APOBEC3G Expression Correlates with T-Cell Infiltration and Improved Clinical Outcomes in High-grade Serous Ovarian Carcinoma

Abstract

Purpose: APOBEC3 DNA cytosine deaminase family members normally defend against viruses and transposons. However, deregulated APOBEC3 activity causes mutations in cancer. Because of broad expression profiles and varying mixtures of normal and cancer cells in tumors, including immune cell infiltration, it is difficult to determine where different APOBEC3s are expressed. Here, we ask whether correlations exist between APOBEC3 expression and T-cell infiltration in high-grade serous ovarian cancer (HGSOC), and assess whether these correlations have prognostic value.

Experimental design: Transcripts for APOBEC3G, APOBEC3B, and the T-cell markers, CD3D, CD4, CD8A, GZMB, PRF1, and RNF128 were quantified by RT-qPCR for a cohort of 354 HGSOC patients. Expression values were correlated with each other and clinical parameters. Two additional cohorts were used to extend HGSOC clinical results. Immunoimaging was used to colocalize APOBEC3G and the T-cell marker CD3. TCGA data extended expression analyses to additional cancer types.

Results: A surprising positive correlation was found for expression of APOBEC3G and several T cell genes in HGSOC. Immunohistochemistry and immunofluorescent imaging showed protein colocalization in tumor-infiltrating T lymphocytes. High APOBEC3G expression correlated with improved outcomes in multiple HGSOC cohorts. TCGA data analyses revealed that expression of APOBEC3D and APOBEC3H also correlates with CD3D across multiple cancer types.

Conclusions: Our results identify APOBEC3G as a new candidate biomarker for tumor-infiltrating T lymphocytes and favorable prognoses for HGSOC. Our data also highlight the complexity of the tumor environment with respect to differential APOBEC family gene expression in both tumor and surrounding normal cell types. Clin Cancer Res; 22(18); 4746-55. ©2016 AACR.

Figure 1. Correlations between APOBEC3 expression and T cell markers in HGSOC

Dot plots illustrating correlations between APOBEC3G (A–F) or APOBEC3B (G–L) expression and the indicated T cell marker (n=354). mRNA expression was determined using RT-qPCR and normalized to the housekeeping gene TBP. Spearman’s correlation coefficients (r_s) and p-values are shown. Best-fit lines are shown for qualitative comparison, and were calculated using linear regression models.

Figure 2. Immunohistochemistry and immunofluorescence of T cell markers in HGSOC

Photomicrographs of immunohistochemistry and immunofluorescence staining performed on HGSOC specimens illustrating the association between levels of T lymphocyte infiltration and the intensity of APOBEC3G expression. Representative staining of one HGSOC specimen with low (patient 6) and three staining sets from two HGSOC specimens with high (patients 3 and 2) levels of T cell infiltration are shown. The images depict hematoxylin (A, F, K, and P), CD3 (B, G, L, and Q), CD4 (C, H, M, and R), CD8 (D, I, N, and S), and APOBEC3G (E, J, O, and T). The dotted box in the 40x hematoxylin images indicates the approximate location of the subsequent panels at 100x magnification. The bottom row shows representative 1000x magnification images of colocalization of CD3 and APOBEC3G by immunofluorescent staining. DAPI-stained nuclei are blue.

Figure 3. Clinical correlates of T cell marker expression in HGSOC

Kaplan-Meier plots illustrating associations between progression free survival (PFS) (A, n=354) or overall survival (OS) (B, n=348) and either one of the conventional T cell markers or APOBEC3G or APOBEC3B expression in the Mayo cohort of patients. Samples were split at the median expression level for each gene with red representing tumors with high and blue representing tumors with low mRNA levels.

Figure 4. Correlations between APOBEC expression and immune cell markers across 22 cancer types

Heatmap of Spearman’s correlation coefficients calculated from the comparison of the T cell marker CD3D (A) or the B cell marker CD20 (B) with the indicated APOBEC family member. Expression levels were determined using TCGA RNAseq data (see Table S3 for the long form for each tumor abbreviation). Dark red squares indicate strong positive correlations, dark blue squares indicate strong negative correlations and white squares indicate a lack of correlation.