- Open Access
Akt and mTORC1 signaling as predictive biomarkers for the EGFR antibody nimotuzumab in glioblastoma
- Michael W. Ronellenfitsch1, 2, 3Email authorView ORCID ID profile,
- Pia S. Zeiner1, 2, 3, 4,
- Michel Mittelbronn4, 5, 6, 7,
- Hans Urban1, 2, 3,
- Torsten Pietsch8,
- Dirk Reuter9,
- Christian Senft10,
- Joachim P. Steinbach1, 2, 3,
- Manfred Westphal11 and
- Patrick N. Harter2, 3, 4Email authorView ORCID ID profile
© The Author(s). 2018
- Received: 10 August 2018
- Accepted: 10 August 2018
- Published: 21 August 2018
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.
- Epidermal growth factor receptor
- Mammalian target of rapamycin
- Targeted therapy
Glioblastoma (GB) is an incurable brain cancer and the most common primary brain tumor in adults . The epidermal growth factor receptor (EGFR) is frequently genetically altered in GB by gene amplification and mutations including a variant where deletion of exons 2–7 causes activated signaling termed EGFRvIII. EGFR gene alterations can be found in 45.1% of GBs , mutations in members of the receptor tyrosine kinase- Ras-PI3 Kinase-AKT signaling network are the most frequent mutations (87.9% of cases) in GB . Further, EGFR signaling is known to enhance proliferative signaling, resistance to cell death and reprogramming of energy metabolism [13, 38, 45]. Therefore, EGFR is a plausible target in GB therapy. Several clinical trials have been performed, with however rather disappointing results . Strategies targeting EGFR in GB include small molecule inhibitors (e.g. erlotinib), antibodies or antibody-drug conjugates (e.g. depatuxizumab mafodotin (ABT-414)) as well as novel immunooncological approaches like a vaccine against EGFRvIII with rindopepimut. The depatuxizumab antibody portion of ABT-414 preferentially binds to cells with amplified EGFR or EGFRvIII . After binding ABT-414 is internalized and can block microtubule formation via its mafodotin part . Currently larger phase II and III clinical trials are underway evaluating ABT-414 in the primary (Intellance 1 phase III trial, ClinicalTrials.gov NCT02573324) and recurrent disease (Intellance 2 phase II trial, ClinicalTrials.gov NCT02343406) setting. In the ACT IV trial, the EGFRvIII vaccine rindopepimut did not prolong survival in GB patients . It is noteworthy that the EGFRvIII mutation if present usually is only found in a fraction of tumor cells within a GB  and that even during the course of standard treatment EGFRvIII is frequently lost . Standard treatment for patients in sufficient clinical condition has been established in 2005 already and involves surgical resection, radiotherapy and chemotherapy with the alkylating agent temozolomide which led to median overall survival times of 14.6 months . Many trials have been conducted in recent years, however, no new drugs have been approved [27, 39]. Histologically, GB is characterized by marked hypoxic areas, with typical histological features of neoangiogenesis and necrosis in a diffusely infiltrating growing glial tumor . These areas reflect the metabolically challenging microenvironment where nutrient and oxygen supply can frequently not match demand of the tumor cells. The transcription factor hypoxia-inducible factor 1α (HIF-1α) is a major cellular regulator of adaptive programs to hypoxia and stabilization occurs when oxygen is low .
The current WHO classification further stratifies GB as either isocitrate dehydrohgenase (IDH) wildtype (wt) or IDH mutant (mut). The vast majority of primary GB harbors IDH wt status . Further, current treatment relevant molecular stratification of GB mainly depends on the methylation status of the O(6)-methylguanine methyltransferase (MGMT)-promoter. MGMT-promoter methylation correlates with reduced expression of the DNA repair enzyme MGMT. Consequently, tumors with methylated MGMT promoter generally respond better to temozolomide treatment whereas MGMT expression in tumors with unmethylated gene promoter is a major mechanism of resistance and indicator for poor prognosis [15, 16, 46].
Nimotuzumab is a blocking monoclonal antibody against EGFR  without intrinsic EGFR activating activity. It has shown promising results as a targeted therapy in the treatment of high grade gliomas in phase II studies  and pediatric brain stem gliomas [28, 57]. Therefore, a two arm phase III clinical trial (OSAG 101-BSA-05) involving 149 patients was performed comparing standard (radiotherapy and temozolomide) treatment with and without addition of nimotuzumab (EudraCT No. 2005–003101-85, ClinicalTrials.gov NCT00753246) . Nimotuzumab was administered once weekly (400 mg) during the concomitant radio-temozolomide phase and afterwards continued biweekly (400 mg) for 12 weeks during the adjuvant temozolomide treatment phase. The trial was negative, and a benefit of nimotuzumab treatment was apparent neither in the whole population studied nor in patients with EGFR amplification. A post-hoc analysis of subgroups, however, revealed a trend for improved survival for MGMT unmethylated patients with residual tumor when treated with nimotuzumab (PFS 6.2 vs. 4.0 months; OS 19.0 vs. 13.8 months). This unplanned subgroup analysis, however, included only 28 patients and failed to reach statistical significance. The results of several recent trials suggest that for an effective targeted therapy, appropriate patients need to be identified . With regard to signal transduction inhibitors it is plausible that genetic heterogeneity in GBs is also reflected by different degrees of dependence on certain signaling cascades . The aim of this study was to analyze EGFR-dependent Akt and mTORC1 signaling in treatment-naïve tumor samples of the OSAG 101-BSA-05 patient cohort as a potential predictive biomarker of nimotuzumab efficacy. We analyzed the response to nimotuzumab therapy of molecular subgroups depending on activation of Akt and mTORC1 signaling, extent of necrosis, HIF-1α staining and MGMT-methylation status. We here report a predictive signature of RPS6 and PRAS40 phosphorylation in MGMT unmethylated patients. Furthermore, we describe a trend for a predictive value of RPS6 phosphorylation in all patients irrespective of MGMT promoter methylation status.
Nimotuzumab as well as the corresponding placebo control solution were provided by Oncoscience (Wedel, Germany). Nimotuzumab is an IgG subtype 1 kappa with a molecular weight of 147.613 kDa. The EGFR inhibitor PD153035  was purchased from Sigma Aldrich (Taufkirchen, Germany).
LNT-229 GB cells have been described previously [38, 50] and were maintained in Dulbecco’s modified eagle medium (DMEM) containing 10% foetal calf serum (FCS) (Biochrom KG, Berlin, Germany), 100 IU/ml penicillin and 100 mg/ml streptomycin (Life Technologies, Darmstadt, Germany).
Immunoblot was performed as described previously . 10 μg of protein per condition were used for SDS-PAGE analysis. Membranes were incubated with antibodies against phospho-RPS6 (Ser 240/244) (D68F8; Cell Signaling), phospho-RPS6 (Ser 235/236) (D57.2.2.E; Cell Signaling), phospho-PRAS40 (Thr246) (C77D7, Cell signaling) or actin (# sc-1616 Santa Cruz Biotechnology, Dallas, Texas, USA). The secondary HRP-conjugated antibodies were purchased from Santa Cruz Biotechnology (Dallas, Texas, USA). A chemiluminescence solution was used for detection .
Patients, sample collection and immunohistochemistry
The OSAG 101-BSA-05 study (EudraCT No. is 2005–003101-85, ClinicalTrials.gov
NCT00753246) cohort included 149 patients with GB . This open label, randomised phase III study was approved by the central and local ethics review boards. Informed consent was obtained from all patients. In case of availability, we obtained tissue sections from these tumors for further immunohistochemistry. We investigated the amount of necrosis (%) in hematoxylin and eosin (HE)-stained slides of the tissue sections (n = 111), HIF-1α expression (%) in the vital tumor centre (n = 106) as well as in perinecrotic areas (n = 98), P-PRAS40-positive cells (%) (n = 101), P-RPS6-positive cells (%) (n = 109) as well as Iba1-positive cells (%) (n = 100) using standard procedures on an automated IHC staining system. Stainings with antibodies against threonine 246-phosphorylated PRAS40 (P-PRAS40) and serine 240/244-phosphorylated RPS6 (P-RPS6) (Cell signaling, #2997 and #5364 respectively) were performed as recently reported . Furthermore, the following antibodies were used: HIF-1α (Novus Biologicals, NB 100–134), Iba1 (Wako, 019–19,741). Samples that consisted of 100% necrosis were excluded from further analysis.
Statistical analyses were performed using JMP version 13 software (SAS Institute, Heidelberg, Germany). A p-value of p < 0.05 was chosen to declare statistical significance. Applied statistical test methods are either mentioned in the figure legend or in the flow content. For dichotomized univariate survival analyses we performed a median split to obtain a high and low group with regard to the investigated factor. The high group includes specimen with values above median, the low group includes specimen with median or below.
Nimotuzumab inhibits EGFR downstream signaling
To test whether nimotuzumab inhibited signaling from the EGFR-downstream kinases Akt and mTORC1 (Fig. 1a), we exposed human LNT-229 glioblastoma cells to nimotuzumab or the intracellular EGFR inhibitor PD153035 . Both substances caused effective inhibition of EGFR downstream signal transduction indicated by a similar degree of reduction in phosphorylation of the corresponding target proteins PRAS40 as well as RPS6 in an immunoblot experiment (Fig. 1b). We chose P-PRAS40 (Thr246) and P-RPS6 (Ser240/244) in our further tissue analysis due to the specificity of the phosphorylation site and the availability of robust, monoclonal antibodies for IHC. Effective Akt inhibition by nimotuzumab had also previously been reported in other cell lines including EGFR overexpressing U87 GB cells, lung and nasopharyngeal carcinoma cells [7, 18, 37].
Phosphorylation of PRAS40 and RPS6 is only detectable in a small proportion of tumor cells and does not correlate with EGFR gene amplification
Information on EGFR amplification and vIII mutation was available for 88 and 81 cases respectively . EGFR gene amplification correlates with increased expression of EGFR  and was found in 43 cases. An inverse effect was detectable on downstream Akt but not mTORC1 signal transduction (Fig. 2l). However, with only 7 cases of vIII mutation in our cohort, the number was too small to derive any conclusions in this regard. Notably, there was also no difference in the end points for patients with and without EGFR amplification or vIII mutation in the OSAG 101-BSA-05 trial .
Necrosis extent and HIF-1α staining is not associated with patient survival
Correlation of histology markers with survival
Parametric survival Weibull p
HIF-1α perinecrotic area
HIF-1α vital tumor
Treatment of hypoxic tumors with nimotuzumab is not detrimental
Unmethylated MGMT promoter status defines a subgroup in which high necrosis, P-RPS6 or P-PRAS40 tumors benefit from nimotuzumab treatment
P-RPS6 expression predicts survival depending on the treatment group in MGMT promoter unmethylated GBs
Increased GAM levels correlate with improved survival in patients treated with nimotuzumab
The experience with targeted therapies in recent GB trials has been overall disappointing highlighting the need for predictive biomarkers. In this retrospective analysis of samples of the OSAG 101-BSA-05 trial , we investigated histological subgroups based on necrosis and hypoxia as markers for a nutrient-deprived tumor microenvironment as well as for phosphorylation of PRAS40 and RPS6 as downstream markers of EGFR signaling. We hypothesized a reduced efficacy of EGFR inhibition therapy in tumors with pronounced necrosis or hypoxia due to potential protective effects of inhibitor therapy in this context [38, 45]. Tumor hypoxia as indicated by HIF-1α staining as well as necrosis were not associated with patient survival (Table 1). When using a median split for necrosis extent, on the contrary to our hypothesis, there was a slight trend towards improved efficacy of nimotuzumab in patients with tumors with above median necrosis (Fig. 3a). No trend was detectable using a median split for perinecrotic HIF-1α staining frequency (Fig. 3b). HIF-1α staining frequency in vital tumor tissue was low with a median of 0%, therefore we did not include a dichotomized analysis in our study. GBs with increased signaling from EGFR and downstream kinases might constitute a collective with oncogene addiction exposing an Achilles heel for targeted therapies. Dichotomizing for P-PRAS40 high and low tumors had no effect on nimotuzumab treatment efficacy (Fig. 3c), in contrast to P-RPS6 where a clear trend towards nimotuzumab efficacy was detectable in tumors with high P-RPS6 (Fig. 3d). Neither P-PRAS40 nor P-RPS6 was associated with patient survival (Table 1). However, when testing for time to progression, P-PRAS40 was associated with a shorter interval (Additional file 4: Figure S4A) similar to a previous report .
The majority of GB (approximately 55–65%) has an unmethylated MGMT promoter defining a subgroup that is especially difficult to treat due to the reduced efficacy of temozolomide [9, 15, 22]. When investigating only MGMT unmethylated tumors, above median P-RPS6 was associated with nimotuzumab efficacy (Fig. 4a) which has already been detectable as a trend in the whole study cohort (Fig. 3a, d). In addition, above median P-PRAS40 was associated with improved survival in patients treated with nimotuzumab (Fig. 4a). The positive correlation between necrosis extent and P-RPS6 (Fig. 2j) was unexpected considering that mTORC1 is also a component of central cellular nutrient sensing pathways and cells with intact nutrient sensing inhibit mTORC1 in nutrient deplete conditions . This indicates a potentially dysregulated mTORC1 sensor in our cohort resulting in higher extent necrosis as has been suggested recently (Additional file 4: Figure S4B) . The efficacy of nimotuzumab in patients with high P-RPS6 (as a trend in the whole study cohort and statistically significant only in MGMT unmethylated GBs) points to a potentially higher degree of addiction to mTORC1 and ultimately EGFR signaling in this subgroup. While the homogeneous patient cohort of a registered randomized phase III trial adhering to central monitoring standards was a major strength of our study, introducing subgroups naturally shrunk patient numbers and our results need to be validated prospectively in a larger patient cohort using our EGFR signaling markers as entry criteria. Additionally, PTEN and PI3 Kinase loss/mutation are frequent events in GB (~ 36% and ~ 6% of GB samples respectively)  and most likely partly impact nimotuzumab efficacy. Therefore, it is remarkable that P-RPS6 dichotomization was sufficient to define a subgroup with a clear trend towards nimotuzumab efficacy in samples of unknown PTEN and PI3 Kinase status (Fig. 3). In an upcoming prospective analysis, it would be important to include these markers and PTEN and PI3 Kinase wildtype status would most likely define an even more nimotuzumab-susceptible subgroup of tumors. Accordingly, in a previously published retrospective analysis of tissue of 26 GB patients treated with the non-antibody EGFR inhibitors erlotinib or gefitinib response in the recurrent disease setting correlated with expression of vIII-mutated EGFR and PTEN . No evaluation of downstream phosphorylation events in the tumor tissue was included in this analysis, still these results suggest that tumors with high EGFR signaling activity and intact signal transduction are sensitive to EGFR inhibitors. In the recent phase II EORTC 26082 trial, similar to our results, mTORC1 activation as indicated by phosphorylation of the mTOR protein itself at Ser2448 was a marker to predict response to treatment with the mTOR inhibitor temsirolimus in MGMT unmethylated GBs . The relevant kinase that mediates phosphorylation of mTOR at Ser2448 is S6 Kinase  which is exactly the same kinase that mediates RPS6 phosphorylation and therefore is responsible for P-RPS6 in our cohort (Additional file 4: Figure S4C). Additionally, in multivariate analyses, PRAS40 phosphorylation was associated with survival in the temsirolimus treatment arm . The authors propose phosphorylated mTOR (Ser2448) and P-PRAS40 as potential biomarkers for mTOR inhibitor therapy in MGMT-promoter unmethylated GBs. Our results confirm this notion with nimotuzumab as an indirect mTORC1 inhibitor (Fig. 1b). Integrating the results of the analyses of predictive signatures for EGFR  and mTOR inhibitors  and our analysis points to a signature where a high (er) degree of activation and an intact EGFR signaling axis defines GBs susceptible to inhibitors of this pathway in general. Accurate analysis of the in vivo phosphorylation status of proteins by IHC to monitor EGFR signaling activity requires special caution. E.g. time to processing and several other factors can have a major influence on phosphorylation and dephosphorylation events . Therefore, for a prospective analysis of biomarkers in a clinical trial, standardized tissue asservation will be an important topic to include in the protocol.
The need for and potential adverse effects of neglecting potential predictive biomarkers is highlighted by the recently published results of the thus far largest randomized phase II trial evaluating the efficacy of the mTORC1 inhibitor everolimus in newly diagnosed GB that randomized 171 patients . Patients receiving everolimus in addition to standard radiochemotherapy in this trial had a reduced survival in comparison to sole standard radiochemotherapy [1, 6]. One potential explanation of these results demonstrating reduced survival when an mTOR inhibitor was added to the therapeutic regimen in GB could be protective effects of mTOR inhibition in the context of the tumor microenvironment that we have previously shown in cell culture models .
Data regarding the prognostic impact of the innate immune system including GAMs in GBs is conflicting [12, 21]. In our study cohort, we found a positive effect on prolonged overall survival in patients treated with nimotuzumab with GBs of above median Iba1 frequency (Fig. 6). Investigating the whole patient cohort irrespective of treatment arm, we found no association with survival when dichotomizing for high vs. low GAM infiltration (Additional file 4: Figure S4D). These results contrast the notion that GAM subpopulations might have negative effects on GB patient survival . However, similar findings as in our cohort regarding the prognostic role of GAMs are described, likewise demonstrating a positive prognostic impact of at least a GAM subpopulation in GB . Currently we can only speculate on the underlying reasons for this positive effect of intratumoral GAMs on overall survival in GB patients treated with nimotuzumab. It is interesting to note that microglia express receptors for binding of the Fc part of antibodies and might therefore react with nimotuzumab-bound GB cells similar to mechanistic hypotheses of antibody mediated plaque clearance in Alzheimer’s models . Further clarifying potential antibody effects on GAMs is beyond the scope of this article and should be investigated elsewhere.
The quest for new treatment options in GB has been cumbersome at best with no new drugs gaining approval since the introduction of temozolomide. In this current study, we investigated tissue samples of yet another negative phase III trial. The EGFR is one of the most plausible treatment targets in this cancer entity. We here report markers for the selection of patients that might benefit from the EGFR-blocking antibody nimotuzumab. Considering the majority of GB patients with unmethylated MGMT promoter status, activation of Akt or mTORC1 signaling was associated with a benefit from nimotuzumab treatment. A clear trend towards a benefit from nimotuzumab therapy was also detectable in the whole study cohort using activation of mTORC1 as a marker for dichotomy. We believe that our results constitute a basis for further investigation of nimotuzumab or other EGFR- and mTOR-inhibitors in selected patient cohorts using the reported criteria as candidate predictive biomarkers.
The Dr. Senckenberg Institute of Neurooncology is supported by the Dr. Senckenberg Foundation and the Hertie Foundation. J.P.S. is “Hertie Professor of Neurooncology”. P.S.Z. has received funding by the Frankfurt Research Funding (FFF) (program “Nachwuchswissenschaftler”). M.W.R. and P.N.H. have received a fellowship by the University Cancer Centre Frankfurt (UCT). M.W.R. has also received funding by the Frankfurt Research Funding (FFF) ‘Clinician Scientists Program’. M.M. would like to thank the Luxembourg National Research Fond (FNR) for the support (FNR PEARL P16/BM/11192868 grant).
Study design and writing of the manuscript: MWR, PSZ, MM, JPS, MW, PNH. Provided material, data collection and data analyses: MWR, PSZ, MM, TP, DR, CS, JPS, MW, PNH. Performed experiments: MWR, PSZ, HU, PNH. All authors read and approved the final manuscript.
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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