An NF-κB signature predicts low-grade glioma prognosis: a precision medicine approach based on patient-derived stem cells

Abstract

Background: While recent genome-wide association studies have suggested novel low-grade glioma (LGG) stratification models based on a molecular classification, we explored the potential clinical utility of patient-derived cells. Specifically, we assayed glioma-associated stem cells (GASC) that are patient-derived and representative of the glioma microenvironment.

Methods: By next-generation sequencing, we analyzed the transcriptional profile of GASC derived from patients who underwent anaplastic transformation either within 48 months (GASC-BAD) or ≥7 years (GASC-GOOD) after surgery. Gene set enrichment and pathway enrichment analyses were applied. The prognostic role of a nuclear factor-kappaB (NF-κB) signature derived from GASC-BAD was tested in 530 newly diagnosed diffuse LGG patients comprised within The Cancer Genome Atlas (TCGA) database. The prognostic value of the GASC upstream regulator p65 NF-κB was assessed, by univariate and multivariate Cox analyses, in a single center case study, including 146 grade II LGGs.

Results: The key elements differentiating the transcriptome of GASC isolated from LGG with different prognoses were mostly related to hallmarks of cancer (eg, inflammatory/immune process, NF-κB activation). Consistently, the NF-κB signature extrapolated from the GASC study was prognostic in the dataset of TCGA. Finally, the nuclear expression of the NF-kB-p65 protein, assessed using an inexpensive immunohistochemical method, was an independent predictor of both overall survival and malignant progression-free survival in 146 grade II LGGs.

Conclusion: This study demonstrates for the first time the independent prognostic role of NF-kB activation in LGG and outlines the role of patient-based stem cell models as a tool for precision medicine approaches.

Fig. 1

(A) Study design. (B) Clinical-pathological features of LGG-bad patients included in the study. Table reports clinical data, histopathological diagnosis, Ki67 expression, and patient prognosis, referred to 3 LGGs with a good prognosis (GOOD) and 3 LGGs with a bad prognosis (BAD). F = female; M = male; D = dead; A = alive; n.s. = not significant. (C–I) GASC phenotype. Representative fluorescence images of the expression of Oct-4 (green, C), Nanog (red, D), Sox2 (yellow, E), vimentin (green, F), nestin (red, G), and glial fibrillary acidic protein (yellow, H) in a GASC line obtained from a patient characterized by a good prognosis. The blue of 4′,6′-diamidino-2-phenylindole identifies nuclei. (I) Quantitative analysis of the fraction of cells expressing the assayed pluripotent state specific transcription factors and intermediate filaments, as assessed by immunofluorescence. Data are presented as mean ± SD. No significant differences between GOOD- and BAD-GASC were detected. (J–K) Anchorage-independent growth of GASC. (J) Representative phase contrast images of colonies acquired 4 weeks after seeding in soft agar. (K) Data are presented as mean ± SD. No significant differences between GOOD- and BAD-GASC were detected.

Fig. 2

Prognostic role of the NF-κB signature in the 530 TCGA lower-grade gliomas. (A) The NF-κB signature has been obtained combining the genes of 3 NF-κB–related signatures enriched in genes differentially expressed in GASC with different prognoses. Kaplan-Meier curves showing OS (B), and PFS (C) in LGG patients stratified according to the NF-κB signature. (D) Level of expression of the NF-κB signature in the 3 molecular classes. Data are presented as box and whiskers. CODEL = gliomas IDH1/2 mutant and 1p/19q codeleted; IDH1/2 MUTANT = gliomas IDH1/2 mutant but not 1p/19q codeleted; IDH1/2 WT = gliomas without IDH1/2 gene mutation.

Fig. 2

Prognostic role of the NF-κB signature in the 530 TCGA lower-grade gliomas. (A) The NF-κB signature has been obtained combining the genes of 3 NF-κB–related signatures enriched in genes differentially expressed in GASC with different prognoses. Kaplan-Meier curves showing OS (B), and PFS (C) in LGG patients stratified according to the NF-κB signature. (D) Level of expression of the NF-κB signature in the 3 molecular classes. Data are presented as box and whiskers. CODEL = gliomas IDH1/2 mutant and 1p/19q codeleted; IDH1/2 MUTANT = gliomas IDH1/2 mutant but not 1p/19q codeleted; IDH1/2 WT = gliomas without IDH1/2 gene mutation.

Fig. 3

Expression of NF-κB-p65 protein in TMA comprising 146 grade II LGGs. (A–D) Representative pictures of the immunohistochemical detection of NF-κB-p65 in the 1-mm cores of LGG presenting the protein only in the cytoplasm (A–B) or in both the cytoplasm and the nucleus (C–D). (E) Table summarizing the quantification of total amount, nuclear expression, and cytoplasmic expression of p65 NF-κB-p65. (F) Nuclear expression of NF-κB-p65 (expressed as nuclear score) in the 3 molecular classes. Data are presented as box and whiskers. CODEL = gliomas IDH1/2 mutant and 1p/19q codeleted; IDH1/2 MUTANT = gliomas IDH1/2 mutant but not 1p/19q codeleted; IDH1/2 WT = gliomas without IDH1/2 gene mutation