Volume 7, Issue 4, Pages 337-350 (April 2005) Expression profiling using a tumor-specific cDNA microarray predicts the prognosis of intermediate risk neuroblastomas  Miki Ohira, Shigeyuki Oba, Yohko Nakamura, Eriko Isogai, Setsuko Kaneko, Atsuko Nakagawa, Takahiro Hirata, Hiroyuki Kubo, Takeshi Goto, Saichi Yamada, Yasuko Yoshida, Misa Fuchioka, Shin Ishii, Akira Nakagawara  Cancer Cell  Volume 7, Issue 4, Pages 337-350 (April 2005) DOI: 10.1016/j.ccr.2005.03.019 Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 1 Schematic diagram of this study Cancer Cell 2005 7, 337-350DOI: (10.1016/j.ccr.2005.03.019) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 2 Posterior probability of survival at 5 years Posterior probability of survival at 5 years for 136 training data samples, output by the leave two out (LTO) crossvalidation without any information leakage. Left panel: Neuroblastoma samples. A red or blue horizontal line denotes survival period after diagnosis for a dead or alive patient, respectively. Red and blue marks denote various clinical properties of patients; see text below the panel for detailed explanation. Background colors show groups determined by stage and MYCN amplification status: red, type III, with MYCN amplification; green, type II, with single copy of MYCN at unfavorable stage (3 or 4); and blue, type I, with single copy of MYCN and at favorable stage (1, 2, or 4s). Right upper panel: The LTO crossvalidated prediction (posterior) for each patient; a red or a blue mark denotes that the patient is dead or alive at 5 years, respectively. Right middle panel: Cumulative smooth histogram of posterior probabilities for patients of dead (red), alive (blue), and unknown (white) at 5 years after diagnosis. Right lower panel: The horizontal and vertical axes denote the posterior and the empirical probability of 5 year survival, i.e., the ratio of the smooth histogram values between dead and alive patients, shown in the middle panel, respectively. Because the border between dead and alive samples is close to the white broken line (x = y), the posterior can be regarded as a 5 year survival chance rate. Cancer Cell 2005 7, 337-350DOI: (10.1016/j.ccr.2005.03.019) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 3 Disease-free survival of patients who were stratified based on the gene expression patterns For each of the four figures, whole objective patients (green) are divided into favorable (blue) or unfavorable (red) based on the posterior values with threshold 0.5, which are calculated from gene expression patterns, and statistical features of their survival times are denoted by the Kaplan-Meier survival curves. The differences of the survival curves between the favorable (blue) and unfavorable (red) groups are evaluated by p values of the log rank test. A and B: Survival analysis of whole and sporadic patients, respectively, divided by the supervised classifier based on microarray data. C and D: Survival analysis of patients in the intermediate risk group with different definitions, divided by the supervised classifier. The intermediate risk group shown in (C) is defined as MYCN single and stage 3 or 4 (type II), and that in (D) is defined as MYCN single, stage 3 or 4, and older than 1 year of age. Cancer Cell 2005 7, 337-350DOI: (10.1016/j.ccr.2005.03.019) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 4 Posterior probability of survival at 5 years for test samples Posterior probability of survival at 5 years for 50 independent test samples measured by newly developed 200 genes chip (the mini-chip system). Left panel: Neuroblastoma samples; see also Figure 2 legend. Center panel: Prediction results when the supervised classifier constructed from 96 training samples is applied to the 50 independent samples (independent test for the classifier’s reproducibility). Right panel: LTO crossvalidation analysis using the new 50 samples (test for the procedure’s reproducibility). Both tests do not introduce any information leakage. Lower panels: Smooth histograms of posterior probabilities for dead (red) and alive (blue) patients. Cancer Cell 2005 7, 337-350DOI: (10.1016/j.ccr.2005.03.019) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 5 Expression profiles of 70 genes selected for predicting neuroblastoma prognosis at 2 years Note that 10 genes for predicting prognosis at 5 years are also included in the 70 genes. The left and lower trees depict hierarchical clustering of the 136 neuroblastoma samples and the 70 genes selected in the present study, respectively. In the left tree, blue, green, and red colors denote “MYCN single and stage 1, 2 or 4s tumor” (type I, favorable), “MYCN single and stage 3, 4 tumor” (type II, intermediate), and “MYCN amplified tumor” (type III, unfavorable), respectively. The blue and red colors in the expression matrix show the high and low expression, respectively. A gene showing high expression level likely for unfavorable samples belongs to the group “UF” (red subtree in the lower tree), while one showing high expression likely for favorable samples belongs to the group “F” (blue subtree in the lower tree). Cancer Cell 2005 7, 337-350DOI: (10.1016/j.ccr.2005.03.019) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 6 Individual survival chance analysis based on posterior probabilities LTO estimation of survival probabilities at 0.5, 1.0, 1.5, …, 5.0 years after diagnosis for 12 typical patients. Left panel: Information of patients (see caption of Figure 2). Right panel: Estimated posterior probabilities at 0.5, 1.0, 1.5, …, 5.0 years after diagnosis, which predict the time course of patient’s survival chance. A blue or a red mark denotes that the patient is alive or dead at that time after diagnosis, respectively. For example, the patient “S108,” who died at 40 months, is predicted as 100% alive at 0.5 year and 52% alive at 5 year, solely from the microarray analysis at the diagnosis Cancer Cell 2005 7, 337-350DOI: (10.1016/j.ccr.2005.03.019) Copyright © 2005 Elsevier Inc. Terms and Conditions