Lale Erdem-Eraslan

Intrinsic Molecular Subtypes of Glioma Are Prognostic and Predict Benefit From Adjuvant Procarbazine, Lomustine, and Vincristine Chemotherapy in Combination With Other Prognostic Factors in Anaplastic Oligodendroglial Brain Tumors: A Report From EORTC Study 26951

PURPOSE Intrinsic glioma subtypes (IGSs) are molecularly similar tumors that can be identified based on unsupervised gene expression analysis. Here, we have evaluated the clinical relevance of these subtypes within European Organisation for Research and Treatment of Cancer (EORTC) 26951, a randomized phase III clinical trial investigating adjuvant procarbazine, lomustine, and vincristine (PCV) chemotherapy in anaplastic oligodendroglial tumors. Our study includes gene expression profiles of formalin-fixed, paraffin-embedded (FFPE) clinical trial samples. PATIENTS AND METHODS Gene expression profiling was performed in 140 samples, 47 fresh frozen samples and 93 FFPE samples, on HU133_Plus_2.0 and HuEx_1.0_st arrays, respectively. RESULTS All previously identified six IGSs are present in EORTC 26951. This confirms that different molecular subtypes are present within a well-defined histologic subtype. Intrinsic subtypes are highly prognostic for overall survival (OS) and progression-free survival (PFS). They are prognostic for PFS independent of clinical (age, performance status, and tumor location), molecular (1p/19q loss of heterozygosity [LOH], IDH1 mutation, and MGMT methylation), and histologic parameters. Combining known molecular (1p/19q LOH, IDH1) prognostic parameters with intrinsic subtypes improves outcome prediction (proportion of explained variation, 30% v 23% for each individual group of factors). Specific genetic changes (IDH1, 1p/19q LOH, and EGFR amplification) segregate into different subtypes. We identified one subtype, IGS-9 (characterized by a high percentage of 1p/19q LOH and IDH1 mutations), that especially benefits from PCV chemotherapy. Median OS in this subtype was 5.5 years after radiotherapy (RT) alone versus 12.8 years after RT/PCV (P = .0349; hazard ratio, 2.18; 95% CI, 1.06 to 4.50). CONCLUSION Intrinsic subtypes are highly prognostic in EORTC 26951 and improve outcome prediction when combined with other prognostic factors. Tumors assigned to IGS-9 benefit from adjuvant PCV.

MGMT-STP27 methylation status as predictive marker for response to PCV in anaplastic oligodendrogliomas and oligoastrocytomas. A report from EORTC study 26951

Purpose: The long-term follow-up results from the EORTC-26951 trial showed that the addition of procarbazine, CCNU, and vincristine (PCV) after radiotherapy increases survival in anaplastic oligodendrogliomas/oligoastrocytomas (AOD/AOA). However, some patients appeared to benefit more from PCV treatment than others. Experimental Design: We conducted genome-wide methylation profiling of 115 samples included in the EORTC-26951 trial and extracted the CpG island hypermethylated phenotype (CIMP) and MGMT promoter methylation (MGMT-STP27) status. Results: We first show that methylation profiling can be conducted on archival tissues with a performance that is similar to snap-frozen tissue samples. We then conducted methylation profiling on EORTC-26951 clinical trial samples. Univariate analysis indicated that CIMP+ or MGMT-STP27 methylated tumors had an improved survival compared with CIMP− and/or MGMT-STP27 unmethylated tumors [median overall survival (OS), 1.05 vs. 6.46 years and 1.06 vs. 3.8 years, both P < 0.0001 for CIMP and MGMT-STP27 status, respectively]. Multivariable analysis indicates that CIMP and MGMT-STP27 are significant prognostic factors for survival in presence of age, sex, performance score, and review diagnosis in the model. CIMP+ and MGMT-STP27 methylated tumors showed a clear benefit from adjuvant PCV chemotherapy: the median OS of CIMP+ samples in the RT and RT-PCV arms was 3.27 and 9.51 years, respectively (P = 0.0033); for MGMT-STP27 methylated samples, it was 1.98 and 8.65 years. There was no such benefit for CIMP- or for MGMT-STP27 unmethylated tumors. MGMT-STP27 status remained significant in an interaction test (P = 0.003). Statistical analysis of microarray (SAM) identified 259 novel CpGs associated with treatment response. Conclusions: MGMT-STP27 may be used to guide treatment decisions in this tumor type. Clin Cancer Res; 19(19); 5513–22. ©2013 AACR.

Mutation specific functions of EGFR result in a mutation-specific downstream pathway activation

BACKGROUND Epidermal growth factor receptor (EGFR) is frequently mutated in various types of cancer. Although all oncogenic mutations are considered activating, different tumour types have different mutation spectra. It is possible that functional differences underlie this tumour-type specific mutation spectrum. METHODS We have determined whether specific mutations in EGFR (EGFR, EGFRvIII and EGFR-L858R) have differences in binding partners, differences in downstream pathway activation (gene expression and phosphoproteins), and have functional consequences on cellular growth and migration. RESULTS Using biotin pulldown and subsequent mass spectrometry we were able to detect mutation specific binding partners for EGFR. Differential binding was confirmed using a proximity ligation assay and/or Western Blot for the dedicator of cytokinesis 4 (DOCK4), UDP-glucose glycoprotein glucosyltransferase 1 (UGGT1), MYC binding protein 2 (MYCBP2) and Smoothelin (SMTN). We also demonstrate that each mutation induces the expression of a specific set of genes, and that each mutation is associated with specific phosphorylation patterns. Finally, we demonstrate using stably expressing cell lines that EGFRvIII and EGFL858R display reduced growth and migration compared to EGFR wildtype expressing cells. CONCLUSION Our results indicate that there are distinct functional differences between different EGFR mutations. The functional differences between different mutations argue for the development of mutation specific targeted therapies.

Abstract 642: A hypermethylated phenotype as predictive marker for response to PCV in anaplastic oligodendrogliomas. A report from EORTC study 26951.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Introduction: In 1995 a large European phase III clinical trial (‘EORTC 26951’) was initiated to examine the effects of adjuvant procarbazine, CCNU and vincristine (PCV) chemotherapy in anaplastic oligodendrogliomas (AOD and AOA). This trial showed that the addition of 6 cycles PCV after 59.4 Gy RT increases overall survival (OS) and progression free survival (PFS) in these tumors. However, some patients appeared to benefit more from the addition of PCV treatment than others. In current study, we aimed to identify the patients in this trial that benefit from adjuvant PCV treatment using genome wide methylation profiling. Methods: Methylation profiles of a total of 115 samples were generated, 49 of which were reported previously. Results: Most (59/66) samples were formalin-fixed and embedded in paraffin (FFPE). Our first experiment was therefore aimed at determining the performance of methylation arrays using such tissue. Paired snap frozen (FF)-FFPE sample analysis on six glioma samples demonstrated that the correlation between FF and FFPE samples was high: 0.961±0.023. Between FFPE technical replicates it was 0.987±0.009. These results demonstrate that methylation profiling can be performed on DNA isolated from FFPE samples. We then performed methylation profiling on an additional 66 samples of the EORTC26951 trial (59 FFPE, 7 FF) and combined the data with those of the 49 FF samples previously analyzed. The cohort analyzed for methylation profiling had similar characteristics as the entire EORTC26951 cohort. However, OS within the RT-only treatment arm of included patients was worse compared to OS in patients not included. Univariate analysis indicated that CIMP (CpG island methylator phenotype) status was a favorable prognostic marker for OS with CIMP+ tumors having a more favorable prognosis than CIMP- tumors (median OS 1.05 v. 6.46 years HR 0.225 95% CI [0.138, 0.369], P<0.0001. Multivariate analysis indicates that CIMP status is a prognostic factor for overall survival that is independent of clinical and histological parameters (age, sex, performance score and review diagnosis). IDH1 mutations (39/51), 1p19q LOH (29/63) and MGMT promoter methylation (45/52) were predominantly identified in CIMP+ tumors whereas EGFR amplification was predominantly identified in the CIMP- subtype (20/42, 48%). When stratified for treatment, CIMP+ tumors showed a clear benefit from adjuvant PCV chemotherapy, both for OS and PFS. Median OS of CIMP+ samples in the RT and RT-PCV arms was 3.27 and 9.36 years respectively (HR: 0.409; 95% CI [0.224,0.746], P=0.0036). There was no such benefit for CIMP- tumors. Conclusion: Our results suggest that CIMP status is predictive for benefit from adjuvant PCV in AODs and AOAs in samples of the EORTC 26951 clinical trial. Further validation of these results is urgently required. Citation Format: Pim J. French, Lale Erdem-Eraslan, Ahmed Idbaih, Wim Spliet, Wilfred den Dunnen, Johannes L. Teepen, Pieter Wesseling, Peter A. Sillevis Smitt, Johan M. Kros, Thierry Gorlia, Martin van den Bent. A hypermethylated phenotype as predictive marker for response to PCV in anaplastic oligodendrogliomas. A report from EORTC study 26951. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 642. doi:10.1158/1538-7445.AM2013-642

Abstract 3142: Whole genome sequencing of oligodendrogliomas identifies genes mutated at a low frequency that contribute to tumor formation.

Background: Causal genetic changes in oligodendrogliomas (OD) with 1p/19q codeletion include mutations in IDH1, IDH2, CIC and FUBP1. Here, we have performed whole genome sequencing on three ODs to determine whether additional genetic changes contribute to tumor formation. Methods: We performed whole genome sequencing of DNA from 3 ODs grade III with 1p/19q codeletion and matched germline DNA. Targeted resequencing of identified genes was performed in an additional 32 ODs with 1p19q LOH (28/32) or partial loss of 1p (2/32), 19q (2/32). All mutations were validated by Sanger sequencing. Constructs of wildtype and mutated genes were subsequently generated (n=13), fused to GFP for visualization. Established cell lines were created to perform functional analysis. Results: Whole genome sequencing identified a total of 55 mutations in coding exons (range 8-32 mutations per tumor), including the known molecular abnormalities in IDH1 (2/3), IDH2 (1/3), CIC (2/3) and FUBP1 (1/3). Mutations in the ATRX gene were not identified. In addition to these known genes, we identified mutations in additional genes, most of which were previously not implicated in ODs. We first examined the mutation frequency of these genes in an additional 32 tumors. No additional mutations were identified. We then performed functional analysis on a subset of these mutations. For ZNF238, we observed a difference in the sub cellular localization between wildtype and mutant contructs; the wildtype protein localized to the nucleus while the mutant protein is present in the cytoplasm. In addition, stably transfected ZNF238 mutant cell line shows increased proliferation compared to wildtype. Conclusion: Our results confirm that mutations in IDH, CIC and FUBP1 are present at high frequency in oligodendrogliomas with 1p/19q loss. Functional analysis of infrequently mutated genes provide evidence that they contribute to oncogenesis. Citation Format: Lale Erdem-Eraslan, Maurice de Wit, Daphne Heijsman, Andreas Kremer, Peter J. van der Spek, Peter A.E. Sillevis Smitt, Pim J. French. Whole genome sequencing of oligodendrogliomas identifies genes mutated at a low frequency that contribute to tumor formation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3142. doi:10.1158/1538-7445.AM2013-3142