Yin Yang Gene Expression Ratio Signature for Lung Cancer Prognosis

<div><p>Many studies have established gene expression-based prognostic signatures for lung cancer. All of these signatures were built from training data sets by learning the correlation of gene expression with the patients' survival time. They require all new sample data to be normalized to the training data, ultimately resulting in common problems of low reproducibility and impracticality. To overcome these problems, we propose a new signature model which does not involve data training. We hypothesize that the imbalance of two opposing effects in lung cancer cells, represented by Yin and Yang genes, determines a patient’s prognosis. We selected the Yin and Yang genes by comparing expression data from normal lung and lung cancer tissue samples using both unsupervised clustering and pathways analyses. We calculated the Yin and Yang gene expression mean ratio (YMR) as patient risk scores. Thirty-one Yin and thirty-two Yang genes were identified and selected for the signature development. In normal lung tissues, the YMR is less than 1.0; in lung cancer cases, the YMR is greater than 1.0. The YMR was tested for lung cancer prognosis prediction in four independent data sets and it significantly stratified patients into high- and low-risk survival groups (p = 0.02, HR = 2.72; p = 0.01, HR = 2.70; p = 0.007, HR = 2.73; p = 0.005, HR = 2.63). It also showed prediction of the chemotherapy outcomes for stage II & III. In multivariate analysis, the YMR risk factor was more successful at predicting clinical outcomes than other commonly used clinical factors, with the exception of tumor stage. The YMR can be measured in an individual patient in the clinic independent of gene expression platform. This study provided a novel insight into the biology of lung cancer and shed light on the clinical applicability.</p></div

Identification and selection of Yin and Yang genes.

<p><b>A</b>. Clustering of gene identification. The probe sets are in rows and the samples are in columns. The expression indexes of all the 12,625 probe sets of the 100 samples were summarized by RMA algorithm and further normalized by itemwise Z-normalization. 74 upregulated genes (bottom half rows) and 108 (top half rows) down regulated genes in cancer tissues were selected from the 2D clustering regions. The preselected 74 and 108 probsets were displayed by clustering again. <b>B</b>. Yin (bottom) and Yang (top) genes selection by functional analysis. The two circles represent the two cores of functional effects of the Yin and the Yang. The genes highlighted by the same color are in the same interaction network.</p

Boxplot of the YMR distributions in normal lung samples and lung cancer samples.

<p>Microarray gene expression data sets from different reports with different platforms were used. The data sets were described as in Table S7.</p

Validation of YMR in four data sets by Kaplan-Meier estimates of the survivor function.

<p><b>A.</b> Free-recurrence time function curve (low risk n = 60; high risk n = 65) of the adenocarcinomas patients from Bhattacharjee <i>et al</i>. <b>B</b>. Overall survival time function curve of the adenocarcinomas patients (low risk n = 27; high risk n = 31) from Bild <i>et al</i>. <b>C</b>. Patient samples (low risk n = 248; high risk n = 194) of the DCC project. <b>D</b>. RNA-seq samples (low risk n = 121; high risk n = 137) from TCGA. Low YMR scores (in green) correspond to the highest predicted survival probability and high YMR scores (in red) correspond to the greatest predicted risk.</p

Yin genes.

<p>Yin genes.</p

Kaplan-Meier estimates of the survivor function of the gYMR signature in different group of patients of the DCC data set.

<p><b>A</b>. Stage I only (low risk n = 122; high risk n = 177). <b>B</b>. Stage I who received chemotherapy (low risk n = 13; high risk n = 28). <b>C</b>. Stage I who did not receive chemotherapy (low risk n = 79; high risk n = 95). <b>D</b>. Stage II & III only (low risk n = 63; high risk n = 78). <b>E</b>. Stage II & III who received chemotherapy (low risk n = 24; high risk n = 23). <b>F</b>. Stage II & III who did not receive chemotherapy (low risk n = 27; high risk n = 31). Low gYMR scores (in green) correspond to the highest predicted survival probability and high gYMR scores (in red) correspond to the greatest predicted risk.</p

Comparison of YMR to different signature models previously reported.

<p>Comparison of YMR to different signature models previously reported.</p

Ovariectomy influences the relationships between bone volume (BV/TV%), adipogenesis (% marrow fat), and hematopoiesis (% cellularity) at Day 30 with or without 16 Gy radiation to the hind limb in mice.

<p>Data of every two measurements are represented as least squares mean values for the group. Panel A: OVX results in a 10 fold reduction in the rate of bone volume loss per unit increase in marrow fat after radiation treatment compared with intact mice. Panel B: OVX blunts the rate of bone volume loss relative to reduced hematopoietic cellularity after radiation compared with intact mice by a factor of 6. Panel C: OVX halves the reduction in hematopoietic cellularity compared with increased marrow fat after radiation treatment compared with intact mice.</p

Comparison of group least squares means (± SEM) of hematopoietic cellularity, adiposity, and bone volume in intact (I) and ovariectomized (OVX) mice without radiation (−R) and with radiation (+R) 30 days after radiation treatment.

<p>Within columns, identical letters indicate relevant statistically significant differences between groups at p≤0.001). Radiation significantly reduced hematopoietic cellularity in intact mice, but the effect was more severe in OVX (interaction F = 4.79, p = 0.0439). OVX alone had no effect on hematopoietic activity in the absence of radiation. Radiation significantly increased marrow adipose only in OVX (interaction F = 10.14, p = 0.0062), and OVX increased marrow fat without and with radiation treatment. Radiation significantly reduced BV/TV% (p = 0.0467), but OVX had no additive effect. All statistical analyses were based on two-way ANOVA.</p

Experimental Plan Schematic.

<p>Sixteen week old BALB/c mice were ovariectomized (OVX) and maintained in the vivarium for 57 days in order to attain skeletal hemostasis. Both intact (I) and OVX mice were then irradiated with 16Gy delivered to the caudal skeleton or maintained as controls. Groups of animals were euthanized at 3, 8, and 30 days post irradiation in order to perform histological evaluations of the distal femur; microCT measurements were conducted at the 30 day time point only.</p