APNG as a prognostic marker in patients with glioblastoma

<div><p>Aim</p><p>Expression of the base excision repair enzyme alkylpurine-DNA-N-glycosylase (APNG) has been correlated to temozolomide resistance. Our aim was to evaluate the prognostic value of APNG in a population-based cohort with 242 gliomas including 185 glioblastomas (GBMs). Cellular heterogeneity of GBMs was taken into account by excluding APNG expression in non-tumor cells from the analysis.</p><p>Methods</p><p>APNG expression was evaluated using automated image analysis and a novel quantitative immunohistochemical (IHC) assay (qIHC), where APNG protein expression was evaluated through countable dots. Non-tumor cells were excluded using an IHC/qIHC double-staining. For verification, APNG was measured by a quantitative double-immunofluorescence (IF) assay. As validation APNG mRNA expression was evaluated using independent TCGA data.</p><p>Results</p><p>Using qIHC, high levels of APNG were associated with better overall survival (OS) in univariate (HR = 0.50; <i>P</i> < 0.001) and multivariate analysis (HR = 0.53; <i>P</i> = 0.001). Patients with methylated MGMT promoters and high APNG expression demonstrated better OS, than patients with methylated MGMT promoters and low APNG expression (HR = 0.59; <i>P</i> = 0.08). Retesting the cohort using IF showed similar results in both univariate (HR = 0.61; <i>P</i> = 0.002) and multivariate analysis (HR = 0.81; <i>P</i> = 0.2). The results were supported by data from the TCGA database.</p><p>Conclusions</p><p>Using two different assays combined with quantitative image analysis excluding non-tumour cells, APNG was an independent prognostic factor among patients with a methylated MGMT promoter. We expect that APNG qIHC can potentially identify GBM patients who will not benefit from treatment with temozolomide.</p></div

Images of APNG staining in different tumor subtypes.

<p>APNG expression visualized using quantitative immunohistochemistry (qIHC). Endothelial cells, leukocytes and microglia/macrophages were excluded using co-stainings. A, C, E: APNG expression in the nuclei is visualized and quantified by the number of red dots. Non-tumor cells were stained with DAB. IHC stainings were evaluated by 1) exclusion of non-tumor cells (DAB) and 2) visualization of APNG expression (red dots) in the tumor cells. B, D, F: Non-tumor cells were automatically excluded and APNG analysis was applied on tumor cells; nuclei are blue and APNG expression visualized as qIHC dots are orange. Scalebar: 100 μm.</p

Survival curves based on APNG and MGMT status.

<p>Median survival in patients with low APNG and methylated MGMT promoter was 2.8/6.1 months (qIHC/IF) compared to 4.5/11.3 months in patients with low APNG expression and a un-methylated MGMT promoter. Patients with high APNG expression and un-methylated MGMT promoter had a median overall survival of 9.8/9.6 months whereas patients with high APNG expression and methylated MGMT promoter (H/M) had a median overall survival of 20.3/20.3 months (<i>P</i> < 0.001). In multivariate analysis patients with high APNG expression and methylated MGMT promoter demonstrated a better overall survival than patients with low APNG expression and methylated MGMT promoter; HR 0.55, <i>P</i> = 0.04 (qIHC) and HR = 0.66, <i>P</i> = 0.1 (IF). Abbreviations: u-MGMT un-methylated MGMT promoter, m-MGMT methylated MGMT promoter. APNG low/high was defined according to the optimal cut point, which was for qIHC was 0.24 and for IF was 0.75.</p

Box-plot and survival curves for patients in the TCGA data set.

<p>The level of APNG mRNA was significantly higher in patients with WHO grade IV tumors compared to patients with WHO grade II and III tumors (A). When patients were divided at the median (B) or at the optimal cut point for qIHC (25% of the patients with the lowest APNG vs. the 75% of the patients with the highest APNG level) (C), APNG mRNA was not associated with overall survival. When divided at the optimal cut point for IF (61% of the patients with low APNG vs. the 39% with high APNG), a trend towards high APNG and improved overall survival (HR = 0.85; <i>P</i> = 0.10) was identified (D).</p

Staining of APNG and the corresponding processed images.

<p>APNG expression visualized using quantitative immunofluorescence (IF). Endothelial cells, leukocytes and microglia/macrophages were excluded using co-stainings. APNG negative (blue) and positive (green) nuclei were identified. Non-tumor cells are red. In B, D and F the image analysis software identifies the cells as follows: APNG-positive tumor cell nuclei (green), APNG-negative tumor nuclei (blue), non-tumor cell nuclei (white), extra-nuclear exclusion cocktail staining (pink), excluded nuclei (red and yellow) and background (turquoise).</p

Box-plots and survival curves.

<p>Box-plots showing the level of APNG and survival curves for APNG qIHC (A+C+E) and IF (B+D+E). <u><i>qIHC</i>:</u> There is no difference between the expression in level in patients with WHO grade IV tumors and patients with WHO grade I, II, and III (A). Divided at median APNG was prognostic (<i>P</i> = 0.02) (C). At optimal APNG cut point (25% vs 75% of the patients), HR improved (<i>P</i> < 0.001) (E). <u><i>IF</i>:</u> The expression in patients with WHO grade IV tumors was higher than the expression in patients with low-grade gliomas, but similar to the expression in patients with WHO grade III tumors (B). Divided at median there was a trend towards APNG being prognostic (<i>P</i> = 0.05) (D). At optimal APNG cut point (61% vs. 39% of the patients), APNG was a prognostic factor in univariate analysis (<i>P</i> = 0.002) (F).</p

Patient characteristics.

<p>Patient characteristics.</p

Multivariate Cox regression analysis of APNG protein levels in glioblastomas including clinical parameters.

<p>Multivariate Cox regression analysis of APNG protein levels in glioblastomas including clinical parameters.</p