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Clinical Trial
. 2018 Aug 21;6(1):81.
doi: 10.1186/s40478-018-0583-4.

Akt and mTORC1 signaling as predictive biomarkers for the EGFR antibody nimotuzumab in glioblastoma

Michael W Ronellenfitsch  1   2   3 , Pia S Zeiner  4   5   6   7 , Michel Mittelbronn  7   8   9   10 , Hans Urban  4   5   6 , Torsten Pietsch  11 , Dirk Reuter  12 , Christian Senft  13 , Joachim P Steinbach  4   5   6 , Manfred Westphal  14 , Patrick N Harter  15   16   17
Affiliations
Clinical Trial

Akt and mTORC1 signaling as predictive biomarkers for the EGFR antibody nimotuzumab in glioblastoma

Michael W Ronellenfitsch et al. Acta Neuropathol Commun. .

Abstract

Glioblastoma (GB) is the most frequent primary brain tumor in adults with a dismal prognosis despite aggressive treatment including surgical resection, radiotherapy and chemotherapy with the alkylating agent temozolomide. Thus far, the successful implementation of the concept of targeted therapy where a drug targets a selective alteration in cancer cells was mainly limited to model diseases with identified genetic drivers. One of the most commonly altered oncogenic drivers of GB and therefore plausible therapeutic target is the epidermal growth factor receptor (EGFR). Trials targeting this signaling cascade, however, have been negative, including the phase III OSAG 101-BSA-05 trial. This highlights the need for further patient selection to identify subgroups of GB with true EGFR-dependency. In this retrospective analysis of treatment-naïve samples of the OSAG 101-BSA-05 trial cohort, we identify the EGFR signaling activity markers phosphorylated PRAS40 and phosphorylated ribosomal protein S6 as predictive markers for treatment efficacy of the EGFR-blocking antibody nimotuzumab in MGMT promoter unmethylated GBs. Considering the total trial population irrespective of MGMT status, a clear trend towards a survival benefit from nimotuzumab was already detectable when tumors had above median levels of phosphorylated ribosomal protein S6. These results could constitute a basis for further investigations of nimotuzumab or other EGFR- and downstream signaling inhibitors in selected patient cohorts using the reported criteria as candidate predictive biomarkers.

Keywords: Biomarker; Epidermal growth factor receptor; Glioblastoma; Mammalian target of rapamycin; Nimotuzumab; Targeted therapy.

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Conflict of interest statement

Competing interests

MWR, JPS and PNH received a grant to purchase materials necessary for immunohistochemistry from Oncoscience, the pharmaceutical company that owns nimotuzumab. DR is an employee and managing director of Oncoscience.

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Figures

Fig. 1
Fig. 1
EGFR signal transduction and effects of EGFR inhibition on downstream targets. a Scheme of EGFR signal transduction. Nimotuzumab and PD153035 are inhibitors of EGFR: Activation of EGFR results in activation of Akt signaling which relieves a TSC1/TSC2 as well as PRAS40 (via phosphorylation of Thr246) -mediated inhibition of mTORC1. RPS6 phosphorylation at Ser235/236 and Ser 240/244 is regulated by mTORC1. b LNT-229 cells were incubated in serum-free DMEM for 90 min with vehicle (DMSO control), PD153035 (dissolved in DMSO), control solution for nimotuzumab (placebo solution of the trial) or 1 μM nimotuzumab as indicated. Cellular lysates were analyzed by immunoblot with antibodies as indicated
Fig. 2
Fig. 2
Histological characterization of the patient cohort. a-f, outlier box plot for the distribution of necrosis, HIF-1α in vital, central or perinecrotic tumor areas, phosphorylated (P-)RPS6, P-PRAS40 and Iba1 in samples as indicated (horizontal line within the box is the median sample value; confidence diamond contains the mean and the upper and lower 95% of the mean; ends of the box represent the 25th and 75th quantiles; bracket outside of the box is the shortest half, which is the most dense 50% of observations). g-k, correlations of histological markers as indicated in a bivariate plot with a linear regression analysis. P and r2 values as indicated. l one way analysis with outlier box plot of P-PRAS40 and P-RPS6 in EGFR amplified vs. non-amplified tumor specimens. P-value calculated using Student’s t-test
Fig. 3
Fig. 3
Survival analyses depending on treatment in histological subgroups. a-d Kaplan-Meier survival curves for patients treated with nimotuzumab (nimo) or placebo (cont) in dichotomized histological subgroups (median split, above median: high, below and equal to median low) for necrosis (a), HIF-1α in perinecrotic regions (b), P-PRAS40 (c) and P-RPS6 (d). P values were calculated using the Wilcoxon test
Fig. 4
Fig. 4
Survival analyses depending on treatment in histological subgroups for the MGMT-promoter unmethylated and methylated tumor cohort. a-b Kaplan-Meier survival curves for patients treated with nimotuzumab (nimo) or placebo (cont) in dichotomized histological subgroups (median split, above median: high, below and equal to median low) for necrosis, P-PRAS40 and P-RPS6 in the MGMT-promoter unmethylated (a) and methylated (b) tumor cohort. P values were calculated using the Wilcoxon test
Fig. 5
Fig. 5
Prognostic relevance of P-RPS6 and P-PRAS40 in treatment groups of MGMT-promoter unmethylated tumors. a-b Kaplan-Meier survival curves for patients with MGMT promoter unmethylated GBs treated with nimotuzumab or placebo (control) for dichotomized histological subgroups (median split, above median: high, below and equal to median low) for P-RPS6 (a) and P-PRAS40 (b). P values were calculated using the Wilcoxon test
Fig. 6
Fig. 6
Survival analysis depending on treatment in subgroups based on microglial prevalence. Kaplan-Meier survival curves for patients treated with nimotuzumab (nimo) or placebo (cont) in dichotomized subgroups based on Iba1 staining frequency (median split, above median: high, below and equal to median low). P values were calculated using the Wilcoxon test
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