Fig 1.
Expression of and survival analysis of CD44 variant 8–10 containing isoforms in the Cancer Genome Atlas Ovarian Serous Cystadenocarcinoma Database.
A) Schematic representation of alternative splice isoforms expressing variable exons v8-v10. B) Distribution of RNAseq2 expression data over 254 samples for all CD44 exons in the TCGA ovarian cancer dataset. Samples with no expression were omitted from this figure. C) Kaplan-Meier plot of the survival curve comparing high and low CD44v8-10 expressing samples and corresponding table with the number of patients at each given time point. We defined the high expressing set (red) and the low expressing set (black) as the samples with the highest and lowest expression respectively using the top/bottom 10% of expressers, with the plot extending to 5 years. Bars represent censored data points.
Table 1.
Multivariate analysis using Cox proportional hazard ratio model in the 255 serous ovarian cancer cohort from The Cancer Genome Atlas examined for CD44v9 expression by RNAseq.
Fig 2.
Immunohistochemical and survival analysis of CD44v8-10 expression in High Grade Serous Ovarian Cancer Tumor Tissue Microarrays (TMAs).
Fixed paraffin embedded tissue microarray cores were stained by immunohistochemistry using an antibody against CD44v9, scanned using an Aperio Scanscope, and analyzed using Aperio Imagescope software positive pixel count algorithm. Representative images taken at high (40x) power show basal epithelial staining in a and c, more diffuse surface epithelial staining in b, and negative stroma with isolated positive epithelial cells in d. e.) Overall Survival of patients with high-grade serous ovarian carcinoma by CD44 variant expression. Left- Highest and Lowest 10% expression variant and Right- Highest and Lowest 20% of expression of variant, demonstrates significant overall survival in the highest expressers with p = 0.0181 and p = 0.0262, respectively.
Table 2.
Patient demographics in a high grade serous ovarian cancer tumor tissue microarrays of 210 patients (TMAs).
Table 3.
Multivariate analysis using a Cox proportional hazard ratio model in the 210 patient cohort analyzed for CD44v8-10 expression by Positive Intensity (PI) score.
Fig 3.
Binary decision tree of clinical variables and CD44v9 staining and their effect on prognosis.
The patients from the tumor array sampleset were stratified using Age, CD44v9 staining intensity (σ of CD44v9 expression), grade, debulking status, and FIGO score. Multivariate analysis was performed and effect sized ranked to generate a binary decision tree to maximize prognostic power in an unbiased manner and assign statistical significance. Only statistically significant variables (age and CD44v9 staining) are shown along with their effect on overall patient survival on Kaplan-Meier plots. Bars represent censored data points.
Fig 4.
Expression of CD44v8-10 mRNA and protein, compared to epithelial markers and mesenchymal markers by Principal component Analysis (PCA) of qPCR and Western Blot.
A) Principal component analysis using qPCR relative quantification of the expression of epithelial, mesenchymal, and pluripotency markers in primary ovarian cancer cell lines. Colored arrows indicate the directions for each group of markers. B) Western analysis of primary cell line protein lysates for expression of CD44v8-10 protein using a rat monoclonal antibody to human CD44v8-10.
Fig 5.
Pattern of expression of CD44v8-10 and EMT markers by immunohistochemistry in PDXa tumors.
Antibody staining of tissue sections from tumors developed in vivo after injection of 1 to 5 million primary patient-derived ovarian cancer cells. Representative sections from tumors derived from OVCAR5, ptAP-sph, Pt W (A), ptAM-sph, ptH, ptAB-sph (B) and ptD (C) are shown. CD44v8-10, and Vimentin immunohistochemical stain is shown with hematoxylin counterstain at 100x magnification. CD44v8-10 labels the surface of epithelial cancer cells (insert), whereas vimentin labels stromal cells in more epithelial tumors and most cells in more mesenchymal tumors (insert). Tumors were grouped into epithelial (A), mesenchymal (B) or mixed phenotype (C) based on the staining.
Fig 6.
Detection of soluble cleaved extracellular domain of CD44v8-10 in patient ascites samples.
A) Ascites samples (N = 28; 6 samples shown as representative) were obtained from patients undergoing therapeutic paracentesis, and probed by western blotting for CD44v8-10, CD44s, and anti human IgG light chain as a loading control. Protein densitometry was performed and samples were analyzed for levels of expression of soluble CD44v8-10 and separated into two equal groups of high and low CD44v8-10 levels. B) Kaplan-Meier survival curve of the patients stratified by CD44v8-10 abundance in the patient ascites shows high expression correlates with decreased survival (p = 0.0481). Bars represent censored data points.
Fig 7.
Surface expression of transmembrane CD44v8-10 protein or soluble cleaved CD44v8-10 extracellular fragments have distinct prognostic significances in ovarian cancer.
High expression of the cell surface transmembrane CD44 containing variant exons v8-10 is associated with improved survival, which may be indicative of a more epithelial tumor. Presence of the cleaved soluble extracellular domain of CD44v8-10 in the ascites fluid is associated with poor survival, which may be indicative of a more metastatic tumor with high expression of proteases (MMP-9, ADAM-10). Higher levels of soluble CD44v8-10 may act as a dominant negative, preventing cells from reestablishing binding with the ECM, contributing to a more mesenchymal and metastatic phenotype.