of the test set. Table S5. Expression of 16 genes in Grade III glioma (n = 27) and GBM (n = 152) samples of the TCGA dataset. Table S6. Expression of 16 genes in AA (n = 19) and GBM (n = 39) samples of GSE1993 dataset. Table S7. Expression of 16 genes in AA (n = 5) and GBM (n = 71) samples of the GSE4422 dataset. Supplementary Figures: Figure S1. Heat map of one-way hierarchical clustering of 16 PAM-identified genes in AA (n = 20) and GBM (n = 54) patient samples in the test set. A dual-color code was used, with red and green indicating up- and down regulation, respectively. Figure S2. Heat map of one-way hierarchical clustering of 16 PAM-identified genes in grade III glioma (n = 27) and GBM (n = 152) patient samples in TCGA dataset. A dual-color code was used, with red and green indicating up- and down regulation, respectively. Figure S3. A. Heat map of one-way hierarchical clustering of 16 PAM-identified genes in AA (n = 19) and GBM (n = 39) patient samples in GSE1993 dataset. A dual-color code was used, with red and green indicating up- and down regulation, respectively. B. PCA was performed using expression values of 16-PAM identified genes between AA and GBM samples in GSE1993 dataset. A scatter plot is generated using the first two principal components for each sample. The color of the samples is as indicated. C. The detailed probabilities of 10-fold cross-validation for the samples of GSE1993 dataset based on the expression values of 16 genes are shown. For each sample, its probability as AA (orange color) and GBM (blue color) are shown and it was predicted by the PAM program as either AA or GBM based on which grade's probability is higher. The original histological grade of the samples is shown on the top. Figure S4. A. Heat map of one-way hierarchical clustering of 16 PAM-identified genes in AA (n = 5) and GBM (n = 71) patient samples in GSE4422 dataset. A dual-color code was used, with red and green indicating up- and down regulation, respectively. B. PCA was performed using expression values of 16-PAM identified genes between AA and GBM samples in GSE4422 dataset. A scatter plot is generated using the first two principal components for each sample. The color of the samples is as indicated. C. The detailed probabilities of 10-fold cross-validation for the samples of GSE4422 dataset based on the expression values of 16 genes are shown. For each sample, its probability as AA (orange color) and GBM (blue color) are shown and it was predicted by the PAM program as either AA or GBM based on which grade's probability is higher. The original histological grade of the samples is shown on the top. Figure S5. A. The detailed probabilities of 10-fold cross-validation for the samples of GSE4271 dataset based on the expression values of 16 genes are shown. For each sample, its probability as AA (orange color) and GBM (blue color) are shown and it was predicted by the PAM program as either AA or GBM based on which grade's probability is higher. The original histological grade of the samples is shown on the top. B. The average Age at Diagnosis along with standard deviation is plotted for Authentic AAs (n = 12), Authentic GBMs (n = 68), Discordant AAs (n = 10) and Discordant GBMs (n = 8) of GSE4271 dataset. C. The Kaplan Meier survival analysis of samples of GSE4271 dataset. Figure S6. PAM analysis of the Petalidis-gene signature in TCGA dataset. A. Plot showing classification error for the Petalidis gene set in TCGA dataset. The threshold value of 0.0 corresponded to all 54 genes which classified AA (n = 27) and GBM (n = 604) samples with classification error of 0.000. B. The detailed probabilities of 10-fold cross-validation for the samples of TCGA dataset based on Petalidis gene set are shown. For each sample, its probability as AA (green color) and GBM (red color) are shown and it was predicted by the PAM program as either AA or GBM based on which grade's probability is higher. The original histological grade of the samples is shown on the top. Figure S7. PAM analysis of the Phillips gene signature in our dataset. A. Plot showing classification error for the Phillips gene set in our dataset. The threshold value of 0.0 that correspond to all 5 genes which classified AA (n = 50) and GBM (n = 132) samples with classification error of 0.159. B. The detailed probabilities of 10-fold cross-validation for the samples of our dataset based on Phillips gene set are shown. For each sample, its probability as AA (orange color) and GBM (blue color) are shown and it was predicted by the PAM program as either AA or GBM based on which grade's probability is higher. The original histological grade of the samples is shown on the top. Figure S8. PAM analysis of the Phillips gene signature in Phillips dataset. A. Plot showing classification error for the Phillips gene set in Phillips dataset. The threshold value of 0.0 that correspond to all 8 genes which classified AA (n = 24) and GBM (n = 76) samples with classification error of 0.169. B. The detailed probabilities of 10-fold cross-validation for the samples of our dataset based on Phillips gene set are shown. For each sample, its probability as AA (orange color) and GBM (blue color) are shown and it was predicted by the PAM program as either AA or GBM based on which grade's probability is higher. The original histological grade of the samples is shown on the top. Figure S9. PAM analysis of the Phillips gene signature in GSE4422 dataset. A. Plot showing classification error for the Phillips gene set in GSE4422 dataset. The threshold value of 0.0 that correspond to all 8 genes which classified AA (n = 5) and GBM (n = 76) samples with classification error of 0.065. B. The detailed probabilities of 10-fold cross-validation for the samples of our dataset based on Phillips gene set are shown. For each sample, its probability as AA (orange color) and GBM (blue color) are shown and it was predicted by the PAM program as either AA or GBM based on which grade's probability is higher. The original histological grade of the samples is shown on the top. Figure S10. PAM analysis of the Phillips-gene signature in TCGA dataset. A. Plot showing classification error for the Phillips gene set in TCGA dataset. The threshold value of 0.0 corresponded to all 8 genes which classified AA (n = 27) and GBM (n = 604) samples with classification error of 0.008. B. The detailed probabilities of 10-fold cross-validation for the samples of TCGA dataset based on Phillips gene set are shown. For each sample, its probability as AA (orange color) and GBM (blue color) are shown and it was predicted by the PAM program as either AA or GBM based on which grade's probability is higher. The original histological grade of the samples is shown on the top. Figure S11. Network obtained by using 16-genes of classification signature as input genes to Bisogenet plugin in Cytoscape. The gene rated network had 252 nodes (genes) and 1498 edges (interactions between genes/proteins). This network consisted of the seed proteins with their immediate interacting neighbors. The nodes corresponding to the input genes are highlighted by the bigger node size as compared to the rest of the interacting partners. The color code is as indicated in the scale.
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