Is CMV a target in pediatric glioblastoma? Expression of CMV proteins, pp65 and IE1-72 and CMV nucleic acids in a cohort of pediatric glioblastoma patients

Abstract

While the 5-year overall survival is better in pediatric than in adult patients diagnosed with glioblastoma (GBM), outcomes in children remain very poor. Understanding the mechanisms of tumorigenesis and tumor propagation can identify therapeutic targets to improve these outcomes. Human cytomegalovirus (CMV) proteins and nucleic acids are present in the majority of adult GBM. Indeed, CMV is emerging as a potential glioma-associated target for anti-CMV agents and cellular therapeutics. Furthermore, CMV appears to contribute to GBM's malignant phenotype, although its role in tumorigenesis is less certain. In this cohort of 25 serially diagnosed pediatric GBMs, the largest described cohort to date, we used immunohistochemical staining and in situ hybridization to show the presence of CMV antigens pp65 and IE1-72 as well as CMV nucleic acids, respectively. Our cohort indicated either CMV antigen pp65 or IE1-72 was present in approximately 67 % of pediatric GBM samples. The majority of samples stained positive for either CMV antigen showing a cytoplasmic pattern in 25-50 % of cells within the sample at a moderate intensity, while a few samples showed nuclear staining and higher grade/intensity. Of 16 samples where in situ hybridization was performed, 13 (81 %) showed specific staining using a CMV genome specific probe cocktail. ISH positive samples showed high concordance with being pp65 or IE1-72 positive. These findings, paired with the association of CMV expression with poor prognosis and overall survival, indicate the need to further investigate how these antigens are promoting tumor growth and preventing cell death. Also, the expression of these antigens in a majority of tumor tissues should be considered for immunotherapeutic targets in cases of pediatric GBM.

Keywords: CMV; GBM; Glioblastoma; IE1-72; Pediatric; pp65.

Fig. 1

IHC staining showing representative grade and intensity scoring. a Sample with 0 % positive staining. b Sample staining positive at Grade: 1 (0–25 %) with Intensity: 2+. c Sample staining positive at Grade: 2 (26–50 %) with Intensity: 1+. d Sample staining positive at Grade: 3 (51–75 %) with Intensity: 2+. e Sample staining positive at Grade: 4 (76–100 %) with Intensity: 3+. Grade and intensity were measured by three independent pathologist for all tested patients. Magnification ×100

Fig. 2

IHC for CMV pp65 and CMV IE1-72. Results from three representative patients are shown for CMV pp65 and CMV IE1-72For CMV pp65. (a) Patient 1 stained negative (b) Patient 2 stained Grade: 2 and Intensity: 2+, and (c) Patient 12 stained Grade: 1 and Intensity: 2+. For CMV IE1-72 (d) Patient 1 stained Grade: 2 and Intensity: 2+ (e) Patient 2 stained Grade: 2 and Intensity: 1+, and (f) Patient 12 stained Grade: 2 and I: 2+. Positive control is from CMV infected lung tissue and negative control has no primary antibody added. Magnification ×200. CMV positive control magnification ×400

Fig. 3

Distribution of grade and intensity of CMV pp65 and IE1-72 staining in a cohort of 25 pediatric GBM. Proportion of positive samples for each Grade (1, 2, 3 and 4) and Intensity (1+, 2+ and 3+) were analyzed for overall staining patterns. Grade and Intensity score is described in Fig. 1. Distribution of (a) Grade and (b) Intensity for samples staining positive for each CMV pp65 and CMV IE1-72 (n = 25 represents the 100 % mark)

Fig. 4

CMV genome-specific in situ hybridization (ISH). ISH was performed on 16 paraffin-embedded primary GBM samples using a CMV DNA probe. Representative results from one positive patient and one control are show. a Patient 12 staining positive for CMV genome using the CMV DNA probe. b Patient 12 staining using a negative control probe. c, d Magnification of the boxed areas in (a) and (b). e Staining of a positive control using the CMV DNA probe. f Staining of a positive control with a negative control probe. g Patient 2 staining negative for CMV genome using the CMV DNA probe. h Patient 12 staining using a negative control probe. Magnification ×100, c, d ×400

Fig. 5

Venn diagram corroborating pp65 and IE1 detection using IHC with CMV genome specific ISH. Of a total of 16 GBMs examined using ISH, 13 were positive for CMV and 3 were negative. Eleven of 13 samples showed concomitant ISH and IE1-72 (n = 10) positivity or concomitant ISH and pp65 positivity (n = 7). Six out of 13 samples were triple ISH, IE1-72 and pp65 positive